Identification of bacteria [link]
Biochemical tests [link]
Overview of Gram-positive cocci [link]
Staphylococcus aureus [link]
Meticillin-resistant S. aureus [link]
Glycopeptide resistance in S. aureus [link]
Coagulase-negative staphylococci [link]
Streptococci – overview [link]
Streptococcus pneumoniae [link]
Streptococcus bovis [link]
Viridans streptococci [link]
Group A Streptococcus [link]
Group B Streptococcus [link]
Other β-haemolytic streptococci [link]
Other Gram-positive cocci [link]
Overview of Gram-positive rods [link]
Bacillus species [link]
Bacillus anthracis [link]
Corynebacterium diphtheriae [link]
Non-diphtheria corynebacteria [link]
Erysipelothrix rhusiopathiae [link]
Rhodococcus equi [link]
Arcanobacterium haemolyticum [link]
Clostridium botulinum [link]
Clostridum tetani [link]
Other clostridia [link]
Actinomadura and Streptomyces [link]
Gram-negative cocci – overview [link]
Neisseria meningitidis [link]
Neisseria gonorrhoeae [link]
Non-pathogenic Neisseria [link]
Anaerobic Gram-negative cocci [link]
Escherichia coli [link]
Other Enterobacteriaceae [link]
Overview of Gram-negative rod non-fermenters [link]
Glucose non-fermenters [link]
Pseudomonas aeruginosa [link]
Stenotrophomonas maltophilia [link]
Burkholderia cepacia [link]
Burkholderia pseudomallei [link]
Burkholderia mallei [link]
Overview of fastidious Gram-negative rods [link]
Haemophilus influenzae [link]
Other Haemophilus spp. [link]
HACEK organisms [link]
Cardiobacterium hominis [link]
Eikenella corrodens [link]
Kingella kingae [link]
Yersinia pestis [link]
Yersinia enterocolitica [link]
Yersinia pseudotuberculosis [link]
Vibrio cholerae [link]
Vibrio parahaemolyticus [link]
Vibrio vulnificus [link]
Other Vibrio Species [link]
Prevotella and Porphyromonas [link]
Spirochaetes – an overview [link]
Treponema species [link]
Borrelia species [link]
Leptospira species [link]
Overview of Rickettsia [link]
Rickettsial diseases [link]
Coxiella burnetii (Q fever) [link]
Bartonella species [link]
Chlamydia trachomatis [link]
Chlamydophila psittaci [link]
Chlamydophila pneumoniae [link]
Mycobacterium tuberculosis [link]
Mycobacterium leprae [link]
Non-tuberculous mycobacteria [link]
Overview of virology [link]
Influenza – introduction [link]
Influenza – clinical features and diagnosis [link]
Influenza – treatment and prevention [link]
Respiratory syncytial virus [link]
Other respiratory viruses [link]
Parvovirus B19 [link]
Human herpesvirus type 6 [link]
Human herpesvirus type 7 [link]
Herpes simplex virus [link]
Varicella zoster virus [link]
Infectious mononucleosis [link]
Epstein–Barr virus [link]
Viral gastroenteritis [link]
Hepatitis A virus [link]
Hepatitis B virus [link]
Hepatitis D virus [link]
Hepatitis C virus [link]
Hepatitis E virus [link]
Hepatitis viruses [link]
HIV virology and immunology [link]
HIV laboratory tests [link]
Lymphocytic choriomeningitis virus [link]
Human herpesvirus type 8 [link]
Human papillomavirus [link]
Human T-cell lymphotropic virus [link]
Yellow fever [link]
Viral haemorrhagic fevers [link]
Arenaviruses (Lassa fever) [link]
Rabies virus [link]
Creutzfeldt–Jakob disease (CJD) [link]
Gerstmann–Sträussler–Scheinker syndrome [link]
Fatal familial insomnia [link]
Overview of fungi [link]
Candida species [link]
Superficial Candida infections [link]
Invasive Candida infections [link]
Malessezia infections [link]
Other yeasts [link]
Cryptococcus neoformans [link]
Pneumocystis jiroveci [link]
Other moulds [link]
Sporothrix schenckii [link]
Histoplasma capsulatum [link]
Blastomyces dermatitidis [link]
Coccidioides immitis [link]
Paracoccidioides brasiliensis [link]
Penicillium marneffei [link]
Plasmodium species (malaria) [link]
Toxoplasma gondii [link]
Trypanosoma cruzi [link]
Trypanosoma brucei complex [link]
Giardia lamblia [link]
Trichomonas vaginalis [link]
Entamoeba histolytica [link]
Free-living amoebae [link]
Ascaris lumbricoides [link]
Trichuris trichiuria [link]
Ancylostoma duodenale and Necator americanus [link]
Strongyloides stercoralis [link]
Enterobius vermicularis [link]
Cutaneous larva migrans [link]
Visceral larva migrans [link]
Trichinella species [link]
Dracunculus medinensis [link]
Loa loa [link]
Onchocerca volvulus [link]
Trematodes (flukes) [link]
Abdominal angiostrongyliasis [link]
Identification of bacteria in the diagnostic laboratory is based on phenotypic characteristics such as:
• Microscopic appearance
• Growth requirements
• Colonial morphology
• Haemolysis pattern
• Biochemical tests
• Antimicrobial susceptibility patterns
Many laboratory technicians are able to make a preliminary identification to genus level based on clinical data, cultural characteristics and a limited range of tests. Commercial identification systems such as the API (Analytical Profile Index) system (Biomerieux) contain a battery of biochemical tests that can identify the organism to species level.
Staining and microscopic examination of samples or cultures reveals the size, shape and arrangement of bacteria and the presence of inclusions e.g. spores. The following stains are commonly used:
• Gram stain – A fixed slide is flooded with 0.5% methyl / crystal violet (30 secs), followed by Lugol’s iodine (30secs), followed by rinsing with 95-100% ethanol or acetone, followed by counterstaining with 0.1% neutral red, safranin or carbol fuschin (2 mins). Gram positive organisms stain deep blue / purple and Gram-negative organisms stain pink / red.
• Acridine orange stain – This is used to identify Trichomonas vaginalis in vaginal specimens. The slide is stained with acridine orange (5-10 secs), decolourised with alcoholic saline (5-10 secs) and rinsed with normal saline. Once dry a drop of saline or distilled water and a coverslip are added and the slide is examined under fluorescence microscopy. Trophozoites ofT. vaginalis stain brick red with green nuclei.
• Auramine stain – This is used to identify mycobacteria in clinical specimens. It is considered more sensitive than the Ziehl Neelsen stain. A heat-fixed slide is flooded with auramine-phenol (1:10 v/v) for 10 mins. It is rinsed with water and then decolourised with 1% acid-alcohol for 3-5 mins (until no further stain seeps from the film). It is rinsed and stained with 0.1% potassium permanganate for 15 secs. It is rinsed and allowed to air dry before examination under fluorescence microscopy. Acid-fast bacilli appear bright yellow/green against a dark background.
• Ziehl Neelsen (ZN) stain – This is used to identify mycobacteria in cultures and provides better morphological detail than an auramine stain. A heat fixed slide is flooded with strong carbol fuschin and heated gently until it is just steaming. It is left to cool (3-5 mins), rinsed with water, and decolourised with a 3% v/v acid-alcohol solution (5-7 mins, until the slide is faintly pink). The slide is rinsed with water and counterstained with 1% v/v methylene blue or malachite green (30 secs). It is allowed to air dry before examination under oil immersion light microscopy. Acid-fast bacilli appear red on a blue or green background. A modified ZN stain is used for identification of Nocardia spp. and cryptosporidia.
• Nigrosin (India ink) stain – This stain is used to identify Cryptococcus neoformans in clinical specimens. A drop of India ink is put on the slide followed by a drop of the specimen and mixed together. A coverslip is applied and the slide is examined under light microscopy. Cryptococcus neoformans is identified by a clear zone (capsule) around the organism.
These can vary considerably and include:
• Atmosphere – Organisms can be divided into categories according to their atmospheric requirements:
• Strict aerobes grow only in the presence of oxygen
• Strict anaerobes grow only in the absence of oxygen
• Facultative organisms grow aerobically or anaerobically
• Microaerophilic organisms grow best in atmospheres with reduced oxygen concentration (e.g. 5-10% C02).
• Capnophilic organisms require additional C02 for growth
• Temperature – Organisms can also be differentiated by their temperature requirements:
• Psychrophilic organisms grow at temperatures of 10-30°C
• Mesophilic organisms grow at temperatures of 30-40°C
• Thermophilic organisms grow at temperatures of 50-60°C
Most clinically encountered organisms are mesophilic.
• Nutrition – Some organisms grow readily on ordinary nutrient media whereas others have particular nutritional requirements e.g. Haemophilus infleunzae requires the specific growth factors such as factor X (haemin) and factor V (NAD).
Bacterial colonies of a single species, when grown on specific media under controlled conditions, are described by their characteristic size, shape, texture and colour. Colonies may be flat or raised, smooth or irregular and pigmented (e.g. Pseudomonas aeruginosa is green/blue or Serratia marcescens is pink) or non-pigmented. Experienced laboratory technicians can often provisionally identify an organism using colonial appearance alone.
Some organisms produce haemolysins which cause lysis of red blood cells in blood containing media. This haemolysis may be:
• β-haemolytic – a clear zone of complete haemolysis around the colony
• α-haemolytic – a green zone of incomplete haemolysis
This feature is often used in the initial identification of streptococci.
For information on the identification of bacteria see the National Standard Methods which are available from the Health Protection Agency at http://www.hpa-standardmethods.org.uk/
A variety of biochemical tests may be used for the identification of bacteria in the diagnostic laboratory.
Many aerobic and facultatively anaerobic organisms are catalase positive whereas streptococci and enterococci are catalase negative. Hydrogen peroxide solution is drawn up into a capillary tube and the tip is then touched onto a colony. Vigorous bubbling indicates the presence of catalase. NB media containing blood may produce a false positive result.
This test is used to differentiate the staphyloocci. Coagulase exists in two forms: bound coagulase/clumping factor (detected by the slide coagulase test) and free coagulase (detected by the tube coagulase test).
• Slide coagulase test – A colony is emulsified in a drop of distilled water on a slide. A loop or wire is dipped into plasma and then mixed into the bacterial suspension. A positive test result occurs if agglutination is seen within 10 secs
• Tube coagulase test – A colony is emulsified in a tube containing plasma and incubated at 37°C for four hours. A visible clot indicates a positive result. If negative at 4 hours the tube should be reincubated overnight. NB Some species e.g. MRSA may give a negative result at 4 hours.
Deoxyribonuclease (DNase) test
This test is used to identify pathogenic staphylococci (e.g. S. aureus and S. schleiferi) which produce large quantities of extracellular DNase. A colony is streaked onto a DNase plate and incubated at 37°C for 18-24 hours. The following day the plate is flooded with hydrochloric acid – unhydrolysed DNA is precipitated producing a white opacity in the agar. Cultures surrounded by a clear zone (hydrolysed DNA) are DNase positive. NB some strains of MRSA are DNase negative and S. epidermidis may be weakly positive.
This test is used to differentiate S. pneumoniae (optochin sensitive) from other α-haemolytic streptococci (optochin resistant). Optochin (ethylhydrocupreine hydrochloride) is a chemical that causes lysis of the cell wall of S. pneumoniae. An optochin disc is placed in the centre of the bacterial inoculum and incubated at 37°C in 5% C02 for 18-24 hours. A zone of inhibition ≥5mm indicates a positive result.
Aesculin hydrolysis test
This test is used to differentiate enterococci (aesculin positive) from streptococci (aesculin negative). It tests the ability of the organism to hydrolyse aesculin to aesculetin and glucose in the presence of 10-40% bile. The aesculetin combines with ferric ions in the medium to form a black complex. The organism is inoculated onto a bile aesculin plate or slope and incubated at 37°C for 24 hours. Presence of a dark brown or black halo indicates a positive result.
The indole test is used to differentiate the Enterobacteriaceae. It detects the ability of an organism to produce indole from the amino acid tryptophan. A coloured product is obtained when indole is combined with certain aldehydes. There are 2 methods:
• Spot indole test – A piece of filter paper is moistened with the indole reagent and a colony is smeared onto the surface. A green / blue colour indicates a positive result
• Tube indole test – The organism is emulsified in a peptone broth and incubated at 37°C for 24 hours. 0.5mL of Kovac’s reagent is added; a pink colour in the top layer indicates a positive result.
ONPG (β-galactosidase) test
This test is used as an aid to differentiate the Enterobacteriaceae. Two enzymes, permease and β-galactosidase are required for lactose fermentation. Late lactose fermenters do not possess permease but do have β-galactosidase. Tubes containing o-nitrophenyl-β-D-galactopyranoside (ONPG) are inoculated with the organism and incubated at 37°C for 24 hours. If present, β-galactosidase hydrolyses ONPG to produce galactose and o-nitrophenol, a yellow compound.
This test is used to differentiate urease-positive Proteus spp. from the other Enterobacteriaceae. Some strains of Enterobacter and Klebsiella spp. are urease positive. Inoculate a slope of Christensen’s medium with the test organism and at 37°C for 24 hours. A pink / purple colour indicates a positive result.
This test determines if an organism has the cytochrome oxidase enzyme and is used as an aid in the differentiation of Pseudomonas, Neisseria, Moraxella, Campylobacter and Pasteurella spp (oxidase positive). Cytochrome oxidase catalyses the transport of electrons from donor compounds (e.g. NADH) to electron acceptors (usually oxygen). The test reagent, N, N, N’, N’-tetra-methyl-p-phenylenediamine dihydrochloride acts as an artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the coloured compound indophenol blue.
For information on the identification of bacteria see the National Standard Methods which are available from the Health Protection Agency at http://www.hpa-standardmethods.org.uk/
Gram-positive cocci are commonly isolated from clinical specimens. They are widely distributed in the environment and are found as commensals of the skin, mucous membranes and other body sites. Because of their ubiquitous nature, recovery of these organisms from specimens should always be interpreted in the context of the clinical presentation.
Gram-positive cocci can be classified into a number of groups
The family Micrococcaceae includes four genera:
• Planococcus – marine cocci, 9 species
• Micrococcus – ‘large cocci’ 1 – 1.8 micrometre diameter, 3 species
• Stomatococcus – upper respiratory tract commensal, 1 species
• Staphylococcus – human and animal commensals and pathogens, 0.5–1 micrometre in diameter, many species.
The other important group of Gram-positive cocci is the streptococci and similar organisms. They belong to a number of families:
• Staphyococcaceae → genus Gemella
• Lactillobacillaceae → genus Pediococcus
• Aerococcaeceae → genus Aerococcus, Abiotrophia, etc
• Carnobacteriaceae → genus Alloiococcus
• Enterococcaceae → genus Enterococcus
• Leuconostocaceae → genus Leuconostoc
• Streptococcaceae → genus Streptococcus, Lactococcus
• Staphylococci are non-motile, non-spore-forming, catalase-positive, Gram-positive cocci.
• They occur as single cells, pairs, tetrads or grape-like clusters (most common).
• Most species are facultative anaerobes, except S. aureus subsp. anaerobius and S. saccharolyticus, which are anaerobic.
• Staphylocci are normally found on the skin and mucous membranes of animals. In some cases their location may be very specific e.g. S. capitis subsp. capitis on the scalp or S. auricularis in the external auditory canal.
• S. aureus, S. epidermidis and S. saprophyticus are the main human pathogens.
• They may be differentiated on the basis of the coagulase test (see Box 4.1 ) into coagulase-positive (e.g. S. aureus) and coagulase-negative (CoNS, e.g. S. epidermidis).
• In addition, S. aureus produces DNase (deoxyribonuclease) while other staphylococci are usually DNase-negative.
• There are 30 or so species of CoNS but it is rarely necessary to identify them at the species level.
• The streptococci are non-sporing, non-motile organisms, catalase-negative Gram-positive cocci that grow in pairs and chains.
• Some species are capsulated.
• They are facultative anaerobes and often require enriched media to grow.
• The streptococci are subdivided on the basis of their ‘classic’ appearance on horse blood agar into α-, β- and non-haemolytic streptococci.
• The α-haemolytic streptococci (incomplete haemolysis on blood agar, resulting in a greenish tinge) include Streptococcus pneumoniae ( p. [link] ), and the viridans streptococci.
• The β-haemolytic streptococci (complete haemolysis/clear zone on blood agar) are grouped on the basis of their Lancefield carbohydrate antigens. The medically important ones are Groups A, B, C, F, and G.
• The enterococci (E. faecalis and others, p. [link] ) were originally called Streptococcus faecalis, and often react with group D antisera, but are now a separate genus.
• Non-haemolytic streptococci make up the remainder and include the viridans streptococci (S. mutans, S. salivarus, S. anginosus, S mitis, and S. sanguinis groups), the anaerobic and the nutritionally variant streptococci.
• For a comprehensive review of taxonomic and nomenclature changes of the streptococci see Facklam 2002). 1
S. aureus is a facultatively anaerobic, non-motile, non-spore-forming catalase-positive, coagulase-positive Gram-positive coccus. It is a major human pathogen and can cause a wide variety of infections ranging from superficial skin infections to severe life-threatening conditions, e.g. toxic shock syndrome
S. aureus is a skin colonizer and is found in the anterior nares of 10–40% of people. Chronic carriage is associated with an increased risk of infection, e.g. in haemodialysis patients. Nasal carriage has contributed to the persistence and spread of meticillin-resistant S. aureus (MRSA)
S. aureus possesses a wide array of virulence factors including:
• biofilm – this is an extracellular polysaccharide network produced by staphylococci (and other organisms) that result in colonization and persistence on prosthetic material. Polysaccharide intracellular adhesin (PIA) is synthesized by the ica operon
• capsule – more than 90% of S. aureus isolates have a capsule, with 11 serotypes reported
• surface adhesins also know as microbial surface components reacting with microbial surface components recognizing adherence matrix molecules or MSCRAMMs. These include protein A, clumping factor A and B, collagen-binding protein, fibronectin-binding protein, serine aspartate repeat protein, plasmin-sensitive protein, and surface proteins A to K
• techoic and lipotechoic acids – these are components of the cell wall. Lipotechoic acids trigger release of cytokines by macrophages
• peptidoglcan is the scaffold for anchoring the MSCRAMMs. It also triggers release of cytokines. Modification of peptidoglycan synthesis is associated with antimicrobial resistance
• haemolysins – S. aureus possesses four haemolysins (α, β, γ, and δ).
• Panton–Valentine leucocidin (PVL) – this is a haemolysin encoded by two genes (lukS and lukF) which are carried on a mobile phage (φSLT). PVL-producing strains are associated with furunculosis, severe haemorrhagic pneumonia, and clusters of MRSA skin infections
• exfoliative toxins – ETA and ETB are encoded by the eta and etb genes respectively. They cause staphylcococcal scalded skin syndrome
• superantigens – this group includes the toxic shock syndrome toxin (TSST-1) and the staphylococcal enterotoxins (SEs). TSST-1 is associated with toxic shock syndrome, whereas the SEs are associated with food poisoning
• pathogenicity (genomic) islands – these are structures that vary in size from 15 to 70 kB and harbour virulence and drug resistance genes, e.g. SaPI1 and SaPI2 carry the gene for TSST-1
• resistance islands – MRSA contains a resistance island called SCCmec which confers resistance to meticillin.
S. aureus can cause a wide spectrum of clinical infections including:
• skin and soft tissue infections, e.g. impetigo, folliculitis, furuncles, and carbuncles, hidradenitis suppuritiva, mastitis, wound infections, erysipelas, cellulitis, pyomyositis, necrotizing fasciitis
• bone and joint infections, e.g. septic arthritis, ostemyelitis
• systemic infections, e.g. bacteraemia, endocarditis, meningitis
• prosthetic device-related infections, e.g. intravascular catheter associated, pacemaker infections, prosthetic joint infections etc
• toxin-mediated diseases, e.g. scalded skin syndrome, toxic shock syndrome.
In some cases, e.g. skin and soft tissue infections, the diagnosis is clinical. In others appropriate samples e.g. pus, tissue, or blood should be taken and submitted to the laboratory for microscopy, culture and identification:
• Gram stain – Gram-positive cocci in clusters
• culture on blood agar or liquid media – growth usually occurs within 18–24 h. Prolonged incubation detects small colony variants
• biochemical tests – catalase-positive, coagulase-positive, DNase positive.
• identification – API Staph (Biomerieux)
• typing methods include PFGE, toxin typing, SCCmec typing and spa typing
• molecular diagnosis, e.g 16S or 23S RNA polymerase chain reaction (PCR) or mecA gene PCR (for meticillin resistance)
• Treatment depends on the type of infection and the drug susceptibility of the organism.
• Flucloxacllin PO (orally) or IV (intravenous) is used for meticillin-sensitive S. aureus isolates.
• Vancomycin IV is used in suspected S. aureus infections where MRSA is a possibility, e.g. hospitalized patient with intravascular catheter.
• Aminoglycosides exhibit synergism and are used in endocarditis to help sterilize the blood cultures.
• Other agents active against S. aureus include clindamycin, teicoplanin, and linezolid.
• Duration of treatment depends on the cause – 7 days for skin and soft tissue infection and up to 4 weeks for endocarditis. In bacteraemia if the source is removeable, e.g. intravascular catheter, 2 weeks of treatment is adequate.
• Prevention of S. aureus infections is based on bacterial decolonization of carriers with local antiseptics, e.g. nasal mupirocin and chlorhexidine soap. This is usually only done for MRSA carriers.
• Vaccines – a conjungate vaccine has been shown to reduce S. aureus bacteraemia rates in haemodialysis patients. 1
Meticillin-resistant S. aureus (MRSA) was first detected in 1961, a few months after meticillin was introduced into clinical practice. However, it was not until the 1980s that endemic strains of MRSA with multi-drug resistance became a global nosocomial problem. The epidemic strains of MRSA have been classified as E-MRSA 1 to 17, and the common ones currently circulating in the UK are E-MRSA-15 and E-MRSA–16. These strains have different genetics compared to the other epidemic strains and produce different toxins. They are also resistant to the macrolides, clindamycin, and ciprofloxacin ± other agents.
Mechanism of resistance
MRSA strains are resistant to all β-lactams due to alteration in the penicillin-binding protein PBP2′ and consequently the structure of the cell wall. Meticillin resistance is due to the mecA gene which codes for the low-affinity penicillin-binding protein PBP2′. mecA is usually located on a mobile genetic element called SCC-mec (staphylococcal cassette chromosome). There are five SCC-mec elements, defined by class of mecA gene and type of ccr complex (cassette chromosome recombinase). SCC-mec types I to III are usually found in hospital strains, while type IV is more common in the community strains.
• Risk factors for MRSA include increasing age, prior antibiotics, indwelling catheters, severe underlying disease, intensive care unit (ICU) stay.
• MRSA rates vary in different parts of the world. Countries like Finland, Denmark and the Netherlands with very low levels of MRSA (<5%) have strictly enforced contact precautions, take surveillance cultures of patients and personnel, and limit the use of broad-spectrum antibiotics. By contrast, some Asian countries (e.g. Japan, China) have high MRSA rates, probably because of antibiotic overuse. Many middle-income (e.g. Turkey) and some high-income countries (e.g. UK, USA) have hyperendemic MRSA and usually focus available resources on high-risk patients. Some countries with endemic MRSA (e.g. Australia, France, Belgium) have managed to stabilize or even lower MRSA prevalence in defined areas.
• Recent changes in the epidemiology of MRSA include:
• the increase in MRSA bacteraemia rates – in the early 1990s, 2% of S. aureus bacteramias in the UK were due to MRSA; the mean figure now is 45%. Reporting of MRSA bacteraemia rates is now mandatory in the UK
• the emergence of new community-associated MRSA strains, which are genetically different from previous healthcare-associated strains, and tend to be more virulent.
• MRSA causes similar infections to meticillin-sensitive strains of S. aureus (see Staphylococcus aureus, p. [link] ). Community-acquired MRSA (CA-MRSA) was originally seen in people with a previous history of hospitalization or who were related to healthcare workers. Since the late 1990s, however, serious infections of caused by CA-MRSA have been reported in previously healthy individuals. CA-MRSA is several times more likely to cause skin and soft tissue infections, often complicated by deep abscesses or necrotizing fasciitis. 1 There have also been community outbreaks of severe CA-MRSA pneumonia. 2 CA-MRSA is defined genetically (type IV SCCmec; distinct PFGE profile) and has evolved from community MSSA, rather than hospital MRSA. This genotype is often sensitive to non-beta-lactam antibiotics (e.g. ciprofloxacin) and Panton–Valentine leococidin toxin-positive. The highly successful USA-300 clone has caused considerable morbidity and mortality in the USA, 3 but is not yet a problem in Europe.
Laboratory diagnosis of MRSA
See National Standard Method. 4
• Criteria for meticillin resistance – presence of mecA gene or oxacillin minimum inhibition concentration (MIC) >2 mg/L or meticillin MIC >4 mg/L or cefoxitin MIC >4 mg/L.
• Molecular detection of the mecA gene is the diagnostic gold standard but is not available in many routine laboratories.
• Conventional methods for detecting MRSA rely on the use of selective media (mannitol salt agar with 7% NaCl, incubated at 37°C for 18–48 h). Broth enrichment in 7% NaCl prior to plating on selective media increases the diagnostic rate but increases the time for diagnosis by 24 h.
• Chromogenic agars (e.g. MRSA-ID) look promising and are recommended by the UK Health protection Agency (HPA) for MRSA screening.
• Isolates from patients with CA-MRSA or suspected toxin-mediated diseases should be submitted to the Laboratory for Healthcare Associated infection (LHCAI) at Colindale.
Treatment of MRSA
• All UK isolates are so far susceptible to the glycopeptides (e.g. vancomycin and teicoplanin), which are the treatment of choice. Vancomycin levels should be monitored, and current practice is tending towards greater serum antibiotic levels (e.g. trough of >15 mg/L).
• There is variable susceptibility to trimethoprim, rifampicin, tetracycline, doxycycline, fusidic acid, aminoglycosides (e.g. gentamicin), and nitrofurantoin (treatment of urinary tract infections (UTIs) only). These may provide alternative treatment choices if oral therapy or a second agent is required.
• Newer agents active against MRSA include linezolid, quinupristin with dalfopristin (Synercid®) and daptomycin. These are all licensed for treating skin and soft tissue infections. In addition, linezolid and synercid may be used for pneumonia.
Infection control issues
The main strategies to control MRSA are isolation/cohorting of patients, appropriate hand hygiene by healthcare workers, and effective cleaning of shared equipment.
1 Miller LG. Necrotizing faciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles. N Engl J Med; 352: 1445–53.
2 Francis S et al. Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Pantone-Valentine leukocidin genes. Clin Infect Dis 2005; 40: 100–107.
3 GJ Moran et al. Methicillin-resistant S. aureus infections among patients in the emergency department. New Engl J Med 2006; 355: 666–74.
Investigation of specimens for screening of MRSA, BSOP 29. Available from http://www.hpastandardmethods.org.uk/pdf-sops. (accessed 23 July 2008).
Mechanism of resistance
The mechanisms of vancomycin resistance in S. aureus are:
• an increase in cell wall turnover that leads to an increase of non-cross-linked D-alanyl-D-alanine side chains that bind vancomycin outside the cell wall and inhibit binding to target peptides
• transfer of the enterococcal vanA determinant from E. faecium to S. aureus,
Strains of S. aureus with homogenously reduced susceptibility to glycopeptides (vancomycin MIC ≥8mg/L) have been reported in several countries, e.g. Japan, France, and the USA. These organisms have been termed vancomycin-intermediate S. aureus (VISA), or glycopeptide-intermediate sensitivity S. aureus (GISA) based on the US Clinical Laboratory Standards Institute (CLSI) vancomycin MIC breakpoints:
• susceptible ≤4 mg/L
• intermediate 8–16 mg/L (VISA)
• resistant ≥32 mg/L (VRSA).
They would be considered resistant to vancomycin, according to the British Society of Antimicrobial Chemotherapy (BSAC) MIC breakpoints (susceptible ≤4 mg/L and resistant ≥8 mg/L).
A much more common situation is for a strain to yield a small proportion of daughter cells (1 in 105) able to grow in the presence of 8 mg/L of vancomycin. Such heterogeneously resistant strains are called hetero-VISA and there is considerable debate about their clinical significance.
The first clinical VRSA infection was reported in the USA in 2002. VRSA (vancomycin MIC >128 mg/L, teicoplanin MIC 32 mg/L) was isolated from a haemodialysis catheter tip and a chronic foot ulcer of a patient in Michigan. Vancomycin-resistant E. faecalis was also isolated from the ulcer, raising the possibility of transfer of the vanA determinant.
Clinical and epidemiological characteristics
• The first GISA infection occurred in 1995 in France in a child with leukaemia and catheter-associated MRSA bacteraemia.
• The second GISA infection occurred in 1996 in Japan in a 4-month-old infant with an MRSA sternal wound infection.
• Seven VISA infections were reported in the USA between 1997 and 2000. These all had several features in common:
• prolonged vancomycin exposure (3–18 weeks)
• prior MRSA infection
• underlying disease, especially renal failure
• prosthetic devices e.g. intravascular catheters, peritoneal dialysis (PD) catheter
• The first VRSA infection was reported in the USA in 2000 and since then six further VRSA infections have been reported in the USA.
The detection of glycopeptide resistance in S. aureus is problematic as both VISA and hetero-VISA isolates appear susceptible to vancomycin by routine disc diffusion tests. Furthermore, there have been conflicting recommendations regarding methods of detection:
• US guidelines – all laboratories should have an algorithm by which they can identify strains of S. aureus that may need additional testing. Laboratories should used acceptable confirmatory testing methods, e.g. 24-h incubation and MIC susceptibility testing method. Any S. aureus with a vancomycin MIC ≥4 mg/L should be referred to the Centers for Disease Control (CDC) for confirmatory testing
• UK recommendations – there are no specific guidelines for the detection of VISA/VRSA in the UK. However, the HPA recommends that reduced vancomycin susceptibility should be confirmed by E test. All vancomycin-resistant strains should be referred to the reference laboratory for confirmation.
Infection control issues
MRSA is known to be highly transmissible in healthcare settings and it seems reasonable to assume that VISA and VRSA will be likewise highly transmissible. Although infection control experience with VRSA is limited, implementation of rigorous infection control procedures is crucial for containing an outbreak of VRSA in a hospital setting. The US Hospital Infection Control Practices Advisory Committee (HICPAC) has published infection control guidelines for all staphylococci with a vancomycin MIC ≥8 mg/L:
• use contact precautions as recommended for multi-drug-resistant organisms (handwashing, gloves, gowns ± masks). Monitor and enforce compliance with contact precautions
• isolate patient in private room. Minimize number of people in contact with/caring for patient. Begin one-to-one care with specified personnel
• initiate epidemiological and laboratory investigations with the help of the state health department and CDC. Determine the extent of transmission within the facility. Assess the efficacy of precautions by monitoring the acquisition of VISA/VRSA by personnel
• educate all healthcare personnel about the epidemiology of VISA/VRSA and appropriate infection control precautions
• consult with state health departments and CDC before transferring or discharging the patient
• inform appropriate personnel about the presence of VISA/VRSA, e.g. emergency department personnel, admitting medical team.
The appropriate use of antimicrobials, especially vancomycin, is paramount in preventing the continued emergence of VISA and VRSA. Several studies have shown that vancomycin is frequently used for inappropriate reasons. Strategies to reduce inappropriate vancomycin use are essential, e.g. minimize use of temporary central venous catheters, use diagnostic techniques to avoid prolonged empiric use of vancomycin, prompt removal of S. aureus-infected prosthetic devices.
The coagulase-negative staphylococci (CoNS) may present as culture contaminants or be true pathogens. Infection is often associated with the presence of prosthetic material, e.g. intravascular catheters, cardiac valves, joint implants. Infections are often indolent but treatment may require removal of the foreign material. These organisms are often resistant to multiple antibiotics, which can make therapy difficult.
CoNS are ubiquitous and are natural inhabitants of the skin. S. epidermidis is the most common species, accounting for 65–90% of all isolates, followed by S. hominis. S. saprophyticus is a urinary pathogen. S. saccharolyticus is the only strict anaerobe. Other less-frequent species include S. haemolyticus, S. warneri, S. xylosus, S. cohnii, S. simulans, S. capitis, S. auricularis, S. lugdunensis (coagulase-positive), S. schleiferi (coagulase-positive).
Plasmid DNA is abundant in all species of CoNS but only a few of theplasmid-encoded genes have been identified. Plasmid-mediated antibiotic resistance to a wide variety of antibiotics is known to occur and may be transferred by conjugation with other organisms. CoNS also produce polysaccharide intracellular adhesin (PIA) resulting in biofilm formation, particularly on prosthetic devices. This biofilm protects the organsisms from antibiotics and host defence mechansisms. S. saprophyticus produces a number of substances that enable it to attach and invade the uroepithelium.
• Nosocomial bacteraemia (most common cause)
• Intravascular catheter-related infections e.g. lines, pacemaker wires
• Cerebrospinal fluid (CSF) shunt infections
• Peritoneal dialysis catheter-associated peritionitis
• Urinary tract infections (S. saprophyticus)
• Bacteraemia in immunocompromised patients
• Sternal osteomyelitis (post cardiothoracic surgery)
• Prosthetic joint infections
• Vascular graft infections
• Neonatal nosocomial bacteraemias
• Endophthalmitis (after surgery or trauma)
Appropriate samples, e.g. blood, pus, tissues should be taken and submitted to the laboratory for microbiological examination. The following tests may be performed:
• Gram stain – Gram-positive cocci in clusters
• culture on blood agar or liquid media – growth usually occurs within 18–24 h
• coagulase-negative (exceptions: S. lugdunensis, S. schleiferi)
• DNase test-negative, or weakly positive
• antimicrobial susceptility testing
• biochemical tests, e.g. API Staph
• typing, e.g. pulsed field gel electrophoresis
• molecular diagnosis e.g 16S or 23S RNA PCR or mecA gene PCR (for meticillin resistance)
Infections usually require the removal of prosthetic material, if present. CoNS are often resistant to multiple antibiotics; >80% are resistant to meticillin. Most CoNS are sensitive to vancomycin, linezolid, quinupristin/dalfopristin, and daptomycin. Sensitivity to teicoplanin is variable and a teicoplanin MIC must be checked before using this antibiotic. S. saprophyticus urinary tract infections may be treated with trimethoprim, nitrofurantoin or a fluoroquinolone.
For an introduction to streptococci see Overview of Gram-positive cocci, p. [link] .
Table 4.1 Classification of streptococci
Pneumococcal pneumonia, bacteraemia, meningitis, otitis media, sinusitis
Dental caries, endocarditis
Invasive (necrotising fasciitis, GAS toxic shock syndrome, bacteraemia etc); tonsillitis, skin infections etc
Neonatal meningitis and bactermaemia
S. dysgalactiae subsp. dysgalactiae
S. dysgalactiae subsp. equisimilis
S. equi subsp. equi
S.equi subsp. zooepidemicus
Sore throat, cellulitis
S. bovis and others
S. bovisendocarditis, S. suis bacteraemia and meningitis
β or non
A, C, F, G
S. milleri (reclassified as S. constellatus, S. intermedius and S. anginosus).
Infective endocarditis, Abscesses
Genera closely related to streptococci
Leuconostoc; Pediococcus; Abiotrophia; Gemella; Aerococcus
S. pneumoniae was first isolated in 1881 by Sternberg in the USA and Louis Pasteur in France. It became recognized as the most common cause of lobar pneumonia and was given the name pneumococcus. S. pneumoniae is an important bacterial pathogen of humans causing meningitis, sinusitis, otitis media, endocarditis, septic arthritis, peritonitis, and a number of other infections. It is a Gram-positive coccus that grows in pairs (diplococci) or chains. It produces pneumolysin which causes α-haemolysis (green discolouration due to breakdown of haemoglobin) of blood agar. It is catalase-negative, inhibited by ethyl hydrocupreine (optochin sensitive), and lysed by bile salts.
• S. pneumoniae colonizes the nasopharynx of 5–10% of healthy adults and 20–40% of healthy children. The rate of colonization is seasonal, with an increase in winter.
• The rate of invasive pneumococcal disease is 15/100,000 persons/year. The incidence is up to 10-fold higher in certain populations, e.g. African-Americans, Alaskans, and Australia aboriginals. Invasive pneumococcal disease is more common at the extremes of age (age <2 years or >65 years).
• Risk factors for pneumococcal infection include antibody deficiencies, complement deficiency, neutropenia or impaired neutrophil function, asplenia, corticosteroids, malnutrition, alcoholism, chronic diseases (liver, renal, diabetes, asthma, chronic obstructive pulmonary disease (COPD), overcrowding).
• Antimicrobial resistance is increasing. Rates are high in European countries, e.g. Spain, Hungary, and in Asia, e.g. Thailand, Hong Kong, Vietnam and Korea. The major source of resistance is the worldwide geographic spread of a few clones that harbour resistance determinants.
A number of virulence factors have been identified:
• capsular polysaccharide >90 serotypes (prevents phagocytosis, activates complement)
• cell wall polysaccharide (activates complement and cytokine release)
• pneumolysin (activates complement and cytokines)
• PspA (inhibits phagocytosis by blocking activation and deposition of complement)
• PspC (inhibits phagocytosis by binding complement factor H)
• PsaA (mediates adherence)
• autolysin (causes release of bacterial components)
• neuraminidase (possible mediates adherence).
Antimicrobial resistance mechanisms
• Penicillin resistance mediated by alterations in PBP2A (low-level resistance) and mutations in PBP2X (high-level resistance).
• Macrolide resistance is mediated by acquisition of ermB (ribosomal methylase) and mefA (efflux pump) genes.
S. pneumoniae may cause infection either by direct spread of the organism from the nasopharynx to contiguous structures (e.g. middle ear, sinuses, and lungs), or by haematogenous spread (to the central nervous system (CNS), heart valves, bones, joints, peritoneum). Clinical syndromes include:
• otitis media
• exacerbation of chronic bronchitis
• septic arthritis
• others – percarditis, epidural abscess, cerebral abscess, skin and soft tissue infections. Unusual infections in young people should prompt investigation for HIV.
• Grows on routine media – causes α-haemolysis of blood agar
• Gram-positive lanceolate diplococci, often with visible capsule
• Identification – catalase-negative, optochin sensitive, soluble in 10% bile salts. Commercial identification tests (e.g. API strep, latex agglutination tests, and serotyping tests) are available
• Penicillin MIC should be determined for invasive isolates
• Depends on the nature and severity of the presenting infection and drug susceptibility results
• Penicillin MIC <0.1mg/L penicillin or amicillin
• Penicillin MIC > 0.1 ≤ 1.0 mg/L – ceftriaxone or cefotaxime for meningitis. High-dose penicillin or ampicillin is likely to be effective for non-meningeal sites of infection, e.g. pneumonia.
• Penicillin MIC ≥ 2.0 mg/L – vancomycin ± rifampicin. If non-meningeal site, also consider ceftriaxone or cefotaxime, high-dose ampicillin, carbapenem, active fluoroquinolone, e.g. moxifloxacin
• Immunization – the 7-valent pneumococcal conjugate vaccine (PCV) was introduced into the UK childhood immunization schedule in 2006, and is given to all children >2 months old in the UK. The 23-valent unconjugated polysaccharide vaccine is given to ‘at-risk’ groups, e.g. adults >65 years old, homozygous sickle cell disease, asplenia/severe splenic dysfunction, chronic renal disease or nephrotic syndrome, celiac disease, immunodeficiency or immunosuppression due to disease or treatment, including HIV infection, chronic diseases (cardiac, respiratory, liver, renal), diabetes mellitus, patients with cochlear implants.
• Antimicrobial prophylaxis – oral penicillin V is recommended for the prevention of pneumococcal disease in asplenic patients.
Enterococci are environmental organisms that are found in the soil, water, food, and the gastrointestinal tract of animals. They are Gram-positive cocci that occur singly, in pairs or in chains and thus resemble streptococci. Until fairly recently they were classified among the Lancefield group D streptococci. In the 1980s they were reclassified as a separate genus, Enteroccoccus, because of different pathogenic, biochemical and serological profiles. At least 12 different species exist. E. faecalis is the most common clinical isolate (80–90%), followed by E. faecium (5–10%). Others include E. avium, E. casseliflavus, E. durans, E. gallinarum, E. hirae, and E. raffinosus
Enterococci are part of the normal gut flora and can cause endogenous or exogenous infections, both in and out of hospital. In the hospital setting, enterococci are readily transmissible between patients and institutions. In the USA enterococci are a common cause of nosocomial infections. Risk factors for nosocomial enterococcal infections include GI colonization, severe underlying disease, prolonged hospitalization, prior surgery, renal failure, neutropenia, transplantation, urinary or vascular catheters, intensive care unit (ICU) admission.
• Enterococci are less intrinsically virulent than organisms such as S. aureus and group A streptococci. They do not have classical virulence factors but are able to adhere to heart valves and renal epithelial cells. Several extracellular molecules play an important role in colonization and adherence, e.g. aggregation factor and extracellular surface protein). Other virulence factors include extracellular serine protease and gelatinase (GelE) and haemolysins.
• Enterococci are frequently found in cultures of intra-abdominal and pelvic infections – their role in this setting has not been clearly defined.
• Enterococcal bacteraemia carries a high mortality (42–68%) but it is not clear whether this is due to the organism itself or a marker of severe debilitation. However, epidemiological studies have calculated an attributed mortality of 31–37% in patients with enterococcal bacteraemia.
• The intrinsic resistance of enterococci to many antibiotics enables them to survive and multiply in patients receiving broad-spectrum agents, and accounts for their ability to cause superinfections.
• Urinary tract infections (most common)
• Bacteraemia and endocarditis
• Intra-abdominal and pelvic infections
• Skin, wound, and soft tissue infections
• Meningitis (associated with anatomical defects, trauma, or surgery)
• Respiratory infections (rare)
• Neonatal sepsis
• Gram stain – elongated Gram-positive cocci (‘cigar-shaped’), often in pairs and short chains
• Culture – facultative anaerobes that can grow under extreme conditions, e.g. 6.5% NaCl, pH 9.6, temperatures of 10°C to 45°C
• Biochemical tests – enterococci hydrolyse aesculin and l-pyrrolidonyl-β-naphthylamide (PYR)
• They usually agglutinate with Group D in streptococcal grouping kits.
• Intrinsically resistant to aminoglycosides (low levels), β-lactams (high MICs), lincosamides (low level), co-trimoxazole (in vivo), and quinupristin/dalfopristin (E. faecalis)
• All isolates should be tested for susceptibility to ampicillin, gentamicin, vancomycin, teicoplanin, linezolid, Synercid, chloramphenicol and nitrofurantoin (UTIs)
• Enterococci are intrinsically resistant to many agents (e.g. cephalosporins, ciprofloxacin), and readily acquire new resistance mechanisms.
• Ampicillin is usual first-line agent for E. faecalis infections, with vancomycin as an alternative. E. faecium is usually resistant to ampicillin.
• When bactericidal therapy is needed, (e.g. endocarditis, meningitis), combination synergistic therapy of a cell-wall agent plus aminoglycoside is standard.
• Ciprofloxacin may be active in vitro, but is not usually recommended clinically (apart from occasionally for UTIs). Newer fluoroquinolones are said to be more active against the enterococci, but not against ciprofloxacin-resistant strains, which may preclude their usefulness.
• E. gallinarum and E. casseliflavus are intrinsically resistant to glycopeptides (pentapeptide terminates D-alanine-D-serine).
• High-level resistance to aminoglycosides and vancomycin resistance (VRE) are increasing problems, particularly on renal units.
• VRE bacteraemia has a worse prognosis than vancomycin-sensitive enterococcal bacteraemia, but this may be related to comorbidity and delay in receiving appropriate antibiotic therapy.
S. bovis bacteraemia and endocarditis are associated with gastrointestinal disease (primarily colonic malignancy). There are two biotypes of S. bovis: S. bovis biotype 1 bacteraemia has a higher correlation with underlying GI malignancy and endocarditis (71% and 94% respectively, in one study) than S. bovis biotype 2.
It is not clear whether S. bovis is a marker for malignancy or has an aetiological role. In some cases, S. bovis bacteraemia is the only pointer to the GI disease. There are also reports of the malignancy being found up to 2 years after the initial S. bovis infection. There seems to be an increase in stool carriage of S. bovis in patients with malignancy or pre-malignancy compared to healthy subjects. Some investigators have suggested biotype I has a type-specific adherence mechanism, which enables adherence to both cardiac valves and abnormal colonic mucosa.
The main clinical infections due to S. bovis are bacteraemia and endocarditis. Occasionally, S. bovis causes other infections such UTIs, meningitis or neonatal sepsis. The GI tract is the usual portal of entry for bacteraemia, and there is a strong association of bacteraemia with endocarditis. Most patients with endocarditis have an underlying valve abnormality or prosthetic valve. They tend to have a subacute course, indistinguishable clinically from endocarditis due to the Streptococcus viridans group, but studies suggest S. bovis endocarditis has a higher mortality rate (45%) compared to non-S. bovis endocarditis (25%).
• S. bovis may be misidentified as enterococci or viridans streptococci (notably S. salivarus).
• Biochemical tests – S. bovis shares a number of properties with enterococci, e.g. they agglutinate with group D antisera, hydrolyse aesculin, and are bile tolerant. However, they differ from enterococci by growing in 6.5% salt and in the results of PYR test.
• Identification – the ‘API Rapid Strep’ reliably identifies S. bovis, and differentiates it to the biotype level, which is important for association with malignancy and endocarditis. Generally, S. bovis biotype 1 strains produce extracellular glucan from sucrose, hydrolyse starch, and ferment mannitol: S. bovis biotype 2 strains are usually negative for these tests. A PCR to differentiate the biotypes has been developed.
• S. bovis is highly susceptible to penicillin (MICs 0.01–0.12 microgram/mL), It is also susceptible to ampicillin, the antipseudomonal penicillins, erythromycin, clindamycin, and vancomycin.
• Penicillin is the treatment of choice for S. bovis infections. Vancomycin is an alternative in β-lactam-allergic patients.
• Although penicillin/aminoglycoside combinations show synergy against S. bovis, combination therapy is no more effective than penicillin alone for treatment of endocarditis.
The viridans streptococci, sometimes known as the oral streptococci, are important in dental caries and endocarditis, bacteraemia, and deep-seated infections. They include S. sanguis, S. mutans, S. mitis and S. salivarus. This heterogenous group has been reclassified in to five distinct groups, on the basis of 16S rDNA analysis:
• the S. mutans group is now divided into seven species and collectively known as the ‘mutans streptococci’. The most common are S. mutans and S. sobrinus
• the S. sanguinis group is now divided into S. sanguinis, S. gordonii, S. parasanguis, and S. crista
• the S. milleri group is now divided into three species: S. constellatus; S. intermedius, and S. anginosus – now called the S. anginosus group/group F
• the S. mitis group includes S. mitis, S. mitior, and S. oralis.
• the S. salivarus group includes S. salivarus and S. vestibularis
The viridans streptococci are commensals of the human upper respiratory tract, femal genital tract, and gastrointestinal tract, with large numbers present in the mouth. Each species has its own particular ecological niche.
• These organisms seem to possess few virulence factors.
• The ability to produce acid, especially by S. mutans, is thought to be important in dental caries.
• Production of various carbohydrates, which aid adherence to tooth enamel and gums, is important in the establishment and maintenance of colonization.
• Extracellular dextran production is important in the adherence of organisms to heart valves and in resistance to antimicrobial therapy.
• Fibronectin production also mediates adherence to heart valves.
• Endocarditis (common cause in patients with abnormal valves)
• Bacteraemia (especially in neutropaenic patients)
• Other infections – abscesses, pericarditis, peritonitis, sialadenitis, odontgenic infections, endophthalmitis
• Facultatively anaerobic Gram-positive cocci, catalase-negative
• Most are α-haemolytic on blood agar; some are non-haemolytic
• Resistant to optochin and lack bile solubility (unlike pneumococci)
• Unable to grow in 6.5% NaCl (unlike enterococci)
• Can be identified by biochemical tests or API STREP
• Community-acquired infections are usually sensitive to penicillin which is the treatment of choice.
• Other β-lactams, e.g. ceftriaxone also have good in vitro acitivity against viridans streptococci.
• Nosocomial infections are associated with increased resistance to pencillin and other β-lactams.
• Some strains, e.g. S. sanguis and S. gordonii exhibit tolerance – inhibited at low concentrations of antibiotic but high levels required for bactericidal activity.
• Often resistant to aminoglyocides (when traditional breakpoints are applied) but exhibit synergy in combination with B-lactam antibiotics. This principle underlies combination treatment for bacterial endocraditis.
• Vancomycin is used in penicillin-allergic patients and penicillin-resistant infections.
Group A Streptococcus (GAS), also known as Streptococcus pyogenes, is responsible for a variety of conditions, ranging from sore throat to severe invasive infections, such as necrotizing fasciitis, which have a mortality approaching 10%. There are also a number of post-infectious ‘immunological’ conditions such as post-streptococcal glomerulonephritis and rheumatic fever.
GAS are upper respiratory tract commensals in 3–5% of adults and up to 10% of children. Transmission is mainly via droplet spread. Some people develop pharyngitis/tonsillitis, others are asymptomatic, and a handful will become carriers of GAS in the throat. In the 1990s, the number of reports of invasive GAS increased globally, probably due to a re-emergence of more virulent strains. Risk factors for sporadic disease include people >65 years old, those with recent varicella zoster (VZV) infection, HIV-positive individuals, those with diabetes, heart disease, cancer, injecting drug use, or those on high-dose steroids. Over time, the epidemiology of GAS infection in terms of clinical manifestation of disease has changed, e.g. scarlet fever and acute rheumatic fever have become less common and toxic shock more common over the last few decades.
Group A streptococci possess a number of virulence factors:
• somatic constitutents – hyaluronic capsule, M protein, serum opacity factor, lipotechoic acid, fibronectin-binding proteins
• extracellular products – streptolysin O, streptolysin S, DNases A to D, hyaluronidase, streptokinase, streptococcal pyrogenic exotoxins (SpeA, SpeB, SpeC, SpeF), C5a peptidase, and streptococcal superantigens (SSA).
• Pharyngitis – most common infection. Suppurative complications include tonsillitis, peritonsillar abscess, retropharngeal abscess, suppurative cervical lymphadenitis, mastoiditis, sinusitis, otitis media
• Scarlet fever – notifiable disease. Similar to pharyngitis but associated with scarlatinal rash due to erythrogenic toxin production
• Rheumatic fever – may occur 1–5 weeks after pharyngitis. Relapses may occur
• Post-streptococcal glomerulonephritis – may occur after throat infections (commonly M types 12, 1, 25, 4, and 3) and skin infections (commonly M types 49, 52, 53–55, and 57–61), and is due to immunological cross-reactions between components of the glomerular basement membrane and cell membranes of nephritogenic streptococci
• Impetigo, erysipelas, cellulitis, necrotizing fasciitis, pyomyositis
• Bacteraemia – recent increase in group A streptococcal bacteraemia in previously healthy adults. Also associated with intravenous drug users (IVDUs)
• Puerperal sepsis – historically associated with group A streptococci
• Streptococcal toxic shock syndrome – fulminant disease with a high mortality is mainly associated with types M1 and 3, but types 12 and 28 are also involved. It is differentiated from the other types of invasive disease by the occurrence of shock and multi-organ failure early in the course of the infection
• Others – meningitis, osteomyelitis, and septic arthritis
• GAS are facultative anaerobic, catalase-negative Gram-positive cocci, which tend to form long chains. They are non-sporing, non-motile and usually non-capsulate.
• Culture on blood agar produces smooth, circular colonies of 2–3 mm diameter, which are usually β-haemolytic. Strains that produce haemolysin O and not haemolysin S will only demonstrate β-haemolysis when cultured anaerobically.
• Lancefield grouping will reliably and accurately identify GAS.
• Most GAS are sensitive to bacitracin.
• Serology – used to diagnose immunological complications, e.g rheumatic fever rather than in caute disease. A rise in anti-streptolysin O titre (ASOT) confirms recent group A streptococcal disease. ASOT is reliable in the throat-associated disease, while antiDNAase B is higher and more frequently raised in pyoderma-associated disease.
• The treatment of choice is oral phenoxymethylpenicillin (mild infections) or IV benzylpenicillin (severe infections). Pharyngitis is treated for 10 days.
• In penicillin-allergic patients, options include azithromycin (which has comparative clinical and bacteriological response rates to phenoxymethylpenicillin, but higher GI side-effects) or erythromycin. In 2003 3–4% of GAS isolates in the UK were resistant to macrolides so sensitivity testing is required.
• Treatment of more-severe infections, e.g. toxic shock syndrome usually requires addition of a second agent to the penicillin – options include clindamycin (prevents toxin secretion).
• Urgent surgical debridement is required in necrotizing fasciitis.
• Infection control – GAS can spread from infected patients to close contacts, so isolate patients with invasive disease and involve the infection control team early.
• Antimicrobial prophylaxis – The available evidence suggests that routine administration of prophylactic antibiotics for close contacts of invasive disease is not justified. 1 All household contacts should be informed of clinical manifestations of invasive disease and instructed to seek medical attention immediately if they develop any symptoms. Antibiotics are only given to certain ‘high-risk’ groups.
Group B streptococci (GBS, Streptococcus agalactiae) were first reported as causes of puerperal sepsis in 1938. By the 1970s, group B streptococci have become the main cause of neonatal sepsis in infants aged <3 months.
• 5–40% of women are colonized with GBS (genital tract or lower GI tract). Colonization of neonates usually occurs via the mother’s genital tract. Risk factors – African-American, diabetes
• Early-onset neonatal GBS disease (≤7 days) – risk factors include GBS bacteriruria, premature rupture of membranes, delivery <37 weeks, intra-partum fever or amnionitis, prolonged rupture of membranes
• Late-onset neonatal GBS disease (7–90 days) – risk factors include overcrowding, poor hand hygiene, increased length of stay
• Over the past 20 years there has been an increase in invasive group B streptococcal disease in non-pregnant adults, most of whom had underlying medical conditions. Risk factors include diabetes, chronic diseases (liver, renal, cardiovascular, pulmonary, GI, urological), neurologic impairment, malignancy, HIV, corticosteroids, splenectomy
Bacterial virulence factors that influence the outcome between exposure and development of colonization/invasive disease include the polysaccharide capsule (in particular high amounts of sialic-acid and type III virulent strains).
• Early-onset neonatal disease (defined as systemic infection in the first 6 days of life; mean age of onset = 12 h, ± pneumonia or meningitis), tends to results from vertical transmission in utero or at the time of delivery.
• Late-onset neonatal disease (onset 7 days to 3 months of age, mean = 24 days) arises from either horizontal transmission (often nosocomial, due to suboptimal nursery conditions) or vertical transmission.
• GBS infection in adults and older children, especially those with underlying disease includes bacteraemia, postpartum infections, pneumonia, endocarditis, meningitis, arthritis, osteomyelitis, otitis media, conjunctivitis, UTI, skin and soft tissue infections, and meningitis.
• GBS are facultative anaerobic, catalase-positive Gram-positive cocci. They are non-sporing, non-motile and usually capsulate.
• Culture on blood agar produces smooth, circular colonies of 2–3 mm diameter, which are usually surrounded by a very small zone of β-haemolysis.
• Selective media containing Todd Hewitt broth and antimicrobials are used to enhance recovery of group B streptococci.
• Identification – Lancefield group B, resistant to bacitracin, hydrolyse sodium hippurate, do not hydrolyse aesculin hydrolysis, production of CAMP factor (results in synergistic haemolysis with the β-lysin of S. aureus on sheep blood agar plate).
• Typing – GBS may be classified as serotypes I to VIII, based on the basis of capsular polysaccharide and surface protein antigens. Other typing methods: multi locus sequence typing (MLST), pulsed field gel elctrophoresis (PFGE).
• Neonatal infections are usually treated with IV ampicillin + gentamicin initially, then penicillin G.
• Adults usually receive 10–14 days of IV penicillin G (+2 weeks gentamicin for endocarditis); vancomycin if penicillin-allergic.
• Routine screening for antenatal carriage not recommended
• Antibiotic treatment of GBS carriage not recommended
• Newborns with signs of sepsis should be treated with broad-spectrum antibiotics that cover GBS
• Infant whose mother is colonized with GBS or has had a previous infant with GBS should be monitored for at least 12 h
• Consider intrapartum antibiotics if two or more risk factors for early-onset GBS disease
• Give intrapartum antibiotics if previous child had GBS disease
• If chorioamnionitis suspected treat with broad-spectrum antibiotics active against GBS
• Vaccines – capsular polysaccharide vaccines are under development, including a vaccine conjugated with tetanus toxoid
1 Green-top guideline no. 36. Prevention of Early Onset Neonatal Group B Streptococcal Disease – available from http://www.rcog.org.uk/index.asp?pageID=520.
• Group C streptococci – there are four species in this group: S.dysgalactiae subsp. dysgalactiae, S. dysgalactiae subsp. equisimilis, S.equi subsp. equi, S. equi subsp. zooepidemicus. They are primarily animal pathogens, but S. equisimilis and S. zooepidemicus can cause a range of infections in humans. The most common problem in humans is outbreaks of tonsillitis, especially in schools and institutions. The group C streptococci can cause syndromes similar to group A streptococci such as postpartum sepsis, septicaemia, meningitis, pneumonia, skin, and wound infections – but group C infections are usually less severe. Group C streptococci are usually sensitive to the penicillins.
• Group F streptococci – these were formerly known as Streptococci milleri, which has a characteristic caramel odour when cultured in the laboratory. However, S. milleri has been reclassified within the viridans streptococcus group into S. constellatus, S. intermedius, and S. anginosus.
• Group G streptococci – these produce infections similar to group A and C streptococci, such as sore throat, erysipelas, cellulitis, bone and joint infection, pneumonia, and septicaemia. Occasionally group G streptococci bacteraemia is associated with underlying malignancy.
Other Gram-positive cocci
Leuconostoc are catalase-negative Gram-positive cocci or coccobacilli, which occasionally cause opportunistic infections. They are usually found on plants and vegetables, or rarely in dairy products and wine. There are only a few case reports of human infections, including bacteraemia (± indwelling line infection), meningitis, and dental abscess.
Note that leuconostoc are intrinsically resistant to the glycopeptides, because the pentapeptide cell wall precursors terminate in D-alanine-D-lactate. The usual agent of choice for these infections is penicillin or ampicillin, but they are generally susceptible to most agents with activity against streptococci.
Abiotrophia is the new name for the nutritionally variant streptococci (NVS). These organisms have been classified in various ways, but 16S rRNA sequencing defined the new genus Abiotrophia to be distinct from the streptococci. NVS are defined by the need for pyridoxal or thiol group supplementation for growth, and thus appear as satellite colonies around bacteria such as S. aureus. Gram staining tends to show pleomorphic variable-staining cells. The two main species, A. defectiva and A. adiacens, are resistant to optochin and susceptible to vancomycin. However, because they grow poorly on solid media, they are easily overlooked if not grown in broth or subcultured appropriately.
Abiotrophia are normal flora in the upper respiratory, urogenital, and GI tract, and are clinically important as they cause approx 5% of cases of endocarditis. Abiotrophia endocarditis responds less well to antibiotics, and has higher morbidity and mortality compared to endocarditis due to other streptococci. Correlation of in vitro antibiotic susceptibility testing and clinical outcome is a specialist field, and the general recommendation is for long-term combination therapy (e.g. penicillin and gentamicin for 4–6 weeks). Bacteriological failure and relapse rates are high.
Anaerobic Gram-positive cocci
Anaerobic Gram-positive cocci have undergone multiple taxonomic changes. Nucleic acid sequencing (particularly 16S rRNA) resulted in most species formerly classified as Peptococcus being transferred to the genus Peptostreptococcus. Other species include Coprococcus, Ruminococcus, Sarcina, and Streptococcus saccharolyticus.
Peptostreptococcus is an obligate anaerobe that is part of normal flora in the mouth, upper respiratory tract, GI tract, vagina, and skin. The most common species are P. magnus, P. micros, P saccharolyticus, and P. anaerobius, and they make up 20–40% of anaerobes isolated clinically. They cause abscesses (e.g. brain abscess, often associated with otitis media, mastoiditis, chronic sinusitis, and pleuropulmonary infections), anaerobic pleuropulmonary disease, and bacteraemia (notably due to oropharyngeal, pulmonary, and female genital tract sources). When mixed with other bacteria, they may be involved with serious soft tissues infections such as necrotizing fasciitis. Peptostreptococcus causes anaerobic osteomyelitis and arthritis at all sites, including bites and cranial infections. Little is known about virulence factors or pathogenesis of infection. Regarding treatment, anaerobic Gram-negative cocci are often mixed with aerobes and anaerobes on culture plates. Obtaining appropriate specimens may be difficult, culture can be prolonged, and anaerobic sensitivity testing can also be challenging. Usually a combination of surgery (e.g. drainage/debridement) and antibiotic therapy is required. Most anaerobic Gram-positive cocci are sensitive to metronidazole, penicillin, and clindamycin.
Aerococcus viridans, and the recently described Aerococcus urinae, are catalase-negative, Gram-positive cocci. They tend to form tetrads and may resemble staphylococci on Gram stain, but their biochemical and growth characteristics are more characteristic of α-haemolytic streptococci.
Aerococcus viridans is generally considered a contaminant on culture, but occasionally may be implicated in bacteraemia and endocarditis. It is a low-virulent organism and only causes systemic infections in the immunocompromised. Optimal treatment of such cases is unclear, so consult an infection specialist.
Aerococcus urinae, first reported in 1989, has been implicated as a cause of approx. 0.5% of UTIs. Most patients were elderly with predisposing conditions. It has also been found in patients with urogenic bacteraemia/septicaemia with or without endocarditis. A. urinae is usually susceptible to penicillin and resistant to sulphonamides and aminoglycosides.
The gram-positive rods can be divided into a number of groups (Table 4.2 ):
• aerobic Gram-positive rods
• anaerobic Gram-positive rods
• branching Gram-positive rods.
Table 4.2 Classification of Gram-positive rods
Bacillus spp. are environmental saprophytes that are found in water, vegetation, and soil. They are Gram-positive (or Gram-variable) aerobic or facultatively anaerobic rod-shaped bacilli with rounded or square ends. They form endospores that tolerate extremes of temperature and moisture. The ubiquitous nature of Bacillus spp. means that isolation from clinical specimens may represent contamination. Members of the group include:
• B. anthracis (see Bacillus anthracis, p. [link] )
• B. cereus
• B. circulans
• B. licheniformis
• B. megaterium
• B. pumilis
• B. sphaericus
• B. subtilis
• B. stearothermophilus.
• Food-poisoning – B. cereus is the most common cause; may also be caused by B. licheniformis and B. pumilis. Occurs within 24 h of ingestion of the preformed toxin in food. The emetic form presents after 1–5 h, with nausea, vomiting, and abdominal cramps. The diarrhoeal form occurs 8–24 h after ingestion of food. Production of a heat-labile toxin results in profuse diarrhoea and abdominal cramps (fever and vomiting are rare). Symptoms usually resolve in 24 h.
• Bacteraemia is the most common systemic infection and is often associated with the presence of an intravascular catheter. B. cereus is the most common isolate but other species, e.g. B. licheniformis have been reported. Bacteraemia or endorcarditis may occur in injecting drug users.
• Disseminated infection has been reported in neonates and young children. Neonatal infection is acquired perinatally. Multisystem involvement may occur. Immunocompromise, e.g. neutropenia is associated with severe and sometimes fatal infections.
• CNS infections may occur following trauma or neurosurgery, or in association with a CSF shunt. Removal of hardware is required. Lumbar puncture may result in Bacillus spp. meningitis.
• Eye infections – endophthalmitis may occur following trauma, eye surgery, or haematogenous dissemination. B. cereus is the most common cause. Keratitis may occur after corneal trauma.
• Soft tissue and muscle infections may occur after injuries or wounds, e.g. road traffic accidents or after orthopaedic surgery.
Bacillus spp. grow readily on ordinary culture media at environmental temperatures (25–37°C). All species may form spores but they vary in their colonial morphology, motility, and nutritional requirements. Microscopically they are large bacteria and are usually Gram-positive (older cultures may be Gram-variable or Gram-negative). Colonies are described as anthracoid as they resemble B. anthracis. However, most Bacillus spp. are β-haemolytic and motile (unlike B. anthracis). They also lack the glutamic acid capsule (thus negative McFadyean’s stain).
• There is no specific treatment for food poisoning syndromes and most cases settle in 24 h.
• For intravascular catheter- or prosthetic device-related infections, removal of the catheter or device is required for cure.
• Most Bacillus spp. isolates are susceptible to vancomycin, clindamycin, fluoroquinolones, aminoglycosides, and carbapenems.
• Serious infections are usually treated with vancomcyin or clindamycin ± an aminoglycoside.
The name anthrax is derived from a Greek word for coal and refers to the eschar seen in cutaneous anthrax. Anthrax occurs most commonly in wild and domestic animals in Asia, Africa, South and Central America, and parts of Europe. Humans are rarely infected and the most common form of infection is cutaneous anthrax, which is associated with occupational exposure to animal products, e.g. wool, hair, meat, bones, and hides. Anthrax was used as an agent of bioterrorism in the United States in 2001 when B. anthracis spores were sent in contaminated letters.
B. anthracis has a number of virulence factors:
• capsule – under anaerobic conditions a polypeptide capsule consisting of poly-D-glutamic acid is produced. Synthesis of the capsule is by three enzymes encoded by the capA, capB, and capC genes on the pX-02 plasmid. A fourth protein, encoded by the dep gene, catalysese the formation of low molecular weight polyglutamates that inhibit phagocytosis
• toxin – two binary toxins (o)edema factor (EF) and lethal factor (LF) bind a third toxin component, protective antigen (PA) before entering the target cell. The three toxin components are also encoded on a plasmid pX-01. The cellular receptor for PA, the anthrax toxin receptor was identified in 2001. LF is a zinc-dependent metallopeptidase that inhibits dendritic cell function. EF converts adenosine monophoshpate (AMP) to cyclic AMP (cAMP), resulting in dysregulation of water and ions.
There are four forms of human disease:
• cutaneous anthrax – >95% of cases, usually acquired by direct contact with infected animals. The incubation period is 1–12 days and the initial lesion is a pruritic papule, which becomes a vesicular or bullous lesion surrounded by non-pitting oedema.The central part becomes necrotic and haemorrhagic and may develop satellite vesicles. Finally there is a classic black eschar which falls off in 1–2 weeks, unless systemic disease ensues
• gastrointestinal anthrax– accounts for <5% of case. Oropharyngeal anthrax presents with febrile neck swelling due to cervical adenopathy and soft tissue oedema after ingestion of contaminated meat. Intestinal anthrax is more common and presents with fever, syncope and malaise followed by abdominal pain, nausea and vomiting. Examination shows abdominal distension and a mass in the right iliac fossa or periumbilical area. The third phase is characterized by paroxysmal abdominal pain, ascites, facial flushing, red conjunctivae, and shock
• inhalational anthrax – very rare. Occurs after inhalation of spores. The incubation period is <1 week. It presents as a flu-like illness with non-productive cough, haemorrhagic mediastinal lymphadenopathy, and multilobar pneumonia ± pleural effusions and bacteraemia. Chest x-ray (CXR) typically shows a widened mediastinum. High mortality rate (45–85%)
• CNS disease – very rare. Presents with a haemorrhagic meningoencephalitis; 95% mortality.
• Specimens – B. anthracis may be isolated from wound swabs (if cutaneous disease); nasal swabs and blood cultures.
• Microscopy – Gram-positive rods 4 × 1 micrometre in ‘box car’- or cigar-shaped chains. The spore is oval shaped and central or subterminal. McFadyean’s stain shows capsulated, dark, square-ended bacilli in short chains.
• Culture – B. anthracis grows readily on ordinary media (optimal incubation temperature 35°C), after 2–5 days incubation. Colonies are white or grey-white with a characteristic ‘medusa head’ appearance. In contrast to most other Bacillus spp., B. anthracis is non-haemolytic and non-motile.
• Identification – B. anthracis can be identified by PCR or phage lysis.
• For flowcharts outlining the clinical evaluation and management of possible anthrax, see www.hpa.org.uk
• Cutaneous anthrax – ciprofloxacin or doxycycline for 60 days
• Inhalational anthrax – initial therapy: intravenous ciprofloxacin or doxycycline and one or two additional antimicrobials (e.g. rifampicin, vancomycin, penicillin, ampicillin, chloramphenicol, imipenem, clindamycin, clarithromycin). This is followed by ciprofloxacin or doxycycline until day 60.
• Vaccines – human and animal vaccines are available to prevent anthrax. Vaccination is recommended for workers at risk of cutaneous anthrax. Vaccination is also recommended post-exposure to inhalational anthrax.
• Antibiotic prophylaxis – oral ciprofloxacin or doxycycline are indicated for post-exposure prophylaxis of inhalational anthrax.
The name for C. diphtheriae is derived from the Greek ‘korynee’ meaning club and ‘diphtheria’ meaning leather hide (for the leathery pharyngeal membrane it provokes). Diphtheria is rare in the UK but remains a common problem in developing countries and the former Russian states. The organism spreads via nasopharyngeal secretions, and can survive for months in dust and contaminated dry fomites. Incidence is highest in young children (>3–6 months old), when protective maternal antibodies wane.
C. diphtheriae exerts its effects by production of a potent exotoxin which inhbits protein synthesis in mammalian cells. It consists of two fragments: fragment A (which inhibits polypeptide chain elongation at the ribosome) and fragment B (which helps transport fragment A into the cell). Inhibition of protein synthesis probably accounts for the toxin’s necrotic and neurotoxic effects, which are mainly on the heart, nerves and kidneys.
• Respiratory tract – asymptomatic upper respiratory tract carriage is common in countries where diphtheria is endemic and is an important reservoir of infection. Anterior nasal infection presents with a serosanguinous or seropurulent nasal discharge often associated with a whitish membrane. Faucial infection is the most common site for clinical diphtheria. Clinical features include fever, malaise, sore throat, pharyngeal injection, development of a pseudomembrane which is initially white, then grey with patches or green or black necrosis. Cervical lymphadenopathy may result in a characteristic ‘bull neck’ and inspiratory stridor.
• Cardiac disease – myocarditis occurs after 1–2 weeks, usually as the oropharngeal disease is improving. Patients should be monitored by electrocardiogram (ECG) which may show ST segment changes, heart block, and arrhythmias. Clinical features include dyspnoea, cardiac failure, arrhythmias and circulatory collapse.
• Neurological disease – local paralysis of the soft palate and posterior pharyax lead to nasal regurgitation of fluids. Cranial nerve palsies and ciliary muscle paralysis may follow. Peripheral neuritis occurs 10–90 days after onset of pharyngeal disease and presents with motor deficits
• Skin infections – in the tropics chronic non-healing ulcers with grey membranes may be due to C. diphtheriae. Outbreaks have been described in homeless alcoholics in the USA.
• Invasive disease – endocarditis, mycotic aneurysms, septic arthritis, and osteomyelitis have been described, caused by non-toxigenic strains.
• Culture – nasopharyngeal, throat, or skin swabs should be immediately transported to the laboratory and cultured on suitable culture media (e.g. Loeffler’s, Hoyle’s tellurite, Tinsdale media). The colonies are black on tellurite media. C. diphtheriae shows a halo effect on Tinsdale’s agar.
• Microscopy – Gram staining of C. diphtheriae shows characteristic palisades, resembling Chinese letters. The beaded appearance obtained by Neisser or Albert stains, whereby the volutin/metachromatic granules are dark purple compared to brown/green counterstain, is characteristic.
• Identification – C. diphtheriae is a non-motile, non-sporing and non-capsulate Gram-positive rod. It is catalase-positive, urease-negative, nitrate-positive, pyrazinamidase-negative and cystinase-negative. It can reliably be identified with the API Coryne. Isolates should be submitted to the HPA reference laboratory at Colindale for toxigenicity testing. Several methods are available: Elek plate, rapid enzyme immunosorbent assay (EIA) or PCR.
• Biotyping – colonial appearance on tellurite, and also biochemical tests (e.g. Hiss serum sugars) subdivide C. diphtheriae into the biotypes var. gravis, intermedius and mitis. These biotypes correspond with clinical severity. Gravis and intermedius (and some mitis) biotypes are usually toxigenic. The fourth biotype, var. belfanti is rare and cannot produce the lethal exotoxin.
• Antibiotics – if high clinical suspicion, treat immediately with IV penicillin for 14 days. Alternatives: erythromycin, azithromycin or clarithromycin. Confirm elimination by nasopharyngeal swab; if cultures are positive give a further 10 days of antibiotics.
• Antitoxin may be given, at different doses depending on site and severity (see guidelines below). Firstly test the patient with a trial dose to exclude hypersensitivity to horse serum.
• Infection control – isolate and barrier nurse the case. Identify close contacts, take nose and throat swabs, and arrange clinical surveillance for 7 days. Provide prophylactic antibiotics (single dose of benzylpenicillin or seven-day course of erythromycin) and booster vaccination for close contacts.
• Notification – diphtheria is a notifiable disease, therefore contact the CDSC.
Diphtheria toxoid is part of the triple vaccine DTP (diphtheria, tetanus, polio) given at 2, 3, and 4 months as part of the UK immunization schedule. Immunity can be assessed by the Schick test, which is no longer used in the UK. Note that diphtheria can occur in immunized individuals.
• Laboratory guidelines for the diagnosis of infections caused by C. diphtheriae and C. ulcerans. Commun Dis Public Health 1999;2:250–7. (also available at www.HPA.org.uk)
• Control of diphtheria: guidance for consultants in communicable disease control. Commun Dis Public Health 1999; 2: 242–9.
Corynebacteria are also known as coryneforms or diphtheroids. They are environmental organisms found in water and soil, and commensals of the skin and mucous membranes of humans and other animals. In the hospital environment they may be cultured from surfaces and equipment. Thus corynebacteria are frequently considered contaminants but may cause severe disease in hospitalized or immunocompromised patients.
Corynebacteria are classified according to cell wall composition and biochemical reactions into the following groups:
• non-lipophilic fermentative, e.g. C. ulcerans, C. pseudotuberculosis, C. xerosis, C. striatum, C. minutissium, C. amycolatum, C. glucuronolyticum
• non-lipophilic non-fermentative, e.g. C. pseudodiptheriticum
• lipophilic, e.g. C. jeikeium, C. urealyticum.
Infections may be classified into two groups:
• community-acquired infections, e.g. pharyngitis, native valve endocarditis, genitourinary tract infections, periodontal infections
• nosocomial infections, e.g. intravascular catheter-associated bacteraemia, endocarditis, prosthetic device-related infections, surgical site infections.
• Microscopy – club-shaped Gram-positive rods. Cells demonstrate variable size and appearance from coccoid to bacillary forms depending on the stage of their life cycle. Corynebacteria typically aggregate to form ‘Chinese letter’ arrangements.
• Culture – corynebacteria grow readily on blood agar and blood culture media. Thioglycolate broth may be used for wound cultures. Special media used for species identification include tryptic soy agar with or without 1% Tween-80 to assess lipid -enhanced growth.
• Identification – corynebacteria are catalase-positive, nitrate-positive, and urease-positive. They can be identified to species level by the API CORYNE system. The CAMP test (named after Christie, Atkins, and Munch–Petersen) may be used. In this test a streak of β-lysin producing S. aureus is plated onto blood agar and the test strain is plated perpendicular to it. A positive reaction is seen if CAMP factor (a haemolysin secreted by some corynebacteria) enhances the haemolysis produced by S. aureus.
• Susceptibility testing is problematic but isolates are uniformly sensitive to vancomycin, teicoplanin, and daptomycin.
Infections caused by various corynebacteria
• C. ulcerans is primarily a cause of bovine mastitis. However, it has the potential to produce diphtheria toxin and cause an exudative pharyngitis indistinguishable from C. diphtheriae. Several reported outbreaks of diphtheria have been found to be due to C. ulcerans.
• C. pseudotuberculosis is an animal pathogen that causes caseous lymphadenitis in sheep. Human disease is rare but granulomatous lymphadenitis has been seen in farm workers and vets.
• C. xerosis is a commensal of the human nasopharynx, conjunctiva, and skin. It may cause severe invasive disease immunocompromised patients.
• C. striatum is a commensal of the skin and mucous membranes. It can rarely cause severe invasive disease in hospitalized patients. It may not be correctly identified by the API CORYNE
• C. minutissimum is a skin commensal which was previously though to cause eryhthrasma. Bacteraemia and endocarditis may occur in patients with indwelling catheters or immunocompromise.
• C. amycolatum is another skin commensal. There are case reports of invasive disease. It may not be correctly identified by the API CORYNE.
• C. glucuronolyticum – normal flora of genitourinary tract. May cause urinary tract infection and prostatitis.
• C. pseudodiphtheriticum – normal flora of upper respiratory tract. Primarily associated with respiratory tract infections in immunocompromised patients.
• C. jeikeium colonizes the skin of hospitalized patients. It may cause severe nosomial infections, e.g. bacteraemia, endocarditis, meningitis, CSF shunt infections, prosthetic joint inections. Risk factors include immunocompromise (malignancy, neutropenia, and AIDS), indwelling catheters and devices, prolonged hospital stay, broad-spectrum antibiotics, and impaired skin integrity. C. jeikeium is resistant to many antibiotics, and vancomycin is the treatment of choice.
• C. urealyticum colonizes the skin of hospitalized patients. It causes chronic and recurrent urinary tract infections mainly in the elderly or immunosuppressed.
Listeria monocytogenes is the main species in this genus, and affects pregnant women, their babies, the immunocom-promised (especially those with impaired cell-mediated immunity) and the elderly. L. ivanovii occasionally causes human infection. Generally L. innocua; L. welshimeri, and L. seeligeri are non-pathogenic to humans. Up to 5% of healthy adults carry Listeria spp. in the gut. While listeria infections are rare in the general population, they can cause life-threatening bacteraemia and meningoencephalitis in susceptible groups. Clinical infections have high mortality rates.
Disease is mainly sporadic, but may be part of an epidemic associated with contaminated foodstuffs such as pâté, unpasteurised milk, chicken, or soft cheese. Hospital outbreaks have been reported. Vets or farmers may become infected through direct animal contact. Human–human transmission occurs vertically (i.e. mother–baby), and cross-infection in neonatal units has been reported.
While the number of pregnancy-associated cases of listeriosis has been relatively stable, there was a dramatic rise in non-pregnancy-associated listeriosis between 2001 and 2004, especially in people over 60 years old. The reasons for this are unclear.
Animal studies have identified listeriolysin O: this is important for bacterial survival after phagocytosis, and its production is related to extracellular iron. In rodents, T lymphocytes are important in protective immunity rather than antibodies. T cells attract monocytes to the infection, acti-vate them and destroy the listeria, resulting in granuloma. The organisms themselves show tropism for the brain itself, particularly the brainstem and meninges. In humans, gastrointestinal disease (e.g. low gastric pH or disrupted normal flora) may help establish listeria infection in the bowel.
• Pregnancy – maternal listeriosis is rare before 20 weeks’ gestation. After this, infection may be asymptomatic or present with mild symptoms such as fever, back pain, sore throat, and headache. Fever may result in reduced fetal movements, premature labour, stillbirth, abortion, or early-onset neonatal disease.
• Neonate – early neonatal disease occurs <5 days post delivery, usually presents with septicaemia, and has a mortality of 30–60%. Late neonatal disease occurs >5 days post delivery, usually presents as meningitis, and may be hospital acquired. Mortality in late disease is lower (approx 10%).
• Adults – the main syndromes are CNS infection and meningitis, septicaemia, and endocarditis. Rare manifestations include other CNS disease (such as encephalitis, cerebritis, CNS abscesses), arthritis, hepatitis, endophthalmitis, continuous ambulatory peritoneal dialysis (CAPD) peritonitis, gastroenteritis, pneumonia. Risk factors include immunosuppression due to steroids, cytotoxic therapy, and HIV. Note that in Listeria meningitis, CSF biochemistry may be indistinguishable from bacterial meningitis. The Gram stain is often negative for organisms, but Listeria may sometimes be cultured from blood.
• Neonatal disease – early = 30–60% (20–40% survivors developing long-term sequelae such as lung disease or CNS defects). Late = 10%.
• Adult disease – CNS = 20–50%; bacteraemia = 5–20%; endocarditis = 50%. Up to 75% survivors of CNS infection have sequelae such as hemiplegia or CNS defects.
• Microscopy – Listeria are short intracellular Gram-positive rods. However, in clinical specimens they may appear Gram-variable, and look like diphtheroids, cocci, or diplococci.
• Culture – Listeria grow on blood agar but selective media are available. Colonies are sometimes β-haemolytic on blood agar, and can be mistaken for streptococci or enterococci.
• Identification – they exhibit tumbling motility at 25°C. They are non-sporulating, and catalase-positive, aesculin-positive, and oxidase-negative. They grow optimally at 30–37°C, but better than most bacteria at 4–10°C (refrigeration temperature). L. monocytogenes and L. seeligeri show enhanced haemolysis in the presence of S. aureus (positive CAMP test). L. ivanovii produces a positive CAMP test.Species can also be differentiated by fermentation of D-xylose, L-rhamnose and α-methyl-D-mannoside.
• Typing techniques in current use include phage typing, serotying, PFGE and multi-locus enzyme electrophoresis (MLEE). Serotyping of L. monocytogenes with rabbit antisera results in 13 serovars: serovar 4 is commonest in human infections (but serovars 1/2a and 1.2b are also important).
Ampicillin ± gentamicin is the usual regimen for meningitis, with co-trimoxazole or meropenem as an alternative for patients who are penicillin allergic. There are no randomized controlled trials to establish the most effective drug or duration of therapy. In meningitis, antibiotics are usually given for at least 14 days (longer in immunocompromised). Most other clinical syndromes should be treated with ampicillin, with consideration given to adding gentamicin for synergy. Vancomycin may be given for bacteraemia, but has been associated with relapse of disease. NB cephalosporins should never be used to treat listeriosis.
E. rhusiopathiae is a thin, pleomorphic, non-sporing, Gram-positive rod. It was first isolated in mice by Robert Koch in 1878 and from swine by Louis Pasteur in 1882. It was identified as a human pathogen in 1909.
E. rhusiopathiae is found in a wide variety of animals and invertebrates – the reservoir is thought to be swine. The organism in transmitted to humans by direct contact. Most human cases are associated with occupational exposure, e.g. fishermen, fish handlers, farmers, vets, butchers, abattoir workers.
There are three clinical presentations:
• erysipeloid – a localized skin lesion. The organism enters the skin by trauma and after an incubation period of 2–7 days, pain and swelling of the affected digit occurs. The lesion is well defined, slightly raised, and violaceous. It spreads peripherally with central fading. Regional lyphadenopathy and lymphangitis may occur
• diffuse cutaneous eruption – this is rare and caused by progression of the primary lesion. Fever and arthralgia may occur. Recurrence is common
• bacteraemia is also rare but frequently associated with endocarditis.
• Microscopy – E. rhusiopathiae is a straight to slightly curved Gram-positive rod (1–2.5 micrometre)– it decolorizes readily and may appear Gram-negative. Rods may be arranged singly, in V-shaped pairs, short chains or non-branching filaments.
• Culture – colonial and microscopic appearances vary with the medium, pH and incubation temperature. Incubation in 5–10% CO2 improves culture.
• Identification – E. rhusiopathiae is catalase-, oxidase-, indole-, Vosges–Proskauer- and Methyl-red-negative.
• Drug susceptibility – E. rhusiopathiae is usually susceptible to penicillins, cephalosporins, clindamycin, imipenem, and ciprofloxacin. It is resistant to vancomycin, teicoplanin, sulfonamides, co-trimoxazole, and aminoglyocisides.
R. equi (previously known as Corynebacterium equi) was identified in 1923 as an animal pathogen causing pneumonia in horses. Since then it has been found in a wide variety of animals. The first human case was reported in 1967 – an immunosuppressed patient who presented with a cavitatory pneumonia. Since the 1980s the rise in the numbers of immunosuppressed patients (AIDS and transplantation) has been mirrored by and increase R. equi infections.
• Necrotizing pneumonia is the most common clinical presentation (80%) and is characterized by a cavitation on the CXR. Blood cultures are positive in 50% of HIV patients and 25% of solid organ transplant recipients.
• Extra-pulmonary infection may affect the brain or present as subcutaneous or organ abscesses. Bacteraemia may also occur, usually associated with intravenous catheters.
• R. equi is a Gram-positive obligate aerobe that is a non-sporing and non-motile.
• Microscopy – it may appear coccoid or bacillary on Gram stain, depending on growth conditions. It can be acid-fast.
• Culture – R. equi grows optimally at 30°C and produces salmon-pink colonies. Selective media include colistin nalidixic acid agar (CNA), phenyl ethanol agar (PEA), or ceftazidime novobiocin agar.
• Identification: R. equi is catalase, lipase, urease and phosphatase-positive. It differs from other coryneforms by its lack of ability to ferment carbohydrates or liquefy gelatin. It can be identified using the API CORYNE, ribotyping or PCR RFLP (restriction fragment length polymorphism).
Optimal treatment has not been determined by clinical trials. R. equi is susceptible to vancomycin, erythromycin, fluoroquinolones, rifampicin, imipenem, and aminoglycosides. Combinations of two or three antimicrobials are usually used until antimicrobial susceptibility results are available.
Arcanobacterium haemolyticum is a β-haemolytic, catalase-negative Gram-positive rod which pits the agar when a colony is removed. Identification can be confirmed by the ‘API CORYNE’. It causes acute pharyngitis, and has also been associated with infective endocarditis and skin sepsis. It is sensitive to most antibiotics, except co-trimoxazole. Treatment is usually with penicillin or erythromycin.
C. botulinum is widespread in the soil and environment. It produces one of the most potent toxins known, which causes botulism.
Toxins A–G have identical pharmacological effects, despite possessing different antigens. All can cause human disease, but A, B, and E are most common. Note that the type-specific antibody must be given to a patient with suspected botulism (see below).
• Food-borne botulism, the preformed toxin is ingested from food (hams, sausages, tinned fish, meat, and vegetables (particularly home-preserved), honey). The food itself may not appear spoiled. Botulinum toxin is absorbed from the human GI tract and blocks the release of acetylcholine mainly in the peripheral nervous system. Initial symptoms include nausea and vomiting, diplopia, and bilateral ptosis (due to oculomotor muscle involvement), followed by progressive descending motor loss with flaccid paralysis. Speech and swallowing become difficult, but the patient maintains consciousness and has in normal sensation. Botulism is fatal in 5–10% cases. Death is usually due to cardiac or respiratory failure.
• Wound botulism causes a similar clinical picture, but is due to growth of the organism. Outbreaks have occurred in intravenous drug users.
• Intestinal botulism is also due to organism proliferation in the gut and toxin production in vivo.
• Infant botulism presents as the ‘floppy child syndrome’ usually in babies <6 months, because the gut is not yet resistant to colonization.
If there is a suspected clinical case, involve experts and always alert lab staff as the toxin is dangerous. Human samples (blood, faeces, vomit) and food should be tested for the organism and the toxin. C. botulinum is a motile strictly anaerobic rod, with optimal growth at 35°C, but some strains able to grow as low as 1–5°C. The oval subterminal spores are very hardy: some spores persist despite boiling at 100°C for several hours. Moist heat at 120°C for 5 min usually destroys spores.
Involve the intensive care unit, as the patient is likely to need organ support. A polyvalent antitoxin is available to neutralize unfixed toxin. In food-borne disease, any unabsorbed toxin should be removed from the stomach and GI tract. In wound botulism, give benzyl penicillin and metronidazole, and surgical debridement. Antibiotics are not recommended for food-borne or intestinal botulism.
C. tetani causes ~10 cases of tetanus/year in the UK. However, this vaccine preventable disease still causes considerable morbidity and mortality in the developing world. Tetanus is a notifiable disease.
Resilient spores survive in soil and GI tract of horses and other animals for a long time. Transmission usually occurs via introduction of spores into open wounds (particularly in injecting drug users), patients with recent abdominal surgery, patients with ear infections (otogenic tetanus), and neonates after cutting the umbilical cord (tetanus neonatorum). C. tetani produces tetanospasmin (powerful neurotoxin which diffuses to the CNS and causes localized or generalized disease), and tetanolysin (oxygen-labile haemolysin).
Localized tetanus involves muscle rigidity and painful spasms near the wound site. Usually a prodrome of generalized tetanus, with symptoms summarised by ROAST (rigidity, opisthotonus, autonomic dysfuction, spasms, and trismus)
Tetanus is a clinical diagnosis. There are three microbiological tests:
• isolation of C. tetani from the infection site. C. tetani is a motile, obligate anaerobe which classically produces ‘drumstick’ terminal spores. It often stains Gram-negative. C. tetani produces a thin spreading film on enriched blood agar, due to the motility by peritrichous flagella. If C. tetani is suspected, involve the HPA Anaerobe Reference Laboratory
• presence of tetanus toxin in serum (performed at HPA Colindale Food Safety Laboratory)
• low/no antibody levels to tetanus toxin is supportive of the diagnosis.
Involve ICU early. Give tetanus immunoglobulin (TIG); wound debridement, and antimicrobials including metronidazole or penicillin. Vaccination with tetanus toxoid following recovery is important to prevent future episodes. See Table 4.4 .
Table 4.4 Recommendations for vaccination
Full, i.e. 5 doses
Only if high risk
Primary immunization complete, boosters incomplete but up to date
Only if high risk
Primary immunization incomplete/boosters not up to date/never immunized/status unknown or uncertain
Yes – 1 dose and plan to complete schedule
Yes – 1 dose and plan to complete schedule
Yes – 1 dose in a different site
Tetanus-prone wound risk factors include the following
• Puncture-type wound,
• contact with soil or manure,
• clinical evidence of sepsis
• significant degree of devitalized tissue
• any wound with delay of >6 h before surgical treatment.
Tetanus immunization, introduced into the UK in 1961, now involves the combined tetanus/low-dose diphtheria vaccine (Td) (previously single antigen vaccines (T) were given). Five doses of tetanus toxoid are considered to give lifelong immunity (usually three as DTP as part of childhood immunizations and two doses of Td later). See the Department of Health’s ‘Green Book’: Immunisation against Infectious Disease (2006) for more information.(http://www.dh.gov.uk/en/PublicHealth/Healthprotection/Immunisation/Greenbook/dh_4097254).
These anaerobic Gram-positive spore-forming organisms are responsible for a variety of conditions, many of which involve toxin production (Table 4.3 ). The rods are pleomorphic, but typically large, straight, or slightly curved, with rounded ends.
Table 4.3 Diseases caused by Clostridium spp.
C. botulinum a
C. tetani a
Antibiotic-associated diarrhoea/pseudomembranous colitis
Toxin A and B
C. perfringens a
Type A causes gas gangrene
C. novyi a
Type A causes gas gangrene
Debate re pathogenicity
a Clusters in injecting drug users in Europe in the last 5 years.
• C. perfringens causes gas gangrene. It is occasionally isolated from blood cultures, and may be associated with food poisoning (enterotoxin production), endocarditis, or a contaminant. In developing countries it may cause enteritis necroticans (‘pig bel’).
• C. histolyticum and C. sordellii may be associated with gas gangrene
• C. novyi gas gangrene is due to C. novyi type A (C. novyi types B, C, and D are differentiated by toxin permutation and soluble antigen production, and do not cause human disease). Compared to C. perfringens, C. novyii bacilli are larger and more pleomorphic. It is a stricter anaerobe, and has peritrichous flagella, but motility is inhibited in the presence of oxygen. The oval spores are central or subterminal. There are at least four toxins which possess haemolytic, necrotizing, lethal, lipase, and phospholipase activities. There was a large outbreak among injecting drug users in Scotland in 1999–2000.
• C. sporogenes is probably not pathogenic in its own right. It is usually encountered in a mixed wound culture containing accepted pathogens, and may have a role in enhancing local conditions and accelerating an established anaerobic infection.
• C. septicum usually lives in the soil, human, or animal gut and can cause gas gangrene in humans and animals. C. septicum bacteraemia is seen with breakdown of gut integrity, e.g. in leukaemia. Gram stain appearance of the organism may be variable, with long, short, and filamentous Gram-positive rods, together with some older Gram-negative cells. Spores start off as swollen Gram-positive ‘citron bodies’ then tend to be oval, bulging, and either central or subterminal. C. septicum grows well on ordinary media at 37°C, and has numerous peritrichous flagella, hence is actively motile. Colonies are often initially transparent and ‘droplet-like’, with projecting radiations, then become grey and opaque with time. The α exotoxin has lethal, haemolytic, and necrotizing properties, and can be demonstrated in cultures.
• C. difficile is an increasing nosocomial infection, which is now subject to mandatory reporting. It can cause C. difficile-associated diarrhoea (CDAD) and pseudomembranous colitis. Clinical features vary, and diagnosis is usually by toxin tests rather than culture. Infection control measures are paramount to control the spread of this organism.
Mobiluncus is a genus of anaerobic Gram-positive rod shaped bacteria. These organisms are found in the female genital tract in association with Gardnerella vaginalis. There are two named species: M. curtisii (smaller, Gram-variable, and slightly bent), and M. mulieris (larger, Gram-negative and crescent-shaped).
Adherence to vaginal squamous epithelial cells is important, and may be caused by a glycocalyx.
Mobiluncus spp. has been detected in >97% of women with bacterial vaginosis (together with mixed anaerobic flora), and ~5% of healthy controls. It has also been found in other sites, such as breast abscesses and mastectomy wounds.
These curved Gram-variable rods grow slowly under anaerobic conditions. Electron microscope studies have described a Gram-positive cell wall. Their characteristic corkscrew motility is due to multiple flagella. They need an enriched media for growth, and are oxidase-, catalase- and urease-negative. Gram stain morphology can reliably differentiate the two species.
Actinomyces species are mouth commensals that may cause the chronic granulomatous infection actinomycosis. The main species of human importance are A. israelii and A. gerencseriae. Others include A. meyeri (isolated from brain abscesses); A. viscosus (found in dental caries); and also A. naeslundii and A. odontolyticus.
Actinomycosis is endogenously acquired, and those with dental caries are at increased risk. It is unclear why males are affected more than females. Historically rural farm workers were affected more than those living in towns, purportedly because of poor dental hygiene. Abscesses, tissue destruction, fibrosis, and sinus formation are typical findings. The masses of mycelia in relatively young lesions may be visible as yellow sulphur granules; later on they form dark brown, hard granules due to calcium phosphate deposition.
Most human cases of actinomycosis are in the cervicofacial area, especially around the jaw. Infection may follow dental procedures. Haematogenous spread to the liver, brain, and other organs is well recognized. In addition to facial disease, clinical presentations include thoracic actinomycosis (due to aspiration of oral actinomyces; characterized by chest wall sinuses and bony erosion of the ribs and spine), appendix or colonic diverticula actinomycosis, pelvic actinomycosis (linked with intrauterine contraceptive devices (IUCDs)), cerebral actinomycosis, and ‘punch actinomycosis’ (knuckle infection due to human bite).
Tissue biopsies of suspect lesions are stained with fluorescein-conjugated specific antisera, to demonstrate characteristic sulphur granules and mycelia. Any sulphur granules available should be crushed and stained with Gram stain – branching Gram-positive rods.
Actinomyces often fail to grow aerobically, so plates should be incubated anaerobically and under micro-aerophilic conditions (i.e. 5–10% CO2). A. israelii form large ‘molar teeth’-shaped colonies, from 2 to 10 days. Further identification can be confirmed at a reference laboratory by biochemical tests, fluorescent antisera staining, or gas chromatography of metabolic products of carbohydrate fermentation. Note that sputum often contains oral actinomyces.
Surgical involvement is vital, and debridement reduces scarring, deformity, and the recurrence rate. Removal of an IUCD is the primary treatment for pelvic disease. Actinomycosis is usually treated with penicillin or ampicillin, for up to six months. Broad spectrum antibiotics e.g. co-amoxiclav, or ceftriaxone and metronidazole may be needed if there are concomitant pathogen. Despite large doses of antibiotics given for long periods, recurrence is common. The issue seems to be one of tissue penetration, rather than drug resistance.
Nocardia species are environmental saprophytes which occasionally cause chronic granulomatous infections in humans and animals. The main organisms responsible for human disease are N. asteroides (colonies appear star shaped), N. brasiliensis, and N. caviae.
Pulmonary nocardiasis is acquired through inhalation of the bacilli. Cutaneous nocardiosis occurs as a result of inoculation injury. Disseminated or CNS nocardiosis occurs following haematogarneous spread. Pulmonary and disseminated disease is more common in immunosuppressed patients.
Pulmonary nocardiasis is more common in the immunosuppressed and those with pre-existing lung disease, particularly alveolar proteinosis. Presentation and clinical/radiological findings are variable, making the diagnosis difficult. Patients tend to develop multiple lung abscesses, and the course may be acute or chronic. Secondary abscesses, mainly in the brain, occur in approx one-third of patients with pulmonary nocardiasis. Other clinical presentations include cutaneous disease (e.g. post trauma) with lymphatic involvement (sporotrichoid), which may progress to a fungating mycetoma.
These branching, aerobic Gram-positive rods are weakly acid-fast when decolourised with 1% sulphuric acid (modified Ziehl–Neelsen stain). Other specialist stains that aid the diagnosis of Nocardia include the Gomori methenamine silver method. Colonies of Nocardia may be coloured (orange/cream/pink) and the surface may be dry or chalky. Nocardia can take up to a month to grow on standard media (e.g. Lowenstein–Jensen media, brain–heart infusion agar, and trypticase–soy agar with blood enrichment). Nocardia organisms can be differentiated from Actinomyces because they are strict aerobes (whereas Actinomyces organisms are facultative anaerobes) and Nocardia grow over a wide range of temperatures (whereas Actinomyces only grow at 35–37°C).
Seek expert advice. Usually a long course (e.g. >3 months in normal host, 6 months if immunocompromised) of a sulfonamide ± trimethoprim, e.g. as co-trimoxazole. Alternatives include minocycline, imipenem and amikacin. In refractory cases, involve the reference laboratory for sensitivity testing.
Actinomadura and Streptomyces species are aerobic filamentous actinomycetes implicated in mycetoma, also known as Madura foot. This is a chronic granulomatous condition that mainly occurs in Africa, Asia, and Central America. Mycetoma can be divided into actinomycetoma (bacterial) or eumycetoma (fungal), which has important treatment implications. The important subspecies of Actinomadura and Streptomyces are Actinomadura madurae, Actinomadura pelletierii, and Streptomyces somaliensis. Other causal organisms include species of Madurella, Exophila, Acremonium, Pseudallescheria, and Nocardia.
Clinically, grains seen within host tissues or in the discharge from sinus tracts, are diagnostic of mycetoma. These grains are colonies of the organism and should be crushed in KOH and Gram stained to distinguish between actinomycetoma (which have Gram-positive filaments) and eumycetoma (septate fungi). These grains should be rinsed in 70% alcohol before culture, to try to eliminate any surface contaminants, and appropriate plates set up at 26°C and 37°C. Macroscopically, grains are often red. Actinomadura spp. show many similar properties to the Actinomyces spp. but strictly Actinomadura are not acid-fast when decolourised with 1% sulphuric acid.
Mycetomas usually involve the hand or foot and arise from traumatic inoculation from soil or plants, usually via thorns or splinters. They are chronic granulomatous infections of the skin, subcutaneous tissue, and bone, and may progress to sinus formation.
The Gram-negative cocci include a variety of pathogenic and non-pathogenic species:
NB Acinetobacter spp. are Gram-negative rods that may appear coccoid or bacillary. Unlike Neisseria spp. they are oxidase negative. They are discussed further on p343.
Epidemic cerebrospinal fever was first described in 1805 by Vieusseaux. In 1887, Weichselbaum isolated N. meningitidis from CSF. In the late 19th century meningococcal carriage was described. In 1909, different serotypes of N. meningitidis were recognized
Humans are the only known reservoir of N. meningitidis, and ~ 20% of the population carry the organism in their throat. However, half of these carriage strains are non-capsulate and thus non-pathogenic. During outbreaks, the carrier rate of an epidemic strain may reach 90%. Risk factors for meningococcal disease:
• lack of bactericidal antibody
• age – bimodal distribution: 3 months to 3 years and 18–23 years
• travel to endemic areas, e.g. Africa, Mecca
• complement deficiencies
• host genetic polymorphisms, e.g. MBL, TNFA, Fc/RIIa and PAI-1.
To cause infection, the organism must cross the nasopharyngeal mucosa and enter the circulation. The Type IV pilus (encoded by pilC) is involved in mucosal colonization. The polysaccharide capsule is important in avoiding host immunity (and defines the serogroup of the isolate Table 4.5 ). Various secretion systems help deliver toxins.
Table 4.5 Gram-negative cocci
Aerobic Gram-negative diplococci, oxidase positive, grow at 37°C on blood and chocolate agar, glucose and maltose positive
Aerobic Gram-negative diplococci, oxidase positive, grow at 37°C on blood and chocolate agar, glucose positive
Non-pathogenic Neisseria spp.
Aerobic Gram-negative diplococci, oxidase positive, grow at 22°C on nutrient agar
Oral commensals – can rarely cause invasive infections
Aerobic Gram-negative cocci oxidase positive, grow at 37°C on blood and chocolate agar
Anaerobic Gram-negative cocci e.g. Veillonella spp.
Anaerobic Gram-negative cocci
• Meningitis and septicaemia
• Other acute infections – purulent conjunctivitis (which occasionally becomes systemic), purulent mono-arthritis, endocarditis, pericarditis and pneumonia
• Chronic septicaemia with joint and skin involvement is also recognized
• CSF examination – in meningitis the CSF pressure is elevated and the CSF appears turbid. The CSF white cell count and protein are normally raised and the the CSF glucose level is low (compared to serum glucose). In very early infection, CSF results may be normal as the meningeal reaction has not had time to take place.
• Microscopy – Gram-negative intracellular diplococci. Note that in meningococcal meningitis, CSF usually has a higher yield than blood cultures. If the Gram stain is negative, a methylene blue stain may pick up scanty meningococci.
• Culture – transparent, non-pigmented, non-haemolytic colonies. May be mucoid if capsule production. Oxidase positive. Identified by API NH.
• Serogrouping – capsular polysaccharide antigens are identified by slide agglutination test using polyclonal antibodies. There are at least 13 xerogroups; the most common ones are summarised in Table 4.6 .
• Serotyping – identification of (PorB) class 2/3 outer membrane protein by a dot-blot enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies.
• Serosubtyping – identification of (PorA) class 1 outer membrane protein by a dot-blot ELISA using monoclonal antibodies.
• Multi locus sequence typing (MLST) is being evaluated for routine surveillance.
• Meningococcal PCR (send to meningococcal reference lab).
Table 4.6 Major serogroups of N. meningitis
Pattern of disease
Epidemic meningitis, associated with different clones
Epidemic strains (and outbreaks)
A meningitis B vaccine is currently undergoing clinical trials
MenC vaccine introduced 1999
Pilgrims returning from the Haj
X, Y, Z, 29E, Z′
N. meningitidis is a fastidious Gram-negative diplococcus. It produces a capsule which forms the basis of the serogroup typing system. There are now at least 13 serogroups but the most common ones are summarized in Table 4.6 .
• See management of acute bacterial meningitis (see Acute meningitis, p. [link] ) and septicaemia. Reduced susceptibility to penicillin in some countries has resulted in empirical therapy for meningitis being a 3rd-generation cephalosporin.
• After treatment, rifampicin (or ciprofloxacin) should be given for nasopharyngeal eradication.
Infection control issues
Inform public health who will arrange chemoprophylaxis of household or kissing contacts of the case. Note that rifampicin is only effective in eradiating carriage in 80–90% of people treated, and rifampicin-resistant strains, which have caused disease in contacts, have been reported. The alternatives are ciprofloxacin or ceftriaxone.
N. gonorrhoeae only infects humans and causes the sexually transmitted infection gonorrhoea (see Gonorrhoea, p. [link] ). This is the second-most common bacterial sexually transmitted infection (STI) in the UK. Increasing rates of antimicrobial resistance, together with its persistence and association with poor reproductive health outcomes have made it a major public health concern.
Gonococci are divided into four Kellogg types, by colonial appearance, ability to auto-agglutinate, and virulence. Kellogg types T1 and T2 are more virulent and possess many fimbriae, while types T3 and T4 are non-fimbriate and avirulent. In gonococci the fimbriae are associated with attachment to mucosal surfaces and resistance to killing by phagocytes. Epidemiological typing of gonococci uses both auxotyping (nutritional requirements of arginine, proline, hypoxanthine, uracil, etc) and monoclonal antibodies against specific proteins.
Gonorrhoea commonly presents as a purulent disease of the urethral mucous membrane and also the cervix in females. Secondary local complications (e.g. epididymitis, salpingitis, pelvic inflammatory disease, p. [link] ) and metastatic complications (e.g. arthritis) may occur if the primary infection is inadequately treated. Other manifestations of disease include disseminated gonococcal infection (skin lesions, painful joints, and fever), ophthalmia neonatorum (purulent conjuncitivitis of the newborn), peri-hepatic inflammation (Fitz-Hugh–Curtis); and rarely endocarditis or meningitis. Rectal or pharyngeal infection is often asymptomatic, and identified through contact tracing. If cultured, gonococcus should always be treated, as it is never a commensal.
• Culture – The only definitive test for legal purposes is culture. Urethral swabs from males and endocervical swabs from females should be Gram stained and then immediately inoculated onto selective media and placed in enriched CO2 conditions. Typical Gram stain appearance of N. gonorrhoeae (Gram-negative diplococci in association with neutrophils) from urethral/endocervical swabs, together with a consistent clinical presentation, is regarded as adequate for treatment in many cases. However, culture is critical for legal cases and for antimicrobial sensitivity testing. After 24–48 h, oxidase-positive colonies appear, and identification can be confirmed by testing for acid production from sugars (APINH). Many laboratories still test for β-lactamase production by the chromogenic cephalosporin (nitrocefin), acidometric, and paper strip methods, although all patients are likely to be treated with a 3rd-generation cephalosporin according to current guidelines
• Non-culture methods – Rapid non-culture tests are increasingly available, mainly based on detection of nucleic acid by hybridization or amplification. These are generally very sensitive and specific, and LCR (ligase chain reaction) has the advantage of being performed on urine.
• If gonococcus is isolated from a prepubertal girl with vulvovaginitis, it may indicate sexual abuse. The case should be dealt with sensitively by a paediatrician and senior laboratory staff should be involved. Thorough documentation is required, since evidence may be needed in court.
Current UK guidelines from the British Association of Sexual Health and HIV (http://www.bashh.org/ceguidelines.htm) recommend ceftriaxone or cefixime as first-line therapy. Spectinomycin can also be given (except for pharyngeal infections). The cephalosporins have replaced the fluoroquinolones due to increasing resistance rates. Ciprofloxacin resistance (MIC ≥1 mg/L) was found to be 14% in 2003 according to the Gonococcal Resistance to Antimicrobials Surveillance Project (GRASP). Rates of ciprofloxacin resistance were even higher in men who have sex with men (MSM). Rates of azithromycin resistance and multi-antibiotic resistance are also rising. Alarmingly, a fluoroquinolone was still prescribed as first–line therapy to almost 25% of patients in 2004 (against recommendations). Many of these patients were likely to be infected with resistant organisms, which would not only result in an adverse clinical outcome for the patient, but also result in transmission of this resistant strain to other contacts.
• Prompt and adequate diagnosis and treatment
• Effective contact tracing
• Prevention – condoms and barrier methods
• Prevent ophthalmia neonatorum by putting 1% aqueous silver nitrate into all newborn babies’ eyes, in areas of high prevalence
• Screening of high-risk individuals
• Sex education/awareness of STIs
The non-pathogenic Neisseria species are upper respiratory tract commensals and include: N. lactamica, N. polysaccharea, N. subshara, N. cirerla (meningitis, endocarditis, bacteraemia, ocular infections, pericarditis, osteomyelitis, empyema). If invasive infection does occur, full susceptibility testing should be performed as penicillin resistance is increasing.
N. lactamica and N. polysaccharea are the species most commonly isolated from nasopharyngeal swabs during meningococcal surveys. Colonies appear similar to N. meningitidis, and they also grow on selective media, unlike the nasopharyngeal commensals. N. lactamica is easy to distinguish as it produces acid from glucose, maltose and lactose, and gives a positive ONPG (orthonitrophenyl-β-D-galactopyranoside) test result for β-galactosidase.
They can occasionally cause invasive diseases such as N. lactamica, N. polysaccharea, N. subflava, N. sicca, N. mucosa, N. flavescens, N. elongata, N. cinerea, and N. weaveri.
Neisseria spp. are naturally competent for DNA uptake, so the pathogenic neisseria can take up DNA encoding for virulence factors or antibiotic resistance from the non-pathogenic neisseria that are part of the normal flora. For example, one mechanism by which N. meningitidis and N. gonorrhoeae have acquired penicillin resistance is the interspecies transfer of penA from the non-pathogenic neisseria in the throat. Studying exactly what constitutes ‘normal flora’ – not just in the throat, but also the GI tract, skin, vagina etc – is likely to increase our understanding of the evolution of pathogens.
For decades, M. catarrhalis was regarded as an upper respiratory tract commensal. However, since the 1970s it has been recognized as an important and common respiratory tract pathogen.
M. catarrhalis grows well on many media including blood and chocolate agar. It shows the ‘hockey-stick’ sign, in that it slides across the agar surface when pushed and can be difficult to pick up onto a loop. M. catarrhalis is oxidase-positive, catalase-positive, DNase-positive, and produces butyrate esterase.
M. catarrhalis causes otitis media, lower respiratory tract infections in COPD patients, pneumonia particularly in the elderly, nosocomial respiratory tract infections, sinusitis and occasionally bacteraemia. Outer membrane proteins (OMPs), lipo-oligosaccharide (LOS), and pili are probably important in pathogenesis.
Anaerobic Gram-negative cocci
Veillonella spp. organisms are part of the normal flora of the gastrointestinal tract of humans and animals. Veillonella may be isolated from a variety of clinical conditions, though their role in causing infection is unclear. The most common species is Veillonella parvula, which fluoresces red under ultraviolet (UV) light. Veillonella are able to use some of the lactic acid produced by streptococci, lactobacilli, and other bacteria that may induce dental caries. They are associated with supragingival dental plaque and also found as part of the tongue microflora. They are generally regarded as minor components of mixed anaerobic infections.
Acidominococcus spp. and Megosphora spp. are other anaerobic Gram-negative cocci found in the human gut. They are considered non-pathogenic.
E. coli is the type species of the genus Enterobacteriaceae, and contains a variety of strains ranging from commensal organisms to highly pathogenic variants. Infections tend to infect the gut and urinary tract but almost any extra-intestinal site may be involved. E. coli is often used as a marker of faecal contamination, e.g. in food and water testing, as it does not otherwise exist outside the animal body.
• O and K polysaccharide antigens protect E. coli from complement and phagocytic killing, unless antibodies are present. Phagocytosis is usually successful if there are antibodies to K antigens present alone, or to both O and K antigens.
• Haemolysin is more commonly produced by strains causing extra-intestinal infections, and is thought to increase virulence.
• The ColV plasmid, harboured by some E. coli, encodes an aerobactin-mediated iron uptake system. This is more common in strains isolated from cases of septicaemia, pyelonephritis, and lower UTIs than in commensal faecal strains.
• Fimbriae – type 1 fimbriae adhere to cells containing mannose residues, possibly contributing to pathogenicity, but their role in UTIs is debated. Other filamentous proteins may cause a mannose-resistant haemagglutination, e.g. CFAs (colonization factor antigens) in human enterotoxigenic E. coli (ETEC), K88 in pigs, and K99 in calves and lambs. P. fimbriae bind specifically to receptors on P blood group antigens of human erythrocytes and uroepithelial cells.
• Other – enteric strains demonstrate specific interactions with the intestinal mucosa, release toxins, and may harbour plasmid-encoded virulence factors.
Serotying of E. coli is based on O (somatic), H (flagellar) and K (surface / capsular) antigens, as detected in agglutination reactions.
• There are >160 O antigens, and cross-reactions occur between E. coli O antigens and O antigens of other species e.g. Citrobacter, Salmonella.
• H antigens are usually monophasic, and are determined from cultures in semi-solid agar.
• K antigens traditionally were those that prevented O agglutination (thus agglutination tests are done on boiled samples). K antigens are the acidic polysaccharide capsular antigens, and divided into groups I and II.
E. coli is the commonest cause of community-acquired uncomplicated UTIs (see Urinary tract infections: introduction, p. [link] ), and also causes nosocomial UTIs. Clinical manifestations range from urethritis and cystitis to pyelonephritis and sepsis. Many uropathogenic strains originate in the patient’s own gut, and cause infection by the ascending route. Specific P fimbriae or ‘pili associated with pyelonephritis (known as the PAP pilus), which attach to uroepithelial cells, are important in pathogenesis. These uropathogenic strains may contain additional virulence factors such as haemolysin, ColV plasmids and resistance to complement-dependent bactericidal effect of serum.
E. coli is responsible for many cases of diarrhoeal disease ranging from acute gastroenteritis, particularly in the tropics (’traveller’s diarrhoea’ Viral gastroenteritis, p. [link] ), to life-threatening haemorrhagic colitis. The strains involved fall into 4–5 groups, with different pathogenic mechanisms (see below and Table 4.7 ).
Table 4.7 Clinical features and pathogenic mechanisms of different E Coli
Enterohaemorrhagic E. coli
Verotoxin-producing E. coli
Shigatoxin-producing E. coli
Haemorrhagic colitis/haemolytic uraemic syndrome (HUS)
Verotoxins (VT1 and 2), also called shiga-like toxins (SLT1 and 2), are phage-encoded toxins thought to target vascular endothelial cells. The A subunit mediates biological activity, while B is responsible for binding and toxin uptake. Risk of developing HUS depends on type of shigatoxin, plus host and environmental factors
Enterotoxigenic E. coli
ST (heat-stable enterotoxin) causes ↑ cGMP, thus altering ion transport and ↑ fluid secretion by mucosal cells of small intestine
LT (heat-labile enterotoxin) – B polypeptide binds to mucosal surface of small intestine, allowing the A polypeptide to enter the cell and catalyse adenosine diphosphate ribosylation of the guanine nucleotide component of adenylate cylase, thus ↑cAMP and ↑fluid secretion (as with V. cholerae)
Colonization/adherence factors – see text
Enteroinvasive E. coli
Disease similar to shigella-like dysentery
Enteropathogenic E. coli
Enteroaggregative E. coli
Traveller’s diarrhoea, especially in Mexico and N. Africa
The usual sources of nosocomial E. coli bacteraemia are the urogenital, gastrointestinal, and respiratory tracts, and foreign bodies such as IV lines and endotracheal tubes. The hallmark of cases of Gram-negative bacteraemia is the systemic reaction to lipopolysaccharide or endotoxin, which may be fatal.
E. coli may cause neonatal meningitis and septicaemia, especially in premature babies. The strains responsible may express the K1 or K5 surface/capsular antigens, which have enhanced virulence.
Other non-enteric infections
E. coli may cause postoperative wound infections and deep abscesses. Respiratory tract infection is usually opportunistic, often in debilitated patients such as diabetics or alcoholics. Nosocomial pneumonia (±empyema) is usually due to aspiration rather than haematogenous spread, and may be associated with high mortality.
E. coli are usually smooth colourless colonies on non-selective media, and may appear haemolytic on blood agar. Most E. coli ferment lactose (and produce acid and gas in 24–48 h), but approximately 5% are non-lactose fermenters (NLFs). E. coli are usually motile, and those responsible for extra-intestinal infections often have a polysaccharide capsule. E.coli are usually positive for indole production, ornithine decarboxylase, lysine decarboxylase and methyl red. They are usually negative for urease, citrate utilization, H2S production and Voges Proskauer test.
The management of E. coli depends on the site and severity of the infection. Simple E. coli UTIs may respond to trimethoprim or ampicillin. Many hospital acquired E. coli infections are due to multi-resistant organisms, and may require treatment with a cephalosporin, fluoroquinolone, aminoglycoside, piperacillin-tazobactam or carbapenem. Susceptibility data often varies geographically (due to prior antibiotic usage) so follow your hospital antibiotic policy. Be guided by antibiotic susceptibility results, and use targeted therapy when possible. Antibiotics may be harmful in cases of E. coli O157.
Klebsiella species are usually harmless colonizers of the human gut. The classification can be confusing, but the main species defined by DNA hybridization studies are Klebsiella pneumoniae subsp. aerogenes (formerly Klebsiella aerogenes), Klebsiella pneumoniae subsp. pneumoniae (formerly Klebsiella pneumoniae) and Klebsiella oxytoca. Other rare respiratory subspecies include Klebsiella ozaenae and Klebsiella rhinoscleromatis. They belong to the tribe Klebsielleae.
Klebsiella organisms that express capsular K antigens are resistant to complement-mediated serum killing. No particular capsular subtype has been linked to a greater risk of infection. Those with O antigens are resistant to phagocytosis. Klebsiella spp. have two iron uptake systems: one system uses aerobactin (related to virulence) and the other uses enterochelin (plasmid encoded).
Common capsular (K) types in the UK are K2, K3 and K21. There are about 80 K antigens recognized overall, some of which cross-react with H. influenzae and S. pneumoniae. There are also five different somatic O antigen types, but these are rarely used for typing. There is an association between antigenic structure, habitat, and biochemical reactivity, for example capsular types 1–6 are most common in the human respiratory tract. For epidemiological investigations, capsular serotyping, bacteriocin typing, and phage-typing may be useful.
Klebsiella infections are rare in the immunocompetent normal host. They tend to cause nosocomial and opportunistic infections, such as UTIs, pneumonia (lobar), other respiratory infections (bronchitis, bronchopneumonia), surgical wound infections, and bacteraemia, in those with risk factors such as diabetes, COPD, or alcoholism. The likely focus in cases of nosocomial bacteraemia is the urinary tract, intravascular lines, lower respiratory tract, biliary tract, and surgical wound site. Severe pneumonia with ‘redcurrant jelly’ sputum and multiple lung abscesses is called Friedlander’s pneumonia, and has a high mortality.
Klebsiella spp. are facultatively anaerobic, catalase-test-positive, oxidase-test-negative, and ferment glucose. Organisms are capsular, which may give colonies a mucoid appearance. The capsule is made of glucuronic acid and pyruvic acid, and there are 80 or so capsular ‘K’ antigens. On Gram stain, organisms may look thicker than other Gram-negative rods, because of the prominent polysaccharide capsule. They are lactose-fermenters and usually fimbriate but non-motile. They are H2S and indole-negative (except K. oxytoca which is indole-positive), Voges–proskauer (VP)-positive, they can grow in KCN, and they can use citrate as a sole carbon source. Different species of Klebsiella are usually recognized by different biochemical tests (Table 4.8 ).
Table 4.8 Biochemical reactions useful to distinguish Proteus, Providencia, and Morganella
Gas from glucose
+ = most strains positive.
– = most strains negative.
V = variable.
Adapted from Greenwood p281.
Greenwood et al. Medical Microbiology; A guide to microbial infections: pathogenesis, immunity, laboratory diagnosis and control. 15th edition (2000) Churchill Livingston.
Most Klebsiella species are inherently resistant to ampicillin and most other penicillins. Many are now multi-resistant, including cephalosporin resistance due to extended spectrum; β-lactameses (ESBLs). Aminoglycoside susceptibility varies between regions. Treat according to local hospital policy and sensitivity data. The carbapenems and fluoroquinolones may be the only options.
P. mirabilis is most commonly isolated from community UTIs, while P. vulgaris and P. myxofaciens tend to cause nosocomial infections. Proteus belongs to the tribe Proteae
Proteus is probably the second most common enterobacteria encountered in many diagnostic laboratories (after E. coli). This is because of the huge numbers isolated from urine samples: approximately 10% of uncomplicated UTIs are due to Proteus (usually P. mirabilis).
Factors that contribute to the ability of Proteus to colonize and infect the urinary tract include:
• production of the enzyme urease which splits urea into ammonium hydroxide. This increases urinary pH and encourages struvite stone formation. These stones act as a nidus for persistent infection and also obstruct urinary flow
• fimbriae help uroepithelial colonization
• flagella-dependent motility helps spread in the urinary tract
• uropathogenic Proteus synthesizes several haemolysins.
In addition to urine infections, Proteus also causes bacteraemia, wound infections and respiratory infections in debilitated hospital patients. The human GI tract is the main reservoir of infection for patients who subsequently become infected.
Proteus organisms rapidly hydrolyse urea. The presence of hundreds of flagella on each organism makes them extraordinarily motile, which appears as ‘swarming’ on agar plates, and can produce the Dienes phenomenon (a line of inhibited growth where 2 strains meet). Proteus organisms give positive methyl-red reactions, are usually VP-negative (except some strains of P. mirabilis), and can grow in the presence of KCN. Most P. mirabilis strains are indole-negative, while the other subspecies are indole-positive (see Table 4.9 ).
Table 4.9 Appearance of Salmonella spp. on different media
Non-lactose fermenters appear white
CLED cysteine lactose electrolyte deficient
Non-lactose fermenters appear blue
Yellow or colourless, often with a dark centre
XLD agar Xylose lysine deoxycholate
Salmonella appear red, some with black centres
SSA Salmonella Shigella
NLF appear colourless, some with black centres
Salmonella are blue-green. S. Typhimurium and others that reduce sulphur produce a black precipitate
Brilliant green agar
Red-pink-colonies surrounded by brilliant red zones
Growth of Salmonella results in a cloudy tube
Tetrathionate-reducing bacteria (Salmonella and Proteus) can grow
Phage-typing, bacteriocin typing and serotyping schemes have been developed. The Dienes phenomenon may be exploited for typing, in that two test organisms are viewed as identical if they show no line of demarcation where the swarming growths meet (after inoculation onto the surface of an agar plate).
Antibiotic resistance is increasing, but the indole-negative P. mirabilis is generally more sensitive than the indole-positive species. Prescribe according to local policy until sensitivity results are available. Some organisms carry the AmpC β-lactamase which is inducible by cephalosporins. Amikacin, new quinolones and carbapenems may be the only options. Note that Proteus is inherently resistant to colistin.
The genus Enterobacter includes E. aerogenes, E. cloacae, E. sakazakii, E. taylorae, E. gergoviae, E. asburiae, E. hormaechei, E. camerogenus, and E. agglomerans. The genus was previously known as Aerobacter species, and belongs to the tribe Klebsiellae.
Enterobacter organisms are common human gut commensals, which rarely cause infection in the normal host.
E. aerogenes, E. cloacae (and occasionally E. taylorae) colonize hospital inpatients and cause nosocomial opportunistic infections, such as wound infections, burn infections, pneumonia, and UTIs. Risk factors for infection include indwelling lines, frequent courses of antibiotics, a recent invasive procedure, diabetes and neutropenia. They can often be isolated from diabetic ulcers. Enterobacter infections have been associated with intravenous fluid contamination. E. sakazakii has been implicated in severe neonatal meningitis (mortality rate 40–80%), and there have been outbreaks associated with dried-infant formula.
In common with the other Enterobacteriaceae, Enterobacter species are facultative anaerobes that give a positive catalase result and a negative oxidase result. They ferment glucose (with the production of acid and gas) and also lactose. They do not produce H2S on triple sugar iron media, they are indole-negative and methyl-red-negative, they are VP-positive, and they can grow in the presence of KCN. They use citrate as a sole carbon source and are ONPG-positive. Unlike Klebsiella, they are usually motile and are less likely to be heavily capsulated. The two most-important clinical species are E. aerogenes (which usually decarboxylates lysine but not arginine) and E. cloacae (usually decarboxylates arginine but not lysine).
Enterobacter organisms (except E. sakazakii) are usually resistant to 1st-generation cephalosporins, and readily develop resistance to 2nd- and 3rd-generation cephalosporins due to inducible β-lactamases such as AmpC. Carbapenems are the mainstay of treatment. E. sakazakii tends to be more sensitive to antibiotics overall, and ampicillin and gentamicin in combination are the usual treatment of E. sakazakii neonatal meningitis.
C. diversus, C. freundii, and occasionally C. amalonaticus are associated with nosocomial respiratory and urinary tract infections. Their role as primary pathogens or secondary infections/colonizers is debated. Note that C. koseri is a synonym for C. diversus. Citrobacter diversus has also been associated with outbreaks of neonatal meningitis.
Animal studies on neonatal meningitis showed that pathogenic strains of C. diversus were more virulent and had an extra outer membrane protein compared to non-pathogenic strains.
The clinical significance of isolation of Citrobacter species from the urinary and respiratory tracts of debilitated hospital patients is often unclear. When isolated from blood cultures, it is usually one of a number of species present, and such polymicrobial infections are often associated with a poor clinical outcome (probably due to the patient’s general debilitated state rather than organisms’ virulence). However, Citrobacter is a recognized cause of endocarditis, and in neonates, Citrobacter organisms (particularly C. diversus) can cause severe meningitis and brain abscesses.
Citrobacter is so named because the organisms can grow on Simmons citrate media. They are usually motile, methyl red-positive, VP-negative and slowly hydrolyse urea. They are usually non-lactose fermenters, but may appear as late lactose fermenters. C. freundii may be mistaken for Salmonella as it is produces H2S. Note there is considerable cross-reactivity with the O antigens of other Enterobacteriaceae.
Like many of the other Enterobacteriaceae that cause nosocomial infections, Citrobacter tend to be multi-resistant, so reliance on laboratory antimicrobial susceptibility testing is paramount. Citrobacter freundii have the inducible AmpC β-lactamase. Plasmid-mediated ESβLs are becoming more common. Treatment options may include aminoglycosides, antipseudomonal penicillins, carbapenems, and quinolones.
There are many named species of Serratia, which belong to the tribe Klebsielleae. S. marcescens is the main one that causes human disease. Infections with S. liquifaciens. S. rubidaea and S. odorifera are very uncommon.
Unlike the other Enterobacteriaceae, Serratia is more likely to colonize the respiratory and urinary tracts of hospital patients (rather than the gut). However, in neonates the GI tract may be the reservoir for cross-contamination.
Serratia species are opportunistic pathogens, particularly in the healthcare setting, and cause respiratory and urinary tract infections, bacteraemias, skin and wound infections. Patients with intravascular catheters and urinary catheters are at increased risk. Serratia infections have been associated with contaminated intravenous therapy, and septic arthritis in patients who have had intra-articular injections. Serratia also causes endocarditis and osteomyelitis in IVDUs, and cellulitis in patients on haemodialysis.
Serratia can be recognized by production of a characteristic red/deep pink pigment. They are slow or non-lactose fermenters, and usually motile. They have the characteristics of the Enterobacteriaeceae. Like Enterobacter, most Serratia do not produce H2S or lactose on triple sugar iron media, are VP-positive, grow in the presence of KCN, and use citrate as a sole carbon source. Serratia can be differentiated from the other Enterobacteriaceae by production of an extracellular DNase.
Serratia are often multi-resistant to antibiotics. Treat according to local epidemiology until sensitivity results are available. Options are often limited to amikacin, piperacillin-tazobactam, and carbapenems. Efforts focused on good infection control practice, especially handwashing, are vital in reducing horizontal transmission between patients. Note that Serratia organisms are inherently resistant to colistin.
Salmonellae belong to the family Enterobacteriaceae. There are seven subspecies and over 2400 serovars. The correct nomenclature is Salmonella enterica, followed by the serotype (e.g. Salmonella enterica serotype Typhimurium). This is commonly abbreviated to S. Typhimurium (serotype not italicized).
Salmonellae are commensals and pathogens of a wide range of domesticated and wild animals. Some species, e.g. S. Typhi and S. Paratyphi are well adapted to humans and have no other host. Others are more adapted to animals and rarely affect humans, e.g. S. Arizonae and reptiles. In humans, salmonellae can be divided into those that cause enteric fever (S. Typhi and S. Paratyphi) and the non-typhoidal Salmonella spp. (NTS). Salmonellae are usually transmitted by the faeco-oral route.
• Infection begins with ingestion of organisms in contaminated food and water.
• Salmonellae express an array of distinct fimbriae that help them to adhere to the intestinal wall.
• They also encode a type III secretion system (T3SS) within salmonella pathogenicity island 1 (SPI-1) that is needed for bacteria-mediated endocytosis and intestinal epithelial evasion.
• A number of SPI-1 translocated proteins (SipA, SipC, SopE and SopE2) promote membrane ruffling and Salmonella invasion.
• Salmonellae are also adapted to survival and replication in the intracellular environment.
• S. Typhi and S. Paratyphi are biohazard group 3 organisms.
• Salmonellae are facultative anaerobic Gram-negative rods, which grow readily on routine media. Their growth on specialized media is summarised in Table 4.9 . They are motile, oxidase-negative, urease-negative, non-lactose fermenters (NLF).
• Salmonellae possess lipopolysaccharide somatic (O) heat-stable antigens, and flagellar (H) heat-labile antigens. Usually the H antigens exhibit diphasic variation, so can exist in phases 1 and 2 (Table 4.10 ).
• S. Typhi, S. Paratyphi C and some strains of S. Dublin and Citrobacter produce the Vi polysaccharide capsule, which may mask the O antigens. If only the Vi antiserum is positive, heat the bacterial suspension in boiling water to remove the capsule and test it again using the same antisera. Rough strains, in which the O antigens are absent, tend to cross-agglutinate with different antisera.
• Most diagnostic laboratories identify the organism as Salmonella by biochemical tests (e.g. API 20E or shorter panel), and partially determine the antigenic structure with different Poly-O and Poly-H antisera (Table 4.10 ). This identifies causes of enteric fever or invasive serotypes.
• All Salmonella should be submitted to the HPA reference laboratory for confirmation of serotype and further epidemiological investigations as necessary.
Table 4.10 Antigenic structure of some Salmonella spp.
H (phase 1)
H (phase 2)
• Enteric Fever. First-line treatment for imported cases of typhoid fever in the UK is now ceftriaxone. When susceptibility results are available, options may include ciprofloxacin, azithromycin, ampicillin or co-trimoxazole.
• Non-typhoidal salmonella. Gastroenteritis does not usually require treatment, except in the immunosuppressed, neonates, the elderly and those at risk of bacteraemia. Suitable antibiotics include ampicillin, ciprofloxacin, trimethoprim or chloramphenicol, depending on susceptibility results. Invasive disease due to NTS (e.g. bacteraemia, meningitis) always requires therapy. Cefotaxime and ceftriaxone penetrate the CSF well so are often used for salmonella meningitis.
• Chronic asymptomatic carriers. Management of chronic asymptomatic carriers is debated. Good personal hygiene should prevent spread of disease. In the absence of biliary disease, prolonged antibiotics (e.g. ampicillin, ciprofloxacin) may cure 80% of carriers. Cholecystectomy may be considered for patients with gallstones or chronic cholecystitis, but there is a risk of spreading the organisms during surgery.
The genus Shigella is divided into four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei, based on serology and biochemical reactions (Table 4.11 ). The organisms cause bacillary dysentery by an invasive mechanism identical to Enteroinvasive E. coli (EIEC). Shigella belongs to the tribe Escherichiaeae, and DNA hybridization studies show that E. coli and Shigella are a single genetic species.
Table 4.11 Biochemical reactions of Shigella
Gas from glucose
V = variable
(+) = positive after incubation for ≥48 hours
ONPG = ortho-Nitrophenyl-β-galactoside
There are 10 serotypes of S. dysenteriae and 15 serotypes of S. boydii. S. flexneri can be divided into six serotypes by group- and type-specific antigens, and each serotype can be further subdivided. S. sonnei must be typed by other means, such as colicine production or plasmids, as they are serologically homogenous. Most cases of shigellosis in the UK occur in young children, although infection occurs in any age after travel to areas where hygiene is poor. S. sonnei is endemic in the UK, while S. boydii and S. dysenteriae, and most S. flexneri infections, originate outside the UK.
The infecting dose of Shigella is only 10–100 organisms, hence the illness can be transferred from person to person (faeco-oral). When one member of a family has acquired the disease, the secondary attack rate is high. Infection can spread rapidly in institutions, especially among young children. It is commonly spread by food and water.
Dysentery results from invasion of the wall of the large bowel, with accompanying inflammation and capillary thrombosis. As the organisms invade and multiply within epithelial cells, cell death results in ulcer formation. Invasiveness is linked to the presence of a 140MDa plasmid. Note that the organisms rarely invade deeper than the mucosa, hence positive blood cultures are uncommon. Some strains also produce an exotoxin, which results in water and electrolyte secretion from the small bowel (and has some similarities with the cholera toxin). This may explain the brief watery diarrhoea that can precede bloody diarrhoea.
see Gastroenteritis, p. [link] . S. dysenteriae usually causes a more-severe illness, possibly with marked prostration and paediatric febrile convulsions. S. dysenteriae may also be associated with toxic megacolon and the haemolytic uraemic syndrome. S. flexneri and S. boydii may also cause severe disease, while S. sonnei usually causes mild symptoms. The severity of S. dysenteriae infection may be due to an exotoxin (previously thought to be a neurotoxin), but its exact role in pathogenesis is uncertain. Shigella rarely invades other tissues, hence septicaemia and metastatic infection is unusual.
Shigella organisms are non-motile, non-capsulated Gram-negative rods. Most appear as non-lactose fermenters after 18–24 h incubation on MacConkey or DCA (desoxycholate citrate) agar, but S. sonnei is the only late lactose fermenter. Shigella is urease, citrate and H2S-negative. S. dysenteriae is the only one that cannot ferment mannitol. Suspicious colonies should be confirmed with species-specific antisera, followed by type-specific antisera for all except S. sonnei. Direct microscopy of a stained faecal smear (usually methylene blue) will reveal numerous polymorphonuclear leucocytes (Box 4.5 ). Shigella dysenteriae type 1 is a biohazard group 3 organism.
Most cases of shigella are mild and self-limiting, so are treated with oral rehydration therapy rather than with antibiotics. Antibiotics may be indicated in severe infections, patients at extremes of age, or the immunocompromised. Options include ciprofloxacin, ampicillin, co-trimoxazole, tetracycline, or cephalosporins, according to in vitro susceptibility testing. Antibiotics are unlikely to reduce the period of excretion.
Hafnia alvei (formerly an Enterobacter) belongs to the tribe Klebsielleae. Hafnia is found in human and animal faeces, sewage, soil, water and dairy products. It usually produces greyish colonies on blood agar and ferments fewer sugars than Enterobacter. All H. alvei are lysed by a single phage, which does not act on any other enterobacteriaceae. H. alvei occasionally causes opportunistic/nosocomial infections, and antibiotic sensitivities are usually similar to those of the Enterobacter group.
Pantoea agglomerans (previously known as Erwinia herbicola or Enterobacter agglomerans) is similar to many plant pathogens. It occasionally causes opportunistic infections in humans (UTIs, bacteraemia, and chest infections), and has contaminated intravenous fluids in the past. Colonies are yellow and may be isolated from superficial skin swabs and respiratory specimens, when they are usually regarded as ‘normal flora’.
Edwardsiella tarda infections in humans probably originate from contact with cold-blooded animals. These organisms are motile and ferment glucose to produce gas. They are H2S-positive, which together with the fact that they do not ferment lactose means they may be mistaken for Salmonella species on enteric media. Edwardsiella species rarely cause disease, but are occasionally associated with a Salmonella-like gastroenteritis, which usually resolves without antibiotics. There are case reports of bacteraemia, liver abscess, soft tissue infection, and meningitis. Treatment should be guided by disc susceptibility testing.
This belongs to the tribe Proteeae. Thus they are motile, deaminate phenylalanine rapidly, give positive methyl-red reactions, are usually VP-negative, and can grow in the presence of KCN. Most are indole-positive and hydrolse urea rapidly (Table 4.8 ). Morganella organisms cause hospital-acquired infections, which are often multi-resistant so treatment is with carbapenems.
Providencia alcalifaciens, Providencia stuartii, and Providencia rettgeri
These also belong to the tribe Proteeae. Thus they are motile and deaminate phenylalanine rapidly. They give positive methyl-red reactions, are usually VP-negative and indole-positive, and can grow in the presence of KCN. Most P. rettgeri hydrolse urea rapidly, while the others are urease-negative. (Table 4.8 ) Providencia causes nosocomial infections in debilitated patients, and treatment is with carbapenems.
These organisms derive energy from carbohydrates by oxidative (rather than fermentative) metabolism.
The pseudomonads are a large and diverse group of aerobic, oxidative Gram-negative rods (GNRs). Most are saprophytes found in soil, water, and moist environments. Pseudomonas aeruginosa is the species most commonly associated with human disease, particularly nosocomial infections. Other opportunistic species of Pseudomonas include P. putida, P. fluorescens (which has been associated with blood transfusions), and P. stutzeri. Organisms which have been allocated to new genera include Burkholderia (B. cepacia and B. pseudomallei), Stenotrophomas (S. maltophilia), Comamonas (below), and Brevundimonas (below).
Formerly known as Comamonas acidovorans or Pseudomonas acidovorans, this rare organism may cause endocarditis in drug users. Confusion arises as it may grow on B. cepacia-selective media, and may be resistant to colistin and gentamicin.
This diverse group is taxonomically distinct from the oxidative pseudomonads and the carbohydrate-fermenting Enterobacteriaceae. They are mainly opportunistic pathogens, and often multi-resistant to antibiotics. Identification difficulties arise because they tend to be biochemically inert.
This oral commensal can cause endocarditis (‘E’ in HACEK, see HACEK organisms, p. [link] ), meningitis, skin and soft tissue infections (particularly from human bites), and pneumonia. It is a facultative anaerobe, requiring incubation in CO2. The colonies pit (‘corrode’) the surface of the agar.
F. oryzihabitans is found in soil, water and damp environments, and most commonly causes central line-associated bacteraemias in immunocompromised patients.
This group of yellow-pigmented organisms is so genetically diverse that many have been re-classified. F. meningosepticum is now Chryseobacterium meningosepticum, and has caused epidemics of adult and neonatal meningitis with high mortality. Other flavobacteria now belong to the genus Sphingobacterium (see below)
Other than C. meningosepticum (see above), isolation of these organisms from clinical samples usually reflects colonization rather than infection. As noted above, C. meningosepticum has caused epidemics of adult and neonatal meningitis with high mortality. In vitro testing may not correlate with antibiotic clinical efficacy. There is evidence for treatment with vancomycin ± rifampicin, or ciprofloxacin, or levofloxacin.
These contain high amounts of sphingophospholipid compounds in their cell membrane. Most human isolates of this genus are S. multivorum and S. spiritivorum, which can cause nosocomial infections in various sites.
S. putrefaciens (formerly Pseudomonas putrefaciens) is commonly isolated from water and the environment, but rarely causes human disease. It is usually found as part of a polymicrobial infection, typically from cellulitis complicating a leg ulcer or burn.
These are also known as the ‘pink-pigmented coccoid’ group. R. gilardii is the most common species isolated from humans, and has been reported to cause community-acquired bacteraemia.
Infection with the rare C. luteola is usually associated with peritoneal dialysis catheters or indwelling lines, and may result in peritonitis, endocarditis, bacteraemia, or meningitis.
Previously called Achromobacter, Ochrobactrum anthropi causes nosocomial opportunistic infections, particularly catheter-related bacteraemia.
O. urethralis (formerly Moraxella urethralis) is a genito-urinary tract commensal, while O. ureolytica is usually found in patients with long-term indwelling urinary catheters. They are of low pathogenicity.
There are three clinically relevant species: A. xylosoxidans (sometimes called Achromobacter xylosoxidans), A. faecalis (formerly Alcaligenes odorans), and A. piechaudii. They are found in soil and water, and the GI and respiratory tracts of hospital patients. Nosocomial outbreaks have occurred (generally, but not exclusively in immunocompromised patients) with a wide range of clinical manifestations. A. xylosoxidans is often multi-resistant to antibiotics, and carbapenems or co-trimoxazole may be required as therapy.
These plant pathogens are usually nonpathogenic to humans, with <50 case reports of human disease in the literature. These are mainly due to A. radiobacter and A. tumifaciens.
P. aeruginosa is widespread in soil, water, and other moist environments. Humans may be colonized with P. aeruginosa at moist sites such as the perineum, ear, and axilla. It is a highly successful opportunistic pathogen, especially in the hospital setting. This success is largely due to its resistance to many antibiotics, ability to adapt to a wide range of physical conditions, and minimal nutritional requirements.
P. aeruginosa is found almost anywhere in the environment, including surface waters, vegetation, and soil. It usually colonizes hospital and domestic sink traps, taps, and drains. It also colonizes moist areas of human skin, leading to ‘toe web rot’ in soldiers stationed in swampy areas, and otitis externa in divers in saturation chambers.
The broad range of conditions caused by P. aeruginosa may be explained by the fact that the pathogen is both invasive and toxigenic. P. aeruginosa has low intrinsic virulence in man and animals. Infection occurs when host defences are compromised or the skin/mucous membranes are breached (e.g. neutropenia, burns patients, intensive care patients, indwelling devices), or when a relatively large inoculum is introduced directly into the tissues. The process can be divided into three stages:
• bacterial attachment and colonization
• local invasion
• dissemination and systemic disease.
Different virulence factors are produced depending on the site and nature of the infections, and include:
• exotoxins – exotoxin A and exo-enzyme S
• lipopolysaccharide (endotoxin)
• cytotoxic substances – proteases (elastase and alkaline phosphatase), cytotoxin (previously called leukocidin), haemolysins, phospholipases, rhamnolipids, pyocyanin
• pili and fimbriae (important in epithelial adherence, e.g respiratory).
P. aeruginosa causes a wide spectrum of conditions:
• Community-acquired infections are rare, and tend to be mild and superficial. Examples include otitis externa, varicose ulcers, and folliculitis associated with jacuzzis.
• Nosocomial infections with P. aeruginosa tend to be more severe and more varied than community infections. P. aeruginosa may account for ~10% of all hospital-acquired infections. Examples include pneumonia, urinary tract infections, surgical wound infections, bloodstream infections, and respiratory infections.
• Cystic fibrosis patients (Box 4.6 ), burns patients and mechanically ventilated patients are at particular risk.
• Other conditions associated with P. aeruginosa include endocarditis (IDUs and prosthetic valves), eye infections, bone and joint infections, postoperative neurosurgical infections, and eye and ear infections.
This non-sporing, non-capsulate, motile Gram-negative rod is a strict aerobe (hence often used in testing anaerobic cabinets). However, note that it can grow anaerobically in the presence of nitrate. It grows on many different culture media, and produces a characteristic ‘freshly cut grass’ odour. The typical green-blue colour is due the diffusible pigments pyocyanin (blue phenazine pigment) and pyoverdin (yellow-green fluorescent pigment; principle siderophore). Other pigments include pyorubrin (red) and pyomelanin (brown). Note that ~10% do not produce detectable pigments even on pigment-enhancing media. P. aeruginosa is oxidase-positive (usually within 10 s), and appears relatively inactive in carbohydrate fermentation tests (only glucose is used). It grows best at 37°C and also at 42°C, but not at 4°C. Confusion occasionally arises in differentiating P. aeruginosa from other Pseudomonas spp. with commercial kits: growth at 42°; flagella stains and differential sugar fermentation tests may prove useful.
For epidemiological studies, serotyping may be useful; however, of the 21 internationally accepted O serotypes, four account for ~50% of clinical and environmental isolates. PFGE may help discriminate between serotypes. Other typing schemes are based on phage susceptibility and bacteriocin production.
Antipseudomonal agents include the fluoroquinolones (these are the only oral option), ceftazidime, ticarcillin, piperacillin, carbapenems (imipenem, meropenem), aminoglycosides (gentamicin, tobramycin, amikacin), polymixins (colistin), and aztreonam. Theoretically, the use of dual therapy should reduce the development of antibiotic resistance and may also have the potential for bacterial synergy, but there is little clinical evidence for this.
Acinetobacter spp. are becoming increasingly important as a cause of nosocomial infections, and are often multi-resistant to antibiotics. Increasing antibiotic-selective pressure and the ability to survive well in the hospital environment (including on curtains and in dust) have contributed to its success as an opportunistic pathogen. There are ~19 genospecies, based on DNA–DNA hybridization studies; seven of these have species names (Table 4.11 ).
In the UK there have been outbreaks of two multi-resistant clones (OXA-23 clone 1 and the ‘SE clone’ which is OXA-51-like). These are now widespread, particularly in London and South-East England. Nosocomial spread in ICUs is common, and may occur via equipment (particularly ventilators), gloves, contaminated solutions, and colonized healthcare workers. There are reports of Acinetobacter baumanii infections in casualties returning from Iraq.
Risk factors include the following:
• community-acquired infections – alcoholics, smokers, chronic lung disease, diabetes, and living in a tropical developing country
• hospital-acquired infections – intensive care, ventilation, urinary catheter, intravenous lines, increased length of stay, treatment with broad-spectrum antibiotics, total parenteral nutrition (TPN), surgery, wounds.
This organism has very few virulence factors, which explains why it only causes opportunistic infections. It occurs naturally as a saprophyte in soil and water, and occasionally colonizes moist human skin. The ability to survive in the environment is probably related to the capsule, the production of bacteriocin, and prolonged viability under dry conditions.
Acinetobacter spp. are able to infect almost every organ system, though it is vital to distinguish true infection from pseudo-infection (e.g. pseudo-bacteraemia due to skin colonization). The most common site of infection is the respiratory tract, where it causes nosocomial pneumonia, particularly ventilator-associated pneumonia (VAP), adult community-acquired pneumonia, and community-acquired tracheobronchitis and bronchiolitis in children. Other sites include the urinary tract, intracranial (usually post-neurosurgery) tissue, soft tissue (burns, wounds, and line-associated cellulitis), eye infections, endocarditis, and bone. Nosocomial bacteraemia is usually associated with the respiratory tract or intravenous catheters, and has a reported mortality rate of 17–46%. A. baumannii bacteraemia tends to be more severe.
Acinetobacter spp. classically appear as Gram-negative coccobacilli, although they may retain crystal violet so appear Gram-positive. They are generally encapsulated, non-motile organisms, which readily grow on routine media as white, mucoid, oxidase-negative, catalase-positive, colonies. Misidentification may arise using API profiles as they are biochemically relatively unreactive, but acidification of glucose, haemolysis of red blood cells, and ability to grow at 44°C are reliable characteristics.
Treatment may require several different approaches:
• localized cellulitis associated with a foreign body or indwelling line may respond to removal of foreign body alone (antibiotics not needed)
• choice of antibiotics for more serious infections is becoming limited – e.g. the OXA-23 clone 1 is resistant to virtually all antibiotics including carbapenems, and many isolates of the SE clone are also carbapenem resistant. Susceptibility testing should include carbapenems, aminoglycosides (including amikacin), sulbactam, polymixins, e.g. colistin, and tigecycline. There is some evidence to support combination therapy with rifampicin and colistin, +/− imipenem
• liason with reference laboratories re typing and antibiotic options
• review of infection control practices and antibiotic prescribing in the case of an outbreak.
Interim Working Party guidelines on the control of multi-resistant acinetobacter (MRAB) outbreaks are available on the HPA website (http://www.hpa.org.uk/infections/topics_az/acinetobacter_b/guidance.htm).
• MRAB is defined as an isolate of Acinetobacter spp. that is resistant to any aminoglycoside and to any third-generation cephalosporin.
• MRAB-C is defined as a MRAB that is also resistant to the carbapenems. These harbour metallocarbapenemases such as VIM and IMP (see Carbapenems, p. [link] ), and are thought to have originated in Korea.
Table 4.11 Genomic species of Acinetobacter
A. species unnamed (>14)
Previously called Pseudomonas maltophilia or Xanthomonas maltophilia, this organism is becoming increasingly seen as a cause of nosocomial infection. It is an opportunistic pathogen, of relatively low virulence, but an amazing ability to survive in a wide range of environments. It is frequently multi-resistant to antibiotics.
Ubiquitous in the environment, S. maltophilia has been isolated from multiple sources in hospitals, including water (tap and distilled), nebulizers, dialysis machines, solutions, intravenous fluids, and thermometers, etc. Transmission of nosocomial infection has been associated with hospital water or contaminated disinfectant solutions. Studies have shown that most outbreaks result from antibiotic-selective pressure (especially the extensive use of imipenem, to which S. maltophilia is resistant) and exposure to multiple environmental strains, rather than cross-infection.
Risk factors for nosocomial infection include: intensive care, increased length of stay, treatment with broad-spectrum antibiotics, malignancy (especially if immunosuppressed), instrumentation (e.g. urinary catheter, intravenous lines, intubation, TPN, CAPD), patients with COPD, IDUs and neutropenia.
Potential virulence factors include those involved in adherence to plastics, and production of exoenzymes such as elastase and gelatinase.
S. maltophilia can cause a variety of infections, ranging from superficial to deep-tissue to disseminated disease. It is most commonly isolated from the respiratory tract, and distinguishing true infection from colonization can be difficult. S. maltophilia pneumonia has a high mortality, especially when associated with bacteraemia or GI obstruction. Other common sites of S. maltophilia infection include skin and soft tissue, intra-abdominal, the urinary tract, the eyes (especially in contact-lens wearers), and implants. Endocarditis is also reported.
This motile non-lactose-fermenting Gram-negative aerobic rod grows readily on standard media. It is often pale yellow on blood agar, with an ammonia-like smell. Most are oxidase-negative, catalase-positive, DNase positive and can hydrolyse aesculin and ONPG. It is the only pseudomonad that gives a positive lysine decarboxylase reaction. Resistance to imipenem may be a useful marker. Note that S. maltophilia is increasingly isolated from sputum from patients with cystic fibrosis. It grows well on colistin-containing media, so may be misidentified as B. cepacia.
Unfortunately, results of antibiotic susceptibility testing correlate poorly with treatment outcome. The drug of choice is co-trimoxazole (see Co-trimoxazole, p. [link] ). Other options to consider include ticarcillin-clavulanate, doxycycline, minocycline, newer-generation quinolones, and third-generation cephalosporins. There is clinical evidence that co-trimoxazole and moxifloxacin are synergistic. Most strains are resistant to aminoglycosides.
Previously classified as Pseudomonas cepacia, this opportunistic pathogen is a particular problem in cystic fibrosis (CF) patients. Other risk factors for infection include chronic granulomatous disease (CGD), and sickle cell haemoglobinopathies. Note that there are ten phylogenetically similar but genomically distinct species, known as the Burkholderia cepacia complex (Table 4.12 ).
Table 4.12 Burkholderia cepacia complex
Common in CF, associated with epidemic spread in a number of CF centres world wide
Most common in CF – see footnote
a Genomovar III has been linked to increased transmissibility between patients, and with a poorer prognosis and higher mortality for some patients.
B. cepacia is ubiquitous in the environment and has been isolated from multiple sources in hospitals. Environmental transmission may occur via contact with respiratory equipment, water supplies, or disinfectants. However, transmission by close contact with colonized patients to other CF patients is more significant, and patients should be segregated into separate groups (e.g. for outpatient clinics and summer camps), which can be a highly emotive issue.
This is a relatively poorly virulent organism, which can survive in a wide range of environments. Virulence factors include adherence to plastics, and production of elastase, gelatinase, adhesin (a mucin-binding protein), siderophores, haemolysin, and exopolysaccharide. Resistance to non-oxidative neutrophil killing may be important. One successful epidemic strain had giant cable pili to help attach to respiratory mucin.
This is a significant pathogen in CF patients, with mortality rates >50% in the first year post infection. The three main patterns of infection are:
• chronic asymptomatic carriage
• progressive deterioration over months, with frequent hospital admissions, recurrent fevers, and weight loss
• necrotizing pneumonia and bacteraemia, associated with rapid deterioration, which is occasionally fatal. Risk factors for this pattern include females with poor lung function and severe CXR changes.
In other patients, B. cepacia can cause a range of other infections, from superficial to deep-tissue to disseminated, but these are rare.
B. cepacia are motile, non-lactose fermenting, Gram-negative, aerobic rods. Selective media are necessary for culture and the colour and shape of the colonies vary with the particular strain and media.
Although agents such as co-trimoxazole, chloramphenicol, minocycline, and the carbapenems show good in vitro activity against B. cepacia, their activity against strains from CF patients is decreased. Also, clearance of these drugs is increased in the CF population, and monitoring of serum levels should be considered to ensure adequate dosing. Use of combination therapy is debated, and specialist units usually produce strict antibiotic treatment protocols. Note that almost all B. cepacia are constitutively resistant to polymyxin. Immunotherapy with specific B. cepacia antigens may prove beneficial.
Burkholderia pseudomallei (formerly known as Pseudomonas pseudomallei) causes melioidosis, which is endemic in parts of SE Asia, Northern Australia, and the Caribbean. It is a major cause of community-onset septicaemia in NE Thailand.
In endemic areas, B. pseudomallei can be cultured from moist soil, surface water (rice paddies), and the surface of many fruit and vegetables. It is also carried by rodents.
B. pseudomallei can survive and multiply within phagocytes, hence a long course of antibiotics is recommended, and antibiotics active in vitro do not always lead to clinical cure.
B. pseudomallei is usually acquired through inhaling contaminated particles, or cutaneously through skin abrasions. This organism has been called the ‘great imitator’ as it can cause suppurative infections of almost any organ. Manifestations range from subclinical infection to localized lung infection (cavitating pneumonia with profound weight loss, resembling TB) to overwhelming septicaemia. Bone and joint infections are common, as is parotid gland infection in children. Symptoms may occur years after exposure, due to the intracellular nature of the organism.
B. Pseudomallei is a Hazard Group 3 organism. Note that laboratory-acquired cases may occur, so all specimens should be handled in Containment Level 3 if meliodosis is suspected clinically.
• Culture – B. pseudomallei colonies grow well on blood or Ashdown medium nutrient agar, after 1–2 days. They appear either wrinkled and dry, or mucoid, and after prolonged incubation may turn orange. Gram staining often shows small bipolar Gram-negative rods. B. pseudomallei is a strict aerobe and oxidizes glucose and breaks down arginine. Isolates are characterstically resistant to gentamicin and colistin. The API 20NE reliably identifies most isolates, but early involvement of the reference laboratory is recommended for confirmatory tests e.g. Indirect haemagglutination, PCR, IgM- and IgG-specific ELISAs, Serology (but note there are problems with sensitivity and specificity).
The antibiotic of choice is ceftazidime IV for 10–14 days, or until clinical improvement occurs (with imipenem or meropenem as alternatives). This should be followed by prolonged oral therapy (e.g. co-trimoxazole plus doxycycline for 20 weeks, plus chloramphenicol for the first 4 weeks) or co-amoxiclav to prevent relapse. Note that resistance to these oral agents may develop during treatment: seek expert advice. In case of β-lactam allergy, this oral regimen may be given IV in cases of sepsis, but is less effective than ceftazidime.
B. mallei causes glanders, which is a rare disease of horses in Asia, Africa, and the Middle East. It is a Hazard Group organism, but has not been isolated in the UK since the 1940s. In humans it causes symptoms similar to meliodosis.
Overview of fastidious Gram-negative rods
These organisms often require specialist supplements or media for culture. They can be divided by appearance on Gram stain as follows:
• HACEK organisms
• Rods with pointed ends
• Curved rods
• Aeromonas, Plesiomonas
Streptobacillus moniliformis causes rat bite fever, as does Spirillium minor.
Haemophilus influenzae is a small, fastidious Gram-negative coccobacillus belonging to the family Pasteurellaceae. It is highly adapted to humans and found in the nasopharynx of 75% of healthy children and adults. It was first isolated in 1890 by Pfeiffer and mistakenly thought to be the cause of influenza. It was also the first living organism to have its genome fully sequenced.
Haemophilus influenzae capsular type 3 (Hib) used to be a common cause of meningitis in childcare. The annual incidence of invasive Hib disease dropped dramatically after introduction of the Hib conjugate vaccine in 1993, but started to rise again in 1999. In 2003 a booster campaign was implemented for children aged 6 months to 4 years.
H. influenzae inhabits the upper respiratory tract of humans; 25–80% of healthy people carry non-capsulate organisms, while 5–10% carry capsulate strains (~50% of which are capsular type b). In addition to the polysaccharide capsule that facilitates invasion, virulence factors of capsular type b include fimbriae (involved in attaching to epithelial cells), IgA proteases (help colonization), and outer-membrane proteins (involved in invasion also). There is evidence that simultaneous viral infection may initiate invasion.
• Invasive infections – e.g. meningitis, epiglottitis, bacteraemia with no clear focus, septic arthritis, pneumonia, cellulitis. These are mostly caused by capsular type b, but types e and f and non-capsulate strains can also cause serious disease. Infections generally occur between 2 months and 2 years of age, as babies < 2 months are protected by maternal antibody.
• Non- invasive infections – e.g. otitis media, sinusitis, purulent exacerbations of COPD. These local infections are usually associated with non-capsulate organisms. There may be an underlying abnormality (anatomical or physiological). Intercurrent virus infection may precipitate infection.
• Culture – These organisms only grow in the presence of X and V factors. X factor (haemin) is needed to synthesize some respiratory enzymes that contain iron (e.g. cytochrome c, cytochrome oxidase, catalase, peroxidase). V factor is NAD(P): nicotinamide adenine dinucleotide (phosphate), and required for oxidation–reduction processes in metabolism. Blood agar (BA) contains both X and V, but H. influenzae grows poorly. NAD supplementation improves growth on BA, as will streaking an organism that excretes NAD, e.g. S. aureus – this phenomenon is called satellitism. H. influenzae grows well on chocolate agar, which is made by heating BA at 70–80°C for a few minutes to inactivate the NADase which normally limits utilization of V factor. Growth is also better in CO2-enriched conditions. Antibiotic susceptibility testing with discs may be unreliable: nitrocefin strips are recommended to test for β-lactamases.
• Antigen detection – As well as culture, H. influenzae may be diagnosed by antigen detection (e.g. with latex agglutination, but beware of cross-reactions with S. pneumoniae and E. coli, so culture is needed for confirmation). Molecular tests, e.g. PCR, are available but not yet widely used.
• Capsule detection – Encapsulated strains of H. influenzae are responsible for most invasive infections (e.g. meningitis and epiglottitis), while respiratory infections and otitis media are usually associated with non-encapsulated strains. The polysaccharide capsule can be demonstrated by the Quellung reaction with type-specific antisera.
• Antigenic type – there are six antigenic types (a–f). H. influenzae type b (Hib), which is a polymer of ribosyl ribitol phosphate, causes the most severe invasive infections.
• Biotypes – there are eight biotypes of H. influenzae (I–VIII), based on indole, ornithine decarboxylase, and urease reactions. The most common are biotypes I–III, and most invasive (type b) organisms are biotype I.
First-choice antibiotics for life-threatening H. influenzae infections are 3rd-generation cephalosporins, e.g. ceftriaxone. They are bactericidal, penetrate the CSF and are clinically effective. Alternatives include co-trimoxazole, ampicillin (but ~25% of UK type b strains produce a β-lactamase), or chloramphenicol. Less-serious H. influenzae infections can be treated with oral ampicillin (but ~20% of UK non-capsulate strains are β-lactamase-positive), co-amoxiclav or clarithromycin.
• Hib conjugate vaccine (capsular polysaccharide and protein) was introduced in the UK in 1992. Given at 2, 3, 4, and 12 months. There is no evidence for serotype replacement in Europe after introduction of this vaccine. For detailed information about use of the Hib vaccine see the Green book guidelines at http://www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/documents/digitalasset/dh_4087384.pdf.
• Household contacts – chemoprophylaxis is no longer recommended to household contacts of an invasive case of H. influenzae. If all children <4 years old in the household have been fully vaccinated against H. influenzae. If one child <4 years old has been unvaccinated or incompletely vaccinated, then ALL household contacts should receive chemoprophylaxis with rifampicin regardless of age or vaccination status.
• Playgroup/nursery school contacts – chemoprophylaxis should be offered to all room contacts (teachers and children) if two cases of Hib occur within 120 days. Unvaccinated children <4 years old should be vaccinated.
• Cases of Hib <4 years old should receive vaccine and chemoprophylaxis before they are discharged from hospital, to eliminate carriage, as there are reports of infection failing to induce immunity.
Haemophilus species other than H. influenzae have been considered rare causes of human disease in the past. However they may cause infections more commonly than was previously believed. Most are normal flora of the human mouth and upper respiratory tract. They are associated with infections such as endocarditis, respiratory tract infection, septicaemia, brain abscess, meningitis, and soft tissue infection.
H. parainfluenzae is increasingly recognized as a cause of human infection. Clinical infections are similar to those caused by H. influenzae, but H. parainfluenzae tends to be less virulent than H. influenzae. H. parainfluenzae has been reported as a cause of pharyngitis, epiglottitis, otitis media, conjunctivitis, dental abscess, pneumonia, empyema, septicaemia, endocarditis, septic arthritis, osteomyelitis, meningitis, abscesses elsewhere, and urinary and genital tract infections. H. Parainfluenzae differs from H. influenzae in that it is V factor dependent only and catalase positive.
H. haemolyticus and H. parahaemolyticus
It is commonly thought that these species rarely cause human disease. However, recent work suggests that standard methods do not reliably distinguish H. haemolyticus from H. influenzae, so it may be more common than previously considered.
The species H. aphrophilus and H. paraphrophilus have been recently reclassified as a single species, Aggregatibacter aphrophilus. Both of these species require CO2 for growth. H. aphrophilus is V factor independent and has been linked with sinusitis, otitis media, pneumonia, empyema, bacteremia, endocarditis, septic arthritis, osteomyelitis, meningitis, soft tissue abscesses elsewhere, and wound infections. H. paraphrophilus is V factor dependent and has been documented as a cause of laryngitis, epiglottitis, endocarditis, brain abscess, hepatobiliary infection, osteomyelitis, and paronychia.
This causes chancroid, a sexually transmitted infection, common in Africa and SE Asia. It presents as a painful penile ulcer associated with inguinal lymphadenopathy. Microbiological diagnosis may be made when Gram-negative coccobacilli are isolated from a lymph node aspirate or from ulcer swabs. Treatment options include tetracyclines, erythromycin, and co-armoxiclar mentin.
H. influenzae biogroup aegyptius
H. influenzae biogroup aegyptius was previously known as H. aegyptius or the Koch–Weeks bacillus. It is very similar biochemically to H. influenzae biotype III, but can be differentiated by PCR. It causes Brazilian purpuric fever (conjunctivitis leading to fulminant septicaemia, with a high mortality) and epidemic purulent conjunctivitis. Combination therapy with ampicillin and chloramphenicol is recommended.
The HACEK organisms (Box 4.7 ) are rare causes of endocarditis (see Infective endocarditis, p. [link] ), which tends to be insidious in onset (mean time to diagnosis ~3 months). Most HACEK organisms are part of normal human mouth flora and are occasionally associated with periodontitis and infections elsewhere (e.g. joints). They grow slowly and may need prolonged incubation (14 days) in CO2 supplementation, so a high index of suspicion and close liaison with the laboratory are crucial.
A. actinomycetemcomitans, a mouth commensal, is the major pathogen of the genus Aggregatibacter. There are two other Aggregatibacter species: Aggregatibacter aphrophilus (includes H. aphrophilus and H. paraphrophilus) and Aggregatibacter segnis (formerly H. segnis).
Aggregatibacter may be difficult to culture, as it is fastidious and grows slowly, so blood cultures should be incubated for at least 14 days. Growth is enhanced by CO2 supplementation (5–10%). Aggregatibacter may form ‘granules’ in blood cultures/broth (the media remains clear). On Gram stain, they look coccoid/coccobacillary, resembling Haemophilus. A. actinomycetemcomitans is urease-negative, indole-negative, catalase-positive and reduces nitrate. They do not grow on MacConkey, and are biochemically similar to Pasteurella spp.
Periodontal disease caused by A. actinomycetemcomitans is associated with the ability to invade and multiply within gingival epithelial cells and the production of a leuokotoxin that lyses neutrophils. Other potential virulence factors include a bacteriocin, endotoxin, chemotaxis-inhibiting factor, and fibroblast-inhibiting factor.
A. actinomycetemcomitans can cause endocarditis, joint infections, and severe periodontal disease. Aggregatibacter has also been found (together with some Haemophilus spp. Fusiforms, and anaerobic streptococci) in actinomycotic lesions. Their contribution to the pathogenesis of actinmycoses is unclear.
A. actinomycetemcomitans is usually susceptible to the third-generation cephalosporins, which are considered the drugs of choice for endocarditis (4 weeks for native and 6 weeks for prosthetic valve endocarditis. Periodontitis requires mechanical debridement with antibiotic treatment (e.g. tetracyclines).
C. hominis is the only species in the genus. It is normal flora in the human mouth, nose and throat and occasionally other mucous membranes and the GI tract. Unlike the other HACEK organisms, it rarely causes diseases other than endocarditis.
This Gram-negative rod has a pleomorphic appearance and may be difficult to decolourise during Gram staining. Culture is enhanced in 5–10% CO2 and high humidity. It grows well on blood agar and chocolate, with slight β-haemolysis, but poorly on MacConkey agar. It is catalse-negative and oxidase-positive. It produces indole (although positivity is weak with many strains), which differentiates it from other HACEK organisms.
Sensitivity testing is difficult because of the slow growth, but it is usually susceptible to β-lactams, tetracycline, and chloramphenicol. However, a β-lactamase-producing isolate that was also resistant to cefotaxime, has been reported. Hence the current first-line recommendation of cefotaxime for HACEK organisms may not be optimal for C. hominis endocarditis: an alternative regimen is co-amoxiclav and gentamicin.
E. corrodens exists as normal mouth and upper respiratory tract flora.
This facultative anaerobic Gram-negative rod is oxidase-positive, catalase-negative, urease-negative, indole-negative, and reduces nitrate to nitrite. About 50% of strains create a depression in the agar (‘corroding bacillus’). As with the other HACEK organisms, culture is slow and enhanced in 5–10% CO2.
E. corrodens causes subacute endocarditis, but is more commonly found as part of mixed infections (e.g. human bite wounds, head and neck infections, respiratory tract infections). It often co-exists with Streptococcus spp. There are reports of E. corrodens causing a variety of other infections. Infections are usually indolent, taking >1 week from time of injury to clinical symptoms of disease. Suppuration is common, and may smell like an anaerobic infection.
There are four species of Kingella, which all colonize the respiratory tract and rarely cause human disease: K. kingae (previously known as Moraxella kingae), K. indologenes, K. denitrificans, and K. oralis. K. kingae is the most common, and a recent increase in cases is likely to be due to increased awareness of the organism and improved diagnostics.
Kingella organisms have been misidentified as Moraxella or Neisseria in the past. They are short Gram-negative rods with tapered ends, which sometimes appear coccoid. They tend to resist decolourisation, so may look Gram–positive. Kingella is catalase-negative, oxidase-positive, urease-negative, and ferments glucose. K. kingae grows on blood and chocolate agar, but not MacConkey. To increase the chance of recovering K. kingae from joint fluid, the fluid should be inoculated into blood culture bottles rather than just plated out directly onto agar plates.
Most cases of invasive disease due to K. kingae occur in children aged between 6 months and 4 years. K. kingae most commonly causes bacteraemia, endocarditis (of native and prosthetic valves), and skeletal infections, e.g. septic arthritis. K.indologenes and K. denitrificans also cause endocarditis. K. oralis is found in dental plaque, but its relationship with periodontal disease is unknown.
Gardnerella vaginalis is found in the female genital tract and is associated with bacterial vaginosis (BV)/non-specific vaginitis (see Bacterial vaginosis, p. [link] ). It is usually classified with GNR, although it is usually susceptible to vancomycin. Of interest, electron microscope studies have noted the cell wall to be either Gram-negative, Gram-positive, or to show an atypical laminated appearance.
There is debate whether specific biotypes have been associated with BV. Newly acquired strains of G. vaginalis may precipitate BV, rather than overgrowth of previously colonizing biopsies.
Adherence of G. vaginalis to vaginal and urinary epithelial cells may play a role in the pathogenesis is of BV and UTIs. Pili have been seen on G. vaginalis, and haemagglutinating activity has been shown. G. vaginalis also produces a cytolytic toxin (haemolysin). It is serum resistant, which may aid survival during bloodstream invasion at childbirth.
• BV – G. vaginalis is almost universally present in women with BV, along with mixed anaerobic flora.
• UTI – G. vaginalis is isolated from <1% of UTIs, and because of its presence in the femal genital tract could represent vaginal contamination. However it has been found from suprapubic aspirates, and also in association with renal disease and interstitial cystitis.
• Bacteraemia – this rare event is associated with female genital tract conditions, such as chorioamnionitis, postpartum endometritis, and septic abortion. Neonatal infection has also been reported.
Gardnerella is a facultative anaerobe, which appears as a pleomorphic Gram-variable rod. It is oxidase- and catalase-negative, non-encapsualted, and non-motile. It needs enriched media for growth. It is also urease-, indole- and nitrate-negative. Note that G. vaginalis is susceptible to SPS (sodium polyanetholesulfonate), which is found in most blood culture bottles, so bacteraemia figures may be underestimated. In clinical practice, BV is diagnosed using the Amsel criteria.
Bordetella pertussis and B. parapertussis cause whooping cough, which is a notifiable disease in England and Wales. The other species only cause human infections under special circumstances: these are B. bronchiseptica (causes kennel cough in dogs and snuffles in rabbits), B. avium (bird pathgoen), B. hinzii, B.holmesii, and B. trematum.
The organism is spread by droplet infection and is highly infectious. Pertussis has the highest incidence in infants but also occurs in adolescents and adults. Morbidity and mortality are higher in females than males, and in those <6 months old.
B. pertussis produces a number of biologically active substances that are thought to play a role in disease:
• Surface components e.g. filamentous haemagglutinin (FHA), pertactin and fimbriae
• Toxins such as pertussis toxin (PT), adenylate cyclase toxin (ACT), tracheal cytotoxin (TCT) and dermonecrotic toxin (DNT).
• Other products e.g. tracheal colonisation factor and BrKA (Bordetella resistance to Killing)
• Classic (severe) pertussis is defined by the World Health Organization (WHO) as ≥21 days cough with paroxysms, associated whoops or post-tussis vomiting, and culture confirmation.
• Mild pertussis is any laboratory-confirmed disease that does not meet the criteria for classic disease.
These tiny coccobacilli occur singly or in pairs. B. pertussis and B. parapertussis are non-motile.
• Bordet–Genou agar: pearly colonies on day 3–4
• CCBA agar (charcoal cephalexin) – B. pertussis produces glistening greyish white colonies on CCBA. It does not grow on nutrient agar, and grows poorly on BA. B. parapetussis colonies are larger and duller, and become visible sooner.
• Culture for B. pertussis lacks sensitivity, and enhanced diagnostic methods are available at the HPA Respiratory and Systemic Infection Laboratory
• PCR – one PCR targets the toxin promoter, while another PCR targets the insertion sequence IS481 (occurs in B. pertussis; B. holmesii, and some B. bronchiseptica). Criteria for reference laboratory PCR: pernasal swab or nasopharyngeal aspirate (NPA) from an acutely ill child age ≤12 months on PICU or paediatric ward, with respiratory illness compatible with pertussis
• Serology – anti-pertussis toxin (PT) IgG antibody levels are determined using an EIA, on paired sera or single samples taken >2 weeks after onset for any individuals with prolonged cough.
A Cochrane review on antibiotics for pertussis (2007) found that short-term antibiotics (azithromycin × 3–5 days, or clarithromycin/erythromycin 7 days) were as effective as long-term (erythromycin × 10–14 days) in eradicating B. pertussis from the nasopharynx, but had fewer side effects. 1 Co-trimoxazole × 7 days was also effective. There were no differences in clinical outcome or microbiological relapse between short- and long-term antibiotics. In 1994, an erythromycin-resistant strain was reported from the USA, but this has not become a clinical problem.
Antibiotic prophylaxis to contacts >6 months old did not significantly improve their clinical symptoms or the number of cases developing culture-positive B. pertussis. The Cochrane authors concluded there is insufficient evidence to determine the benefit of prophylactic treatment of pertussis contacts.
There were major epidemics of whooping cough in 1977/79 and 1981/83, after immunization coverage dropped from >80% to 30%, following a report linking the vaccine to brain damage. Coverage is now back up at ~95%. Acellular pertussis (aP) vaccine is given in the primary immunization course as DTaP/IPV/Hib, aged 2, 3, and 4 months. A further booster as dTaP–IPV, is given with preschool boosters, as vaccine immunity wanes over time.
Altunaiji S, Kukuruzovic R, Curtis N, Massie J. Antibiotics for whooping cough (pertussis). Cochrane Database Syst Rev 2007;Issue 2:CD004404. http://www.cochrane.org/reviews/en/ab004404.html.
Brucella spp. (Table 4.13 ) cause brucellosis, which is also called undulant fever, Mediterranean fever or Malta fever (or contagious/infectious abortion in cattle). This zoonosis is transmitted via contaminated or untreated milk and milk derivatives, or direct contact with infected animals or their carcasses. Brucella species survive well in aerosols and resist drying, so are candidates for agents of bioterrorism (see Bioterrorism, p. [link] ).
Table 4.13 Main species of Brucella – note that the host relationship is not absolute, and man and domestic animals may be susceptible to infections by different species
Cattle; bison and elk in N America
Pigs (swine brucellosis)
Dogs (mainly beagles in USA)
Mild disease only
Sheep (Australia and New Zealand)
No evidence this species infects man
Brucellosis is still endemic in Africa, the Middle East, central and south-east Asia, south America, and in some Mediterranean countries. It has been virtually eliminated from most developed countries. Human–human transmission has been documented but is rare – methods include breast-milk, sexual transmission, and congenital disease. Most human cases seen in the UK are due to B. melitensis from unpasteurized goats milk and cheeses. Brucellosis also occurs through occupational exposure of laboratory workers, vets, and slaughterhouse workers. A careful epidemiological patient history is crucial, regarding travel, dietary habits, and possible exposure.
After ingestion (or entry via skin abrasions, or inhaling infected dust), the bacteria live in the regional lymph nodes during the incubation period (usually 2–8 weeks). They then enter the circulation, and subsequenlty localize in different parts of the reticulo-endothelial system, forming granulomatous lesions which may result in complications in many organs. Brucella organisms surviving within granuloma may cause relapses of actue disease, or result in chronic brucellosis.
Brucellosis has a wide variety of clinical presentations. The ‘undulant’ or wave-like fever rises and falls over weeks in ~90% of untreated patients. Malodorous perspiration is said to be pathognomonic. Osteoarticular disease occurs in ~20%, with epididimo-orchitis in ~6%. Other symptoms include weakness, headaches, depression, myalgia, and bodily pain. Sequelae are also variable, and include granulomatous hepatitis, anaemia, leukopenia, thrombocytopenia, meningitis, uveitis, and optic neuritis.
• Hazard Group 3 organism
• Culture from blood or bone marrow. These coccobacilli or short bacilli may occur singly, in chains, or in groups, and can take up to 8 weeks to grow. They are non-motile, non-sporing, and non-capsulate. They are aerobic, and B. abortus requires 5–10% CO2 to grow. The three main species (B. melitensis, B. abortus, B. suis) can be differentiated biochemically and by antigenic structure. Each of these three species can be further divided into biotypes: there are >9 biotypes of B. abortus, >3 of B. melitensis, and >5 of B. suis
• Molecular techniques/real-time PCR has been developed
• Serology – raised (1:160) or a rising antibody titre in symptomatic patients suggests the diagnosis of active brucella. Demonstration of antibodies with various tests including the SAT (standard agglutination test), mercaptoethanol test, classic Huddleson, Wright, and/or Bengal Rose reactions. At the HPA Reference Laboratory, Liverpool, all sera are screened with a brucella antibody assay and specific IgG/IgM enzyme immunoassays. Positive samples then undergo further testing with in-house micro-agglutination and complement fixation in-house assays
• Histological evidence of granulomatous hepatitis (hepatic biopsy)
• Radiological alterations in infected vertebrae – the Pedro Pons sign (preferential erosion of antero-superior corner of lumbar vertebrae) and marked osteophytosis are suspicious of brucellic spondylitis
• Dye inhibition test (basic fuschin and thiamin dyes) can differentiate individual Brucella spp.
Drugs must penetrate macrophages and be active in an acidic environment. Doxycycline and one of the following agents (gentamicin, streptomycin, rifampicin) are suitable regimens, and the combination is usually given for at least 6 weeks. Fluoroquinolones or tetracyclines may also be used in combination. Intensive treatment of the acute disease is recommended, to prevent progression to chronic forms which are more difficult to treat. Antibody levels may be measured to monitor response to therapy. A triple combination of doxycycline, together with rifampin and co-trimoxazole has been used succefully to treat neurobrucellosis.
Good standards of hygiene in the production of raw milk and its products, or pasteurization of all milk, will prevent brucellosis acquired from ingestion of milk. Also avoid contact with infected animals. Vaccination of young cattle helps to protect animals against B. abortus, but is not completely effective. However it helps to limit the spread of disease, and thus aids eradication. Only by testing all animals, and slaughtering those with positive results, can the disease be truly eradicated.
Y. pestis causes plague. There are 3 clinical syndromes: bubonic, pneumonic and septicaemic (Table 4.14 ).
Table 4.14 Features of the main clinical forms of plague
Rat flea bites
Respiratory aerosols from rat fleas
Person-to-person spread in crowded unhygienic conditions, during epidemics
Complication of bubonic or pneumonic plague
May arise as a complication of bubonic or septicaemic plague
Fluid from buboes
Blood culture/blood films
Fever, painful buboes, inguinal lymphadenopathy
Cough or haemoptysis ± bubo
Fever, hypotension, no buboes
1–4 days (maximum 6 days)
Mortality if untreated
High mortality (approaching 100%)
High mortality (approaching 100%)
Y. pestis occurs worldwide, but most cases of plague are reported from developing countries of Africa and Asia. There are ~10 cases annually from rural areas of the USA. The last outbreak of plague acquired in the UK was in 1918.
The somatic (heat-stable) and capsular (heat-labile) antigens are important in virulence and immunogenicity. Somatic antigens V and W help resist phagocytosis, and the capsular antigen containing the immunogenic fraction (F1) is antiphagocytic also. Other virulence factors include a lipopolysaccharide endotoxin, the ability to absorb iron as haemin, and temperature-dependent coagulase and fibrinolysin.
• Hazard Group 3 organism
• Culture – this short Gram-negative coccobacillus occurs singly or in pairs (or as chains in fluid culture). Old cultures are pleomorphic and may even resemble yeast cells. They are non-sporing and non-motile, and often capsulate at 37°C. Methylene blue shows bipolar staining. Yersinia spp. grow between 14°C and 37°C, with optimal growth at 27°C. Small non-haemolytic colonies are seen on blood agar at 24 h, which are catalase-positive and oxidase-negative. Although Y. pestis grows on MacConkey, it tends to autolyse after 2–3 days. Organisms are citrate-, indole- and urease-negative. It is usually cultured from a bubo aspirate, but may also grow from blood, CSF, or sputum. Cefsulodin Irgasan Novobiocin (CIN) agar is selective for Yersinia (and Aeromunas species).
• Direct immunofluorescence is a more-rapid diagnostic method.
• Serological tests for yersiniosis (acute and convalescent) include the complement fixation test and haemagglutination of tanned sheep red cells to which F1 capsular antigen has been adsorbed.
Early antibiotic therapy for suspected cases (e.g. streptomycin, gentamicin, or doxycycline) reduces the otherwise high mortality to ~10%. Contacts may also be given antibiotic prophylaxis. Patients with pneumonic plague should be isolated until they are sputum smear-negative (usually ~3 days since starting treatment). There is no vaccine currently available.
Flea and rodent control are important. See the HPA website for guidelines for action in the event of a deliberate release of plague.
Y. enterocolitica resembles Y. pestis and Y. pseudotuberculosis on culture and morphologically, but differs antigenically and biochemically. The most common serotypes causing human infection in Europe are 3 and 9. Serotypes cultured from healthy individuals are probably non-pathogenic.
Y. enterocolitica is acquired from eating infected meat or milk. Patients with conditions associated with iron-overload (e.g. haemochromatosis) and the immunosuppressed are at increased risk of Yersinia infections.
see Yersinia, p. [link] .
Y. enterocolitica usually presents as a febrile illness associated with bloody diarrhoea, and may mimic salmonellosis, shigellosis, or appendicitis. Other presentations include mesenteric lymphadenitis and septicaemia, which may be fatal in the elderly. Secondary complications include erythema nodosum, polyarthritis, peritonitis, Reiter’s syndrome, meningitis, osteomyelitis, and hepatic, renal, and splenic abscesses. Y. enterocolitica has been cultured from pseudotuberculous lesions in animals.
Gastroenteritis usually resolves without antibiotics. In severe infection, the recommended regimen is doxycycline plus an aminoglycoside. Alternatives include cefotaxime, co-trimoxazole and fluoroquinolones. Note resistance to penicillin. If the patient is on desferrioxamine this should be stopped as it may increase the severity of infection.
Strains of Y. pseudotuberculosis can be differentiated by somatic and flagellar antigens, some of which are shared with Y. pestis. Most human infections with Y. pseudotuberculosis are due to serotype 1.
see Yersinia, p. [link] .
Y. pseudotuberculosis causes a fatal septicaemia in animals and birds. Humans usually acquire the infection from contact with water polluted by infected animals, or eating contaminated vegetables – infection due to direct contact with animals is rare. In humans, yersiniosis infection ranges from asymptomatic to a fatal typhoid-like illness with fever, purpura, and hepatosplenomegaly. Mesenteric adenitis ± erythema nodosum may mimic appendicitis and tends to infect males aged 5–15 years.
This small Gram-negative rod is slightly acid fast. It grows poorly on MacConkey (like Y. pestis), but can be differentiated from Y. pestis because Y. pseudotuberculosis can produce urease and is motile at 22°C.
The genus Pasteurella includes the species P. multocida, P. haemolytica, P. canis, P. stomatis, and P. pneumotropica. Pasteurella live in the mouth, and GI and respiratory tracts of many animals (especially dogs and cats) ± humans. P. multocida, the most frequent species causing human infections, usually causes skin and soft tissue infections.
Fifteen serotypes of P. multocida have been identified, based on four capsular antigens and 11 somatic antigens. PFGE can be used to compare strains. In addition to acquiring infection through animal bites, humans can also become infected through inhaling air which has become contaminated by infected animals’ coughing.
In animals, P. multocida causes hemorrhagic septicaemia, which is usually fatal. Most virulent Pasteurella strains have a polysaccharide capsule, which is antiphagocytic and protects against intracellular killing by neutrophils. Also, some strains produce a leukotoxin and some bind transferrin.
• P. multocida causes skin and soft tissue infections after animal bites, most commonly a localized abscess with cellulitis and lymphadenitis. P. multocida has also been associated with upper and lower respiratory tract infections. Other sites of infection are uncommon – these include meningitis post head injury, bone and joint infections, septicaemia, endocarditis, and intra-abdominal infections.
• P. haemolytica is non-pathogenic for humans. It causes pneumonia in sheep and cattle, septicaemia in lambs, and also infects poultry and domestic animals.
• P. pneumotropica may be isolated from the respiratory tract of laboratory animals. There are reports of it causing human infections, e.g. animal bite wound infections, septicaemia, and upper respiratory tract infections.
P. multocida is a facultative anaerobic Gram-negative coccobacillus, which appears pleomorphic in culture and does not grow on MacConkey agar. At 37°C, organisms are capsulate, non-sporing, and non-motile. They show bipolar staining with methylene blue. Most are fermentative, and oxidase-positive, catalase-positive, and indole-positive. P. haemolytica can be differentiated from P. multocida as it is haemolytic on blood agar and can grow on MacConkey agar.
Penicillin is the mainstay of treatment, and there is a wealth of clinical experience to support this. It is resistant to oral first-generation cephalosporins, flucloxacillin, clindamycin, and erythromycin. It is sensitive in vitro to fluoroquinolones, which may be appropriate in penicillin-allergic patients.
Francisella tularensis is primarily an animal pathogen (rabbits and hares), which occasionally infects humans as accidental hosts. The resulting infection is called tularaemia, and may be either ulceroglandular or typhoidal/pulmonary. Only two of the four F. tularensis subspecies are clinically important (type A is highly virulent and type B less virulent). It is also a potential agent of bioterrorism (see Bioterrorism, p. [link] ).
Tularaemia is endemic in N America and parts of Europe, Asia, northern Australia, and Japan. Most cases in man are sporadic, though outbreaks have been reported. It may survive for days in moist soil and in water polluted by infected animals, and for years in culture at 10°C. Organisms are killed in 10 min after exposure to moist heat at 55°C. For epidemiological investigations, the most discriminatory typing system is based on VNTR (multiple-locus variable-number tandem repeats).
There is evidence from animal experiments of intracellular multiplication of F. tularensis. Virulence has been associated with the capsule and also citrulline ureidase activity.
Infection ranges from asymptomatic to septic shock, depending on virulence of particular strain, host immune response, route of entry, and degree of systemic involvement. There are two main forms:
• ulceroglandular form of tularaemia (due to direct contact with infected animal) – acute-onset fever, headache, and rigors, usually followed by glandular lesions and skin ulceration, ± eye involvement.
• typhoidal/pulmonary form of tularaemia (results from indirect contact through bites from ticks/mosquitoes/biting flies, inhaling infected dust, or eating contaminated food or water) – acute-onset fever, headache, and rigors, usually followed by respiratory or typhoid-like symptoms.
F. tularensis is a Hazard Group 3 organism. It is a small, non-motile, non-sporulating, capsulate Gram-negative cocco-bacillus, which shows characteristic bipolar staining with carbol fuchsin (10%). It stains poorly with methylene blue. It is a strict aerobe, and culture requires the addition of egg yolk or rabbit spleen to agar. After culture, the bacilli may appear filamentous and larger. Traditional microbiological methods are slowly being replaced by immunological and molecular tests, including ELISA and immunoblots for antibodies (but tests relying on antibody detection are limited in early clinical stages of disease). If a case is suspected, involve the HPA Special Pathogens Reference Unit (SPRU) at Porton Down, who will process tissue biopsies, wound swabs, or specimens from bacterial culture.
Seek expert advice. Streptomycin or gentamicin are antibiotics of choice, with the addition of chloramphenicol for meningitis. Relapse is more common if tetracycline or chloramphenicol are used, as these are bacteriostatic for F. tularensis. The live vaccine (LVS) is based on an attenuated strain of F. tularensis. Post-exposure prophylaxis with doxycycline or ciprofloxacin may be considered after potential inhalation.
This organism is named after the outbreak of pneumonia affecting >180 members of the American Legion at a convention in Philadelphia in 1976. The Legionellaceae naturally live in water and only accidentally infect humans. This may result in either Legionnaires’ disease or Pontiac fever. There are 52 different genetically defined species of Legionella, of which ~50% of infect humans. Legionella pneumophila serogroup 1 is the most pathogenic, and accounts for ~95% of human cases.
Legionella is acquired via inhalation of contaminated aerosols (e.g. from spas, showers, air-conditioning systems, water-storage tanks, nebulizers) or drinking water. Water systems are more likely to be contaminated with Legionella if the temperature is outside the recommended range (it should be <20°C or >55°C), if the flow is obstructed, or if biofilms have formed. Note that Legionella is an intracellular organism and can survive in amoebae, within the environment. The incubation period is 2–10 days, and occasionally symptoms may develop up to 3 weeks post exposure. It is not transmitted person to person. Most cases are isolated, but outbreaks can occur.
After the infection is established, pneumonic consolidation develops, characterized by proteinaceous fibrinous exudates pouring into the alveoli. The mechanism of distant toxic changes (e.g. confusion, hallucinations, focal neurology) is poorly understood. Legionella organisms are engulfed by monocytes, and may survive intracellularly for prolonged periods of time.
In addition to the two main clinical syndromes below, rare conditions (e.g. prosthetic valve endocarditis, wound infections) have been reported:
• Legionnaires’ disease – this rapidly progressive pneumonia is characterized by fever, respiratory distress, and confusion, and has a mortality rate of >10% in healthy people. Risk factors include age >50 years, hospital admission, immunosuppression, and smoking. Men are affected more than women
• Pontiac fever – this is a brief flu-like illness, which has a high attack rate but low mortality.
• Microscopy/culture – these short rods/coccobacilli may be difficult to see by Gram stain, so fluorescent antibody stains or silver impregnation may help. Legionella grows best on media such as BCYE (buffered charcoal yeast extract), which contains iron plus cysteine as an essential growth factor. Some strains prefer 2.5–5% CO2 at 35–36°C. L. pneumophila colonies usually appear by day 5, but other species may require 10 days. Colonies may autofluoresce under ultraviolet (UV) light. Serogroups can be differentiated by slide agglutination or fluorescent antibody tests, which are available at reference laboratories.
• Antigen detection – Legionella urinary antigen test (ELISA) only detects serogroup 1 of L. pneumophila.
• Antibody detection – FAT (fluorescent antibody test), RMAT (rapid micro-agglutination test), or ELISA (enzyme-linked immunosorbent assay). A >4-fold rise, or titre 1:256 is usually diagnostic. Remember that antibody may take >8 days to develop after onset of infection, and may persist for months/years post infection. Also note some cross-reactivity with Campylobacter.
Conventional susceptibility tests in broth and agar are unreliable, and methods have not been standardized. In addition, many antibiotics with excellent in vitro activity against Legionella (eg, β-lactams and aminoglycosides) are ineffective. Essentially, the macrolides, quinolones, tetracyclines, and rifampicin are effective as they have good intracellular penetration. Preferred treatment is intravenous erythromycin or oral clarithromycin, with ciprofloxacin as an alternative. In severe infections, the dose of erythromycin may be doubled, or rifampicin added.
Control and prevention
This relies on good design and maintenance of water systems to prevent growth of Legionella organisms, and subsequent treatment of the source (e.g. contaminated water systems) if a case occurs. The main approaches to control are:
• physical: – heat, UV light, sonication: use of compressed air to drain and flush pipes
• chemical – inhibit scale formation, use of biocides to kill the amoebae (such as sodium hypochlorite, ozone), use of charcoal filters
• plumbing – regular maintenance, no dead legs in the system, pumps should be in series and not in parallel, no dead spaces in the heaters, regular flushing of the system, use of correct components.
Guidelines on legionella investigations and control are available (Box 4.8 ).
The genus Capnocytophaga in the family Flavobacteriaceae may be divided into two groups:
• species associated with dog-bite infections (and occasionally bites from other animals such as rabbits or cats), and normally found in dogs’ mouths: C. canimorsus (formerly known as dysgonic fermenter, 2 DF2) and C. cynodegmi
• species found in the human mouth – C. ochracea (These were the dysgonic fermenter group 1 (DF1)), C. gingivalis, C. sputigena, C. haemolytica, and C. granulosa.
While C. canimorsus and C. cynodegmi are most commonly associated with bites, there are reports of infections occurring merely after exposure to dogs, with no bites or scratches.
Species found in the human mouth produce a variety of enzymes that help in the invasion of periodontal tissue (e.g. acid and alkaline phosphotases, aminopeptidases, IgA, proteases, and trypsin-like enzymes). These are thought to be important in periodontitis.
All species may cause a wide range of infections in normal and immunocompromised hosts. Among animal bite infections, C. canimorsus is more common and more severe than C. cynodegmi, with a mortality approaching 30%. Particular risk factors include asplenic patients, alcoholics, and those on steroids. Asplenic patients with C. canimorsus infection may present with shock, disseminated purpuric lesions, and disseminated intravascular coagulation (DIC). Fulminant infections with C. canimorsus may also occur in healthy people, although infections tend to be milder. Meningitis, endocarditis, pneumonia, corneal ulcer, cellulitis, and septic arthritis due to C. canimorsus have also been reported.
Species found in the human mouth may be important in localized juvenile periodontitis. They have also been found in the female genital tract and associated with intrauterine infection, amnionitis, and neonatal infections in premature babies. Rarely, they cause severe infections as opportunistic pathogens (e.g. endocarditis, eye infections, hacteraenis, peritonitis), in both immunocompent and immunosuppressed patients.
These long thin delicate GNRs are typically fusiform, but older cultures often show pleomorphic sizes and shapes. They are facultative anaerobes, and grow best with CO2 enrichment. On blood or chocolate agar they may appear yellowish, with a spreading edge with finger-like projections due to the typical gliding motility. Note they do not grow on MacConkey agar and they do not produce indole. Differentiating each individual species is more difficult and generally requires reference laboratory assistance. In general, species from the human mouth are oxidase-negative and catalase-negative, while those from animals’ mouths are oxidase-positive and catalase-positive.
C. canimorsus is more fastidious than the others, and may be difficult to grow from blood cultures (even when organisms have been seen on Gram stain). In this situation, culture on enriched agar (e.g. heart infusion agar with rabbit or sheep blood) for 14 days in 10% CO2 may help.
Co-amoxiclav is usually recommended for these infections. Asplenic patients should be given penicillin or co-amoxiclav prophylaxis after a dog bite, as the organism may take a while to grow and be identified, and the mortality rate is high. While the animal-bite-associated species are sensitive to penicillin, resistance to the β-lactams has been reported in the human-mouth species. For instance, C. haemolytica and C. granulosa are often resistant to β-lactams and aminoglycosides. All species are usually sensitive to clindamycin, erythromycin, tetracyclines, and the quinolones.
The genus Vibrio (family Vibrionaceae) includes over 30 species. The most important ones that result in human infections are V. cholerae, V. parahaemolyticus and V. vulnificus. Other species, such as V. alginolyticus, V. damsela, V. fluvialis, V. hollisae, and V. mimicus, occasionally cause opportunistic infections.
There are ~20 cases of cholera (see Cholera, p. [link] ) imported into the UK every year. These are most commonly O1-El Tor. In the mid-1990s a new serogroup (O139) appeared in the Bay of Bengal – this was the first time a non-O1 serogroup had resulted in epidemic cholera.
Cholera is prevalent in Central and South America, Africa, and Asia. There are more than 130 different O (somatic antigen) serogroups of V. cholerae. Serogroup O1 (the ‘cholera vibrio’) causes epidemic cholera, and some strains of non-O1 (the ‘non-cholera or non-agglutinable vibrios) can also cause diarrhoea. Serogroup O1 is usually acquired by the faecal-oral route, while non-O1 V. cholerae may be associated with consumption of seafood or exposure to saline environments. The two biotypes of serogroup O1 (El Tor, which is the most common, and Classical) can be distinguished by susceptibility to phage, and the fact that El Tor is haemolytic and resistant to polymixin B. The subtypes of serogroup O1 are Ogawa (most common), Inaba, and Hikojima (which possesses determinants of both other subtypes).
The potent cholera enterotoxin, produced by serogroup O1 and some non-O1 strains, comprises five B (binding) subunits and one A (active) subunit. Insertion of the B subunits into the host cell membrane forms a channel for subunit A to enter the cell. By causing the transfer of ADP ribose from NAD to another protein, adenylate cyclase is irreversibly activated and cAMP is overproduced. The resulting hypersecretion of Cl− and HCO3− causes loss of massive water and electrolytes (rice-water stool). Other features important in pathogenesis of serogroup O1 include production of mucinase and other proteolytic enzymes (which help the organism reach the enterocytes), the motility of the organism, and adhesive haemagglutinins (aid close adherence to enterocyte surface). Non-O1 strains may produce other enterotoxins, cytotoxins, haemolysins, and colonizing factors.
Cholera is transmitted by contaminated food or water, and requires a large infective dose. Humans are the only host. Only a handful of those infected are symptomatic (ratios quoted are 40 asymptomatic carriers:1 symptomatic individual for El Tor and 5:1 for classical), which underscores the need for good hygiene.
V. cholerae usually causes the typical profuse watery diarrhoea of cholera, which may rapidly lead to hypovolaemic shock and death from dehydration. Milder cases are similar to other causes of secretory diarrhoea, and asymptomatic infections also occur. Non-O1 V. cholerae usually causes mild, sometimes bloody diarrhoea, but may occasionally be severe and resemble cholera. Patients exposed to aquatic environments may suffer from wound infections, and bacteraemia and meningitis have been reported.
During an epidemic, cholera is a clinical diagnosis. Otherwise, diagnosis is based on high clinical suspicion together with culture or dark-field microscopy of stool (comma-shaped organisms are seen moving around, which ceases when diluted O1 antisera is added). Vibrios are short, curved or ‘comma-shaped’, aerobic Gram-negative rods, which are motile by a single polar flagellum. They ferment both sucrose and glucose but not lactose, and reduce nitrate to nitrite. Most are oxidase-positive and produce indole. The growth characteristics of vibrios are summarised in Table 4.15 . V. cholerae is non-halophilic (i.e. can grow on media without added salt), provided the necessary electrolytes are present. V. cholerae can grow at 42°C (along with V. parahaemolyticus and V. alginolyticus). Vibrios are tolerant to alkali but have a low tolerance to acid. V. cholerae is usually VP-positive. Vibrios accumulate on the surface of alkaline peptone water. If a loopful is inoculated onto TCBS (thiosulphate citrate bile salts),V. cholerae appear as a yellow sucrose-fermenter, which is oxidase-positive. V. cholerae is killed by most detergents and by heating at 55°C for 15 min. However, it can survive for up to 2 weeks in salt water at ambient temperatures, and also on chitinous shellfish for 2 weeks, even if refrigerated.
Table 4.15 Growth characteristics of Vibrio spp.
Growth at 42°C
Oxidase-, nitrate-, lysine-, ONPG-positive
Not halophilic (0–3% NaCl)
Halophilic (3–6% NaCl)
Green (85%), yellow (15%)
Lactose-, lysine-, salicin-positive
Halophilic (3–5% NaCl)
Halophilic (3–10% NaCl
Rehydration is key. Antibiotics (e.g. azithromycin, ciprofloxacin) reduce the duration of disease and period of excretion of V. cholerae in the stool of infected patients. In the UK, a killed oral vaccine is licensed for relief workers and travellers to remote endemic areas. Results of trials of a live oral vaccine (‘Peru-15’) are promising. However, the most important preventative strategies are improvement of sanitation and food and water standards.
V. parahaemolyticus is ubiquitous in fish and shellfish, and the waters they inhabit. Outbreaks of diarrhoea occur infrequently in the UK.
V. parahaemolyticus infection is common in SE Asia, particularly Singapore and Japan. However it also occurs in the UK and USA, particularly during summer months.
Kanagawa-positive strains (see below) of V. parahaemolyticus adhere to human enterocytes and produce a heat-stable cytotoxin.
V. parahaemolyticus is usually acquired through ingesting seafood, and causes acute explosive diarrhoea. Extra-intestinal infections arise from handling contaminated seafood or exposure to the aquatic environment, the most common being wound infections.
This organism is halophilic, hence will not grow on CLED agar. Clinical strains of V. parahaemolyticus usually appear as green, non-sucrose fermenting colonies on TCBS agar, but isolates from estuary and coastal waters may ferment sucrose. Stool samples should be enriched in alkaline peptone water containing 1% NaCl. The Kanagawa phenomenon refers to haemolysis of human erythrocytes on Wagatsuma’s agar, by strains of V. parahaemolyticus which cause gastroenteritis.
V. vulnificus has been called the ‘terror of the deep’ due to the severe fulminant infection it can cause.
Infections are most common in areas with higher water temperatures, such as the mid-Atlantic and Gulf coast states of the USA. Septicaemia arises from eating contaminated raw shellfish, while wound infections are due to injuries sustained in aquatic environments.
The polysaccharide capsule helps resist phagocytosis and bactericidal effects of human serum. The association with liver disease (with increased serum iron levels) may be explained by the ability of virulent strains to use transferrin-bound iron. Toxin production is also important.
There are 3 main infections associated with V. vulnificus:
• fulminant septicaemia, followed by cutaneous lesions. This is associated with a high mortality (50%). Immunosuppressed patients are at increased risk, particularly elderly male alcoholics with liver dysfunction
• wound infection, rapidly progressing to cellulitis, oedema, erythema, and necrosis. Patients may develop septicaemia, and it may be fatal
• acute diarrhoea, usually in those with mild underlying conditions. Mortality is rare.
This organism is also halophilic (see V. parahaemolyticus above). See Table 4.15 for further growth characteristics.
Other Vibrio Species
• V. alginolyticus is the most common vibrio organism found in seafood and seawater in the UK. It is a halophilic organism, which will not grow on CLED but grows in 10% NaCl. Colonies are large and yellow (sucrose-fermenting) on TCBS agar, and there is swarming on non-selective media. It can cause opportunistic wound infections, with mild cellulitis and a seropurulent exudate. Most infections are associated with exposure to seawater and are self-limiting. Little is known about the pathogenic mechanisms.
• V. fluvialis is phenotypically similar to Aeromonas hydrophila (see Aeromonas, p. [link] ). It has been implicated in outbreaks of diarrhoea, and is acquired from contaminated seafood.
• V. damsela is a halophilic, marine organism that can cause severe wound infections. It is acquired in warm coastal areas.
• V. hollisae has been associated with diarrhoea and bacteraemia in areas of warm seawater in the USA. It is acquired from raw seafood.
• V. mimicus is associated with gastroenteritis from eating raw oysters. There are also reports of ear infections. It occurs in environments similar to V. cholerae.
Aeromonas spp. are aquatic organisms, which have been implicated in causing diarrhoea. They also cause soft tissue infections and sepsis in the immunocompromised. A. hydrophila, A. sobria, and A. caviae are the main species, and A. salmonicida is an economically important fish pathogen. The genus Aeromonas has undergone a number of taxonomic and nomenclature revisions recently, and been moved from the family Vibrionaceae to the new family Aeromonadaceae.
Aeromonas diarrhoea is more common in the summer months when water concentrations of aeromonads are higher. Outbreaks may occur, and Aeromonas infection is being increasingly recognized as a cause of traveller’s diarrhoea.
Gastroenteritis is the most common disease associated with Aeromonas, but its role is debated (Box 4.9 ). It is unclear whether most faecal isolates recovered from symptomatic patients actually cause diarrhoea. It may be that only specific subsets of Aeromonas are pathogenic, and new biotyping schemes are needed to differentiate environmental from clinical strains.
Diarrhoea tends to be watery and self-limiting, but is occasionally more severe. Chronic colitis following diarrhoea has been reported. In addition to gastroenteritis, there are reports of aeromonas septicaemia in the immunocompromised, and wound infections in healthy people and those undergoing leech therapy. The main skin-associated aeromonad is A. hydrophila. There are rare reports of nosocomial bacteraemia, peritonitis, meningitis, and eye and bone and joint infections.
This facultatively anaerobic GNR is usually β-haemolytic on blood agar, and ferments carbohydrates to produce acid and gas. It grows readily on MacConkey agar, and lactose fermentation is variable. Growth on TCBS agar is also variable. It is oxidase-positive, so can be distinguished from the oxidase-negative Enterobacteriaceae. Selective techniques are needed to isolate it from a mixed culture. Suitable plates for detection of Aeromonas from stool include cefsulodin-irgasan-novobiocin (CIN) agar or blood agar containing ampicillin. Note that not all laboratories routinely culture stools for Aeromonas, and some enteric media actually inhibit its growth.
Plesiomonas shigelloides, the only species in the genus, is associated with outbreaks of gastroenteritis in warm climates. In the literature it has been known as Pseudomonas shigelloides, C27, Aeromonas shigelloides, and Vibrio shigelloides. The taxonomic status has varied – it is related to Proteus, but currently placed in the family Vibrionaceae.
P. shigelloides is found in soil and water (mainly fresh water, but also salt water in warm weather). It is usually transmitted to humans via water or food (e.g. shrimp, chicken and oysters), and also colonizes many animals. Most patients recently travelled abroad.
There is no animal model, and no pathogenic mechanism has been identified. Volunteer studies have been largely unsuccessful in causing disease. Hence it has been difficult to prove a causal relationship.
Symptoms vary from mild self-limiting diarrhoea to mucoid bloody diarrhoea with features of entero-invasive disease. It has occasionally resulted in serious extra-intestinal infection such as osteomyelitis, septic arthritis, endophthalmitis, spontaneous bacterial peritonitis, pancreatic abscess, cellulitis, cholecystitis, and neonatal sepsis with meningitis. Bacteraemia is rare and usually in the immunocompromised.
This motile, facultatively anaerobic GNR does not ferment lactose. It grows readily at 35°C on most enteric agars, such as MacConkey, but does not grow on TCBS. It appears non-haemolytic and is oxidase-positive. Selective techniques are needed to isolate it from a mixed culture.
Campylobacter organisms are spiral-shaped flagellate bacteria belonging to rRNA superfamily VI. C. jejuni is the commonest cause of diarrhoea in most developed countries. C. coli also causes diarrhoea. C. fetus is the type species of the genus and causes abortion in sheep and cows. It occasionally causes septic abortions in humans and bacteraemia in the immunocompromised. Some species including C. lari and C. upsaliensis cause diarrhoea in children in developing countries, while species such as C. concisus and C. rectus are associated with periodontal disease.
Campylobacter organisms are ingested (faeco-oral transmission), then colonize (and usually invade) the jejunum, ileum, and terminal ileum, occasionally extending to the colon and rectum. Mesenteric lymph node involvement and transient bacteraemia may occur. Histological findings of acute inflammation ± superficial ulceration are the same as in Salmonella, Shigella or Yersinia infections.
Campylobacter gastroenteritis is variable in terms of symptoms and severity. In severe cases, GI haemorrhage, toxic megacolon, and Haemolytic Uraemic Syndrome (HUS) have been reported. Other complications include meningitis, deep abscesses, cholecystitis, and reactive arthritis. Approx. 25% cases of Guillain-Barré syndrome (GBS) have documented preceding Campylobacter gastroenteritis – the LOS cell surface structures act as critical factors in triggering GBS through ganglioside mimicry.
This small, spiral GNR has a single unsheathed flagella at one or both poles and is extremely motile. It is micro-aerophilic and grows best at 42°C. Like Helicobacter, Campylobacter organisms undergo coccal transformation under adverse conditions and are biochemically inactive. However they are oxidase-positive. C. jejuni is the only species that hydrolyses hippurate. Typing methods include serotyping (Penner scheme for O antigens and Lior scheme for heat-labile surface and flagellar antigens), biotyping, phage typing, and newer molecular methods.
Rehydration and symptom relief is usually adequate, as Campylobacter infection is usually self-limiting in 5–7 days. However, in severe dysenteric disease, erythromycin or ciprofloxacin may be prescribed. Resistant strains, especially C. coli may respod to trimethoprim or co-trimoxazole. Good hygiene standards are important in prevention. Infective organisms may be excreted in the stool for ~3 weeks after resolution of diarrhoea. There is no vaccine.
The genus Helicobacter contains up to 17 species, which colonize the stomachs of different animals. H. pylori is a spiral-shaped flagellate bacteria belonging to rRNA superfamily VI, which colonizes humans (it is found in approx 50% of the world population). H. pylori was discovered in 1983 in Australia by Warren and Marshall, who went on to receive the Nobel Prize for Medicine in 2005. Its importance in the pathogenesis of peptic ulcer disease soon became clear. H. cinaedi and H. fennelliae are associated with proctitis in homosexual men.
As with other bacteria in rRNA superfamily VI, H. pylori is adapted to colonizing mucous membranes (in this case the gastric mucosa only) and penetrating mucus. The cagA protein is important in virulence. After phosphorylation by tyrosine kinase, cagA is injected into epithelial cells by a type IV secretion system. This alters signal transduction and gene expression in host epithelial cells
H. pylori is associated with 95% of duodenal and 70% of gastric ulcers. Epidemiological studies have highlighted the association of H. pylori and gastric cancer, and WHO classifies H. pylori as a group 1 carcinogen.
This GNR is shaped like a helix (hence its name) and has a tuft of sheathed unipolar flagella. It is strictly micro-aerophilic and requires CO2 for growth. It is relatively inactive biochemically, except for strong urease production. Under adverse conditions, it undergoes coccal transformation. Options for testing patients are as follows:
• Serology – if positive, this indicates the patient has been infected
• biopsy of stomach or duodenum for histology ± urease test ± culture
• urea breath tests – the patient drinks 14C- or 13C-labelled urea, which is metabolized by H. pylori, producing labelled CO2 that can be detected in the breath. This test is also used to assess effectiveness of treatment
• rapid urease test (the enzyme urease produced by H. pylori catalyses the conversion of urea to ammonia and bicarbonate, which is reflected by a rise in pH.) This is usually performed on a biopsy sample
• faecal antigen tests.
The urea breath test or stool antigen test have greater sensitivity and specificity than serology for diagnosis, and can also be used to confirm eradication. The patient should receive no antibiotics for 4 weeks before the tests, and no proton pump inhibitor (PPI) for 2 weeks before the tests. Molecular typing of H. pylori is more useful than serotyping.
NICE has issued clinical guidelines on Managing dyspepsia in adults in primary care (August 2004). 1 Triple therapy is given in specific circumstances and consists of a proton pump inhibitor (PPI) e.g. omeprazole and two antibiotics (e.g. amoxicillin, clarithromycin, or metranidende).
Eradication of H. pylori is beneficial in duodenal and gastric ulcers and low-grade MALToma (mucosal associated lymphoid tissue), but not in gastro-oesophageal reflux disease (GORD). In non-ulcer dyspepsia, 8% of patients benefit. Triple treatment achieves >85% eradication. Essentially, any dyspeptic patient with no alarm symptoms should receive a PPI for one week. If symptoms relapse they should be tested and treated for H. pylori, using the breath test or stool antigen test. First-line ‘triple therapy’ is with a PPI for 1 week, and two antibiotics (amoxicillin or metronidazole, together with clarithromycin). Clarithromycin or metronidazole should not be given if they have been used for any infection in the previous year. Approximately 10% of patients fail treatment, possibly due to antibiotic resistance (Box 4.10 ).
A Cochrane review (2006) of eradication therapy for peptic ulcer disease in H. pylori-positive patients found that treatment had a small benefit in initial healing of duodenal ulcers, and a significant benefit in preventing the recurrence of both gastric and duodenal ulcers, once healing had been achieved. 2 Other treatment includes probiotics (which improved eradication rates and reduced adverse events in a recent meta-analysis), and bismuth compounds.
1 NICE. Managing Dyspepsia in Adults in Primary Care. London: NICE. Marygedi P et al. Eradication of Helicobacter pylori for non-ulcer dyspepsia. Cochrane Database of Systematic Reviews 2006, Issue 2. Art no. CD0002026. DOI:10:1002/14651858. CD002096.pub4.
2 Moaygedi P. et al. Eradication of Helicobacter pylon for non-ulcer dyspepsia. Cochrane Database of Systematic Reviews 2006. Issue 2. Art. no CD002026. DOI: 10:1002114651858.CD002096.pub4.
More than 30 genera of anaerobic GNR are recognized but human infections are largely restricted to four of these: Bacteroides, Prevotella, Porphyromonas and Fusobacterium (Table 4.16 ). These organisms are found in the mouth, gastrointestinal tract and vagina and are among the most important constituents of ‘normal flora’. They may cause a variety of infections in humans, particularly polymicrobial infections and abscesses. Bacteroides fragilis is the most important species; it is found in the gastrointestinal tract and is associated with a wide variety of infections.
Table 4.16 Characteristics of anaerobic Gram-negative rods
Growth in 20% bile
Penicillin Vancomycin Kanamycin Colistin
Colistin Penicillin Kanamycin
Erythro-mycin Rifampicin Colistin Penicillin
Erythro-mycin Rifampicin Penicillin Vancomycin
Virulence factors of Bacteroides spp. include:
• capsular polysaccharide – inhibits opsonisation / phagocytosis, promotes abscess formation and promotes adherence to epithelial cells
• pili and fimbriae – promote adherence to epithelial cells and mucus
• succinic acid – inhibits phagocytosis and intracellular killing.
• Enzyme production – contribute to tissue damage and /or promote invasion and spread e.g heparinase, fibrinolysin, hyaluronidsase, neuraminidase
• Synergy between anaerobic and facultative bacteria – see Box 4.11
• Intra-abdominal infections – B. fragilis is the most common anaerobic isolate in intra-abdominal abscesses
• Diarrhoea – enterotoxin-producing strains have been implicated in diarrhoea in children.
• Bacteraemia – B. fragilis is the most common isolate in anaerobic bacteraemias. The source is usually intra-abdominal and associated with abscesses, malignancy, bowel performation or surgery. Septic shock is less common in B. fragilis bacteraemia than in bacteraemia caused by aerobic Gram-negative bacilli; this is presumably related to the absence of lipid A in the endotoxin of B. fragilis.
• Endocarditis – associated with large vegetations and high frequency of thromboembolic complications
• Skin and soft tissue infections – often found as part of mixed flora in diabetic and decubitus ulcers. B. fragilis has also been isolated from cutaneous abscesses of the lower limbs
• Bone and joint infections – B. fragilis may rarely cause osteomyelitis and septic arthritis.
• CNS infections – anaerobic meningitis is rare and most laboratories do not culture CSF anaerobically. In the cases of anaerobic meningitis that have been described, B. fragilis is the most common isolate. In contrast, anaerobes are frequently implicated in brain abscesses.
• Bacteroides are non-spore forming, non-motile anaerobic Gram-negative rods.
• On blood agar, Bacteroides appears as glistening, non-hemolytic colonies which are aerotolerant.
• Gram stain may reveal pale pink, pleomorphic coccobacilli, with irregular or bipolar staining.
• They can be differentiated from the other anaerobic GNR by growth in 20% bile.
• The MASTRING™ ID (Mast Diagnostics) may be used to identify B. fragilis in the laboratory. This is a ring containing 6 antibiotic discs that is placed on the culture plate and incubated anaerobically at 37°C for up to 3 days. B. fragilis is usually sensitive to erythromycin and rifampicin and usually resistant to penicillin G, vancomycin, kanamycin and colistin.
• Drainage of abscesses and debridement of necrotic tissue is the mainstay of treatment for anerobic infections. However, some abscesses (e.g. brain, liver and tubo-ovarian) have been managed with antimicrobial therapy alone.
• The choice of antibiotics to treat anaerobic infections is usually empirical, as most of the infections are polymicrobial and require broad spectrum therapy
• Bacteroides is usually sensitive to antimicrobials such as metronidazole, clindamycin, chloramphenicol, carbapenems, cefoxitin and β-lactam/β-lactamase inhibitor combinations (e.g. co-amoxiclav, piperacillin-tazobactam).
Prevotella and Porphyromonas formerly belonged to the genus Bacteroides ( p. [link] ) but were reclassified in 1990.
• The genus Prevotella includes P. melaninogenica, P. bivia, P. oralis and P. bucalis.
• The genus Porphyromonas includes P. gingivalis P. endodontalis, and P. asaccharolytica.
• Virulence in P. melaninogenica is associated with the capsular polysaccharide, which inhibits opsonophagocytosis, promotes abscess formation and also promotes adherence to epithelial cells.
• In P. gingivalis, pili and fimbriae aid adherence to epithelial cells and mucus.
• Production of various enzymes may also aid evasion of the host immune response, or promote tissue destruction.
Prevotella and Porphyromonas contribute to the formation of abscesses and soft tissue infections in various parts of the body. They also cause infections of the oral cavity (such as periodontal and endodontal disease), female genital tract infections, osteomyelitis of the facial bones and human-bite infections.
• These are non-spore forming, non-motile anaerobic GNR.
• They are usually isolated (along with other anaerobes) from abscesses and soft tissue infections.
• Prevotella and Porphyromonas may both appear pigmented – usually brown/black
• Young unpigmented colonies can show brick-red fluorescence under UV light.
• Gram stain reveals small, pale pink cocco-bacilli.
• Prevotella and Porphyromonas are both inhibited by 20% bile.
• Prevotella are moderately saccharolytic whereas Porphyromonas are asaccharolytic.
• The mainstay of treatment for anaerobic infections is surgical drainage of abscesses and debridement of necrotic tissue.
• Prevotella and Porphyromonas are usually sensitive to agents such as metronidazole, clindamycin, chloramphenicol and cefoxitin. Penicillin resistance is common, but isolates are usually susceptible to co-amoxiclav and other β-lactam / β-lactamase inhibitor combinations
Fusobacterium spp. colonise the mucous membranes of animals and humans, and occasionally cause infections of the oral cavity and head and neck. Clinically, the most important species are:
• F. nucleatum (subspecies nucleatum, polymorphum and fusiforme)
• F. necrophorum (subspecies necrophorum and fundiliforme).
Fusobacteria are commensals of the oral cavity. As with other obligate anaerobes, the significance of these organisms is being increasingly recognized. However, Fusobacterium infections are relatively rare in the UK.
Fusobacterium spp. produces lipopolysaccharide (LPS) endotoxin which is biologically active. They also produce metabolites that are important to oral spirochaetes.
• F. necrophorum causes severe systemic infections such as Lemierre’s disease (see p. [link] ), post-anginal sepsis and necrobacillosis.
• Lemierre’s disease is a severe systemic disease which occurs in previously healthy young adults, and usually presents initially as severe sore throat, followed by fever, cervical lymphadenopathy and unilateral thrombophlebitis of the internal jugular vein. Metastatic infection with spread to the lungs or bones or brain may occur. If untreated the condition leads to death in 7-15 days.
• Other species commonly isolated from oral infections include F. periodonticum, F. alocis, F. sulci and F. naviforme.
• Species found in the gastrointestinal or genitourinary tracts (e.g. F. mortiferum, F. necrogenes F. varium and F. gonidiaformans) may cause intra-abdominal infections, osteomyelitis, ulcers and skin / soft tissue infections.
• F. ulcerans was originally isolated from tropical ulcers, but may be found in other sites.
• Fusobacterium are long, thin, GNR with pointed ends (‘fusiform’) that are often arranged in pairs. They are non-spore forming and non-motile
• They may be haemolytic on blood agar and may grow in the presence of 20% bile.
• They can be identified using commercial tests e.g. MASTRING™ ID (Mast Diagnostics) and the API 20A or Rapid ID 32A (Biomerieux).
• Susceptibility testing is vital as there have been reports of penicillin resistant strains.
Molecular techniques (e.g. PCR) have been developed.
• The mainstay of treatment for anaerobic infections is surgical drainage of abscesses and debridement of necrotic tissue.
• Lemierre’s syndrome and other severe invasive disease is usually treated with a combination of penicillin and metronidazole, for 2-6 weeks.
• Alternatives include clindamycin monotherapy or chloramphenicol.
The spirochaetes are a group of helical organisms sharing many properties with Gram-negative bacteria. The vast majority are non-pathogenic but a few are important causes of disease in humans (see Table 4.16 ). There are aerobic and anaerobic species, both free living and parasitic. Axial filaments, fixed at each end of the organism, run along the outside of the protoplasm within the outer sheath and give the characteristic coiled appearance. These are similar to bacterial flagella and are capable of constricting, warping the cell bod, and enabling the bacterium to move by rotating it in space.
Table 4.16 Overview of spirochaetes of clinical significance
T. pallidum subsp. pallidum
Morpho-logically identical. Thin helical cells 10 micrometre by 0.15 micrometre. Visible on dark-field microscopy
Cannot be cultured in vitro; remain motile in specific enriched media at 35°C for several days
Direct detection only means in primary syphilis; mainstay is serology; cross-reactivity between species
T. pallidum subsp. pertenue
T. pallidum subsp. endemicum
Louse-borne relapsing fever
Helical. 3–20 micrometre by 0.25 micrometre. Can be stained with aniline dyes
Can be cultured but not practical
Demonstration of spirochaetes in peripheral blood smears; immunological and PCR-based tests available
B. hermsii and others
Tick-borne relapsing fever
Culture possible from biopsy of rash.
Serology; can remain positive for years
Motile, 10 micrometre by 0.1 micrometre. Stain poorly – visible on dark-field or phase contrast
Specialized media. Allow minimum 6 weeks.
Serology; molecular techniques available.
Four members of the genus Treponema cause human disease: Treponema pallidum subsp. pallidum (syphilis) and three ‘non-venereal’ treponematoses.
Morphologically identical, Treponema species appear as motile helical rods on dark-field microscopy. They are thin, helical cells around 10 micrometre long and 0.15 micrometre wide. They cannot be cultured in vitro (unlike the non-pathogenic treponemes), but remain motile in specific enriched media for several days at 35°C. Organisms remain viable after freezing. The organisms all share a significant degree of DNA homology and are very similar antigenically, thus all cause positive serological tests for syphilis.
Epidemiology and clinical features
• Treponema pallidum subsp. pallidum – the causative agent of syphilis. An increasing incidence in the UK, beginning in the 1960s, plateaued in the mid-90s but several large outbreaks between 1998 and 2003 saw diagnoses of infectious syphilis in men rise 15-fold. Transmission: sexual contact, direct vascular inoculation (IVDU, transfusions), direct cutaneous contact with infectious lesions or transplacental infection (congenital syphilis, p. [link] ). Interacts with HIV in both acquisition and diagnosis. see p. [link] for clinical features.
• Treponema pallidum subsp. pertenue – the causative agent of yaws, a chronic non-venereal disease endemic in the humid tropics (Central Africa, South America, South-East Asia, and parts of the Indian subcontinent). Acquired in childhood through contact with infectious skin lesions. Incubation: 3 weeks. Affects the skin (papular skin lesions which may ulcerate) and bones (periosteitis, dactylitis). Primary stage: lesion at inoculation site; secondary stage: dissemination of treponemes causing multiple skin lesions; latent stage: usually asymptomatic (most patients remain non-infectiously latent for their lifetime); tertiary stage (<10% patients 5–10 years later): bone, joint, soft tissue deformities.
• Treponema pallidum subsp. endemicum – the causative agent of non-venereal endemic syphilis or ‘bejel’. Endemic in dry subtropical or temperate areas of the Middle East, India, Asia and parts of Africa. Infection occurs in childhood and is associated with poor standards of hygiene. Transmission: contact with mucosal lesions or contaminated eating utensils/water. Incubation: 10–90 days. Primary lesions (1–6 weeks): patches in mouth followed by skin lesions resembling the chancres of venereal syphilis; secondary stage (6–9 months): macerated patches on lips and tongue, anogenital hypertrophic condyloma lata, painful osteoperiostitis of long bones; tertiary stage: destruction of cartilage and bone, gummata of skin, bones and nasopharynx. CNS/CVS (cardiovascular system) disease is very rare.
• Treponema carateum – the causative agent of pinta, the most benign of the endemic treponematoses affecting only the skin. Endemic to South/Central America. Spread by contact with infected skin. Incubation 2–3 weeks. Primary lesion: papule or erythematous plaque on exposed surfaces of the legs, foot, forearm, or hands which slowly enlarges becoming pigmented and hyperkeratotic. May be associated with regional lymphadenopathy. Secondary lesions: disseminated lesiosn of similar appearance appearing 3–9 months later. Late/tertiary pinta: disfiguring pigmentary changes, and atrophic lesions.
• Direct detection – culture (the gold standard) is expensive and time-consuming and used primarily in research. Direct detection of organisms via dark-field microscopy, or preferably immunofluorescence of material scraped from a lesion, is the only means of diagnosis in primary syphilis.
• Serological diagnosis – the mainstay. Serological tests fall into two groups. Both show cross-reactivity between the four Treponema species:
• non-treponemal tests (e.g. Venereal Disease Research Laboratory (VDRL)) – detect antibodies to cardiolipin produced as a response to treponemal infection and are not specific but are very sensitive. Samples with very high antibody titres may give false-negative results (the ‘prozone’ phenomenon), e.g. in early infection or HIV. Poor sensitivity in late-stage infection. Antibody titre tends to decline to negativity, with adequate therapy
• treponemal tests (e.g. treponema pallidum haemagglutination assay TPHA) – use specific treponemal antigens and are consequently more specific. They are able to detect late-stage infection and tend to remain positive after adequate therapy.
• Traditional WHO recommendations for diagnosis are a sensitive non-treponemal screening test, with positive samples followed up using a more-specific and ideally equally sensitive treponemal assay. In the UK, a combination of sensitive screening tests for treponemal antibody – VDRL and TPHA – is used initially, allowing sensitive and specific screening of all but very early primary disease. Positive samples are followed up using fluorescent treponemal antibody tests. Enzyme-immunosorbent assays (EIAs) for treponemal IgG and IgM are beginning to supersede this technique: more easily automated, more objective, at least as good as the VDRL/TPHA combination, and a useful method for detecting antibody in patients with HIV. Positive EIAs are confirmed using TPHA. Discrepant results are repeated using the fluorescent treponemal antibody-absorption test (FTA-Abs). Seronegative patients at recent risk of acquiring disease should be followed up because of the seronegative window in early primary syphilis (see Syphilis, p. [link] ).
• Molecular techniques – PCR tests are available but not yet widely used.
• Early syphilis and pinta/yaws/bejel – prolonged antibiotic therapy is required due to the slow dividing rate of T. pallidum (averages one doubling in vivo per day). Highly sensitive to penicillin and a long-acting depot injection of benzathine benzylpenicillin is the standard therapy. A single dose is sufficient for early infection. Alternative: 15-day course of azithromycin (increasing reports of resistance) or doxycycline.
• Late syphilis – weekly doses of benzathine penicillin over three weeks. Alternatives: doxycycline, ceftriaxone. Consider the possibility of re-infection in cases of treatment failure.
• Neurosyphilis – IV penicillin, as benzathine benzylpenicillin achieves no measurable CSF levels and there are a number of reports of patients treated with benzathine benzylpenicillin developing neurosyphilis.
These are helical bacteria around 0.25 micrometre in diameter and between 3 and 20 micrometre long. Those causing human disease are transmitted by insect vectors.
• Louse-borne relapsing fever – B. recurrentis
• Tick-borne relapsing fever – variety of species
• Lyme disease – B. burgdorferi
Relapsing fever is caused by several Borrelia species transmitted by arthropods, characterized by recurring episodes of fever. Two distinct clinical forms were recognized as far back as ancient Greece: epidemic louse-borne and endemic tick-borne relapsing fever. The presentation of abrupt fever, muscle aches, and joint pains with crisis, remission, and then relapse are similar for both but the periodicity tends to be characteristic (e.g. 5.5 days for louse-borne versus 3.1 days for tick-borne). The recurrent nature is thought to be due to antigenic variation of the spirochaetal outer membrane proteins.
• Tick-borne relapsing fever is worldwide and transmitted by soft-bodied Ornithodorus ticks. Most tick species carry a distinctive borreliae. Epidemiology depends on the local vector, e.g. O. hermsii is the commnest vector in California and Canada and lives in dead trees and on rodents and transmits B. hermsii. Infection is passed down the tick generations, thus disease tends to be endemic.
• Louse-borne relapsing fever has occurred in Africa, Middle East, and Asia. Human body-louse inhabits only humans and B. recurrentis is not transmitted vertically within lice thus is maintained by passage from louse to human and then back to another louse, which remains infective for its entire life. Therefore infection is associated with poverty and overcrowding, and disease tends to be epidemic.
• Incubation and symptoms are similar in both conditions – 3–8 days after exposure there is the abrupt-onset fever, headache, myalgia, arthralgia, chills, weakness, anorexia, epistaxis, cough/haemoptysis, and weight loss. Examination findings include hypotension, hepatosplenomegaly, lymphadenopathy, nuchal rigidity, jaundice, photophobia, injected conjunctiva, and iritis.
• Tick-borne disease – the primary episode lasts 3–6 days and is followed by a critical episode than may cause fatal shock. The first relapse occurs 7–10 days later. Subsequent relapses are less severe. The average number of relapses experienced is 3 but can be as many as 10.
• Louse-borne disease – there are fewer relapses than with tick-borne infection, and hepatic or splenic involvement is more common as are neurological manifestations (coma, hemiplegia, meningitis, seizures).
Organisms can be cultured but isolation is not practical. Serological tests are not diagnostically useful. Approx 5% of patients have positive VDRL. Most useful is demonstration of spirochaetes in peripheral blood smears (and other body fluids – marrow aspirates, CSF). Unlike the other spirochaetes, borreliae stain well with acid aniline dyes such as Giemsa. They are most likely to be found during febrile episodes when the sensitivity of blood smears is around 70% for louse-borne fever (less for tick). Multiple thick and thin smears may need to be examined. Immunological and PCR-based tests are available. Other lab findings iclude deranged clotting tests, elevated liver function tests (LFTs).
• Tick-borne relapsing fever – tetracycline is the drug of choice, given for 7–14 days. Other: doxycycline 7 days, erythromycin 10 days.
• Louse-borne relapsing fever – a single dose of doxycycline (preferred), tetracycline, erythromycin, or penicillin G.
• Jarisch–Herxheimer reactions can occur (usually within the first 2 h after antibiotic administration), particularly in louse-borne relapsing fever. Features: sweating, tachycardia, hypertension followed by profound hypotension. It can be fatal and appears to be mediated partly by tumour necrosis factor (TNF)-α. Preadministration of steroids does not appear to limit the reaction significantly. Anti-TNF-α antibodies may help.
Caused by infection with, and the host immune response to Borrelia burgdorferi. Acquired by the bite of ixodes (hard) ticks and co-infection with other tick-borne organisms can occur (e.g. babesiosis).
Ticks acquire a spread infection through feeding on infected animals (particularly deer). A tick must be attached to a person for 2–3 days to pass on infection, as only small numbers of bacteria are present in the tick until it feeds – the act of feeding sees bacteria multiply and pass to the salivary glands. Until this multiplication occurs ticks are rarely able to pass on infection; 85% of human infections occur while the tick is in the nymph stage (spring to summer), 15% when it is in the adult stage (autumn). Cases are commonest in children aged 5–9 years and adults aged 60–69 years – only 40% give a definite history of tick bite. Cases occur across Europe, China, Japan, Australia, and parts of the USA. It is relatively rare in the UK, most cases occurring in the south (New Forest and Salisbury Plain), East Anglia, Cumbria, and the Scottish highlands.
Like its cousin, syphilis, it is a great imitator. Clinical features may be a result of direct bacterial infection (particularly in the early stages of disease), or a consequence of an immune response leading to symptoms in many organs (e.g. arthritis). They differ with the strain of Borrelia involved (see below). Asymptomatic infection occurs – <10% of those in endemic areas with no history of infection are seropositive. Features may be seen in three overlapping stages:
• early localized – around 7 days after tick bite <75% of patients develop erythema chronicum migrans; an expanding painless annular skin lesion centred on the bite with or without local lymphadenopathy may occur. It is probably a result of the inflammatory response to the organism in the skin. Multiple lesions can occur and do not necessarily represent multiple bites. Lasts 2–3 weeks untreated
• early disseminated – weeks to months after the bite patients develop more severe constitutional symptoms, malaise, generalized lymphadenopathy, hepatitis, arthritis (50% – initially intermittent and migratory it may evolves into a chronic monoarticular arthritis in 10% of those affected), neurological features (15% – include meningitis, meningo-encephalitis, cranial nerve lesions, and neuropathy), cardiac features (10% – atrioventricular (AV) block, pericarditis, congestive cardiac failure (CCF))
• late persistent (but can occur within the first year) – arthritis, late neurological manifestations including focal deficits, fatigue, and neuropsychiatric problems. Acrodermatitis chronica atrophicans is a decolouration of the skin, seen in the extremities and similar to the skin changes of peripheral vascular disease.
Three members of the Borrelia burgdorferi sensu lato complex cause Lyme disease: Borrelia garinii and afzelii in Asia, and Borrelia burgdoferi sensu stricto in North America. Borrelia garinii and afzelii are the commonest European clinical isolates. These differences account for the variation in clinical manifestations across the world (B. garinii associated with neurological disease, B. afzelli cutaneous manifestations, and B. burgdorferi sensu stricto, joint symptoms).
• Culture of the organism is possible from skin specimens taken from those patients with erythema migrans. It is difficult to identify spirochaetes in histological section.
• Serology – the diagnostic method of choice – CDC recommend a screening ELISA for anti-Lyme antibody with positive titres confirmed by Western blot. Specific response is sensitive but develops late (30% positive in the acute phase, 70% at 2–4 weeks, 90% at 4–6 weeks). Prompt antibiotic therapy may prevent a good antibody response. Some patients remain positive for years after illness, thus active and inactive infection cannot be distinguished. Tests must be interpreted with caution in those without a positive travel history or a presentation consistent with Lyme disease. Serology is nearly always positive in those with extracutaneous lesions. False positives may be seen in association with rheumatoid disease, infectious mononucleosis, and syphilis.
• PCR for Borrelia burgdorferi DNA is more sensitive than culture or microscopy in the examination of blood or joint fluid, but has not been standardized for routine diagnosis.
• CSF – spinal fluid should be obtained in those patients with neurological symptoms in whom the diagnosis is not obvious. CSF serology may be helpful.
• Patients with a good history and classic erythema migrans should be treated regardless of serology results. Patients probably remain at risk of infection even after treatment for one episode.
• Early-stage skin manifestations, arthritis, or Bell’s palsy – doxycycline or amoxicillin PO for 28 days. If arthritis persists repeat course or consider IV ceftriaxone for 14–30 days.
• 3rd-degree heart block – IV ceftriaxone for 14–28 days.
• Neurological disease – cranial nerve palsies: 30-day oral regime as above; parasthesia/radiculopathy: 14 days IV ceftriaxone; encephalitis/encephalopathy: 28 days IV ceftriaxone.
Leptospira are motile, obligately aerobic spirochaetes measuring 0.1 micro-metre in diameter and around 10 micrometre long. They stain poorly but can be visualized on dark-field or phase-contrast microscopy. Two species are identified: L. interrogans (includes all human pathogens), and L. biflexa (a saprophytic species). L. interrogans has many serotypes, and antigenically related organisms are grouped into serovars (a synonym for serotype) for classification. Although more-recent DNA analysis does not correlate well with serological classification, serological classification will continue to be used for the foreseeable future. The ‘type’ strain is L. interrogans serovar ictero-haemorrhagiae, and the type disease, leptospirosis.
A biphasic disease with initial septicaemia and a secondary phase characterized by immune phenomona (vasculitis, aseptic meningitis). Weil’s disease is a severe form characterized by jaundice and acute renal failure.
Leptospira are found worldwide. The primary reservoirs of most leptospiral serovars are wild mammals. These continually re-infect domestic populations and at least 160 mammalian species are affected. The organism has been recovered from rats, pigs, dogs, cats, and cattle among others, but rarely causes disease in these hosts. Rodents are the most-important reservoir, and rats the commonest worldwide source. There are associations between particular animals and serovars (e.g. L. interrogans serovar ictero-haemorrhagiae and rats). Humans are incidental hosts and onwards transmission is rare. Transmission occurs when people come into contact with infected animal urine, e.g. canoeing, swimming in lakes and rivers, farming. It is primarily a disease of tropical and subtropical regions, and infection in temperate regions is uncommon.
After gaining entry via the skin or mucous membranes, the organism replicates in blood and tissue. Leptospiraemia particularly affects the liver and kidney causing centrilobular necrosis and jaundice, or intersititial nephritis and tubular necrosis respectively. Renal failure may occur, exacerbated by hypovolaemia. Other organs affected include muscle (oedema and focal necrosis), capillaries (vasculitis), eye (chronic uveitis).
Incubation is 7–12 days. The majority of patients (90%) develop mild disease without jaundice; 5–10% develop the severe form, Weil’s disease. Disease is biphasic. The first phase (‘septicaemic’ – the organism can be cultured from blood, CSF and most tissues) lasts 4–7 days and is characterized by a flu-like illness, fever, chills, weakness, myalgia, cough, haemoptysis, rash, meningism, and headache. A 1–3-day period of improvement follows and patients may become afebrile. The second stage then starts (‘immune’ or ‘leptospiruric phase’ in which antibodies may be detected and the organism isolated from urine). Features are due to the immunological response to infection and may last up to a month. Disease may be anicteric or icteric. Aseptic meningitis is the most-important feature of anicteric disease and is seen in 50% of cases. Death is rare in this group. Icteric disease is characterized by jaundice, hepatosplenomegaly, nausea/vomiting, anorexia and diarrhoea/constipation. Organisms can be isolated from the blood <48 h after jaundice onset. Other features: uveitis (<10% – can occur up to a year after initial illness), subconjunctival haemorrhage is commonest ocular complication (92% of patients), renal impairment (uraemia, pyuria, haematuria, oliguria), pulmonary manifestations. Weil’s disease is characterized by jaundice, renal failure, hepatic necrosis, lung disease, and bleeding. It starts at the end of stage one. Overall mortality is 10%, up to 40% in those with hepatorenal involvement.
• Direct examination – dark-field examination of blood, CSF, or urine may demonstrate leptospira but there is a high false-positive rate (misinterpretation of fibrils and red cell fragments). It is not recommended.
• Culture – there has been little change in culture techniques over the years. It is difficult, insensitive and requires several weeks of incubation. Specialized culture media are required (e.g. Ellinghausen-McCullough-Johnson-Harris (EMJH) which contains 1% bovine serum albumin and Tween 80, a fatty acid source). They should be inoculated within 24 h of specimen collection (either blood or CSF in heparin or sodium oxalate). Leptospiral culture can be established by subculture of routine blood culture samples. Organisms can be isolated from blood and CSF in the first week of illness. In the second phase of illness they can be found only in the urine where they may be isolated for up to 1 month. Cultures can be reported as negative after a minimum of 6 weeks – continuing for as long as 4 months is preferable.
• Molecular techniques – quantitative PCR assays to detect leptosiral DNA have been developed. They are sensitive, can distinguish different species, allow early diagnosis, and organisms can be detected after antibiotic therapy has been initiated.
• Serology – the mainstay of diagnosis. Commercial tests using genus-specific antigens are used to screen sera, and positive reactions confirmed in a reference laboratory using the microagglutination test (MAT) with live leptospira (killed have lower sensitivity). The MAT detects agglutinating antibodies in patient serum and is relatively serovar specific so a large number of antigens must be tested. Interlaboratory variation is high. A positive MAT is considered to be a fourfold increase in antibody titre, or a switch from seronegative to a titre of 1/100 or over. Early samples tend to cross-react; convalescent samples are more specific and diagnostic. Enzyme immunoassay for IgM is useful for diagnosing current infection but cross-reactions occur.
• Mild disease – doxycycline, ampicillin, amoxicillin
• Severe disease – ampicillin, penicillin G, ceftriaxone
• Prophylaxis – doxycycline reduces morbidity and mortiality in endemic areas, but has no impact on infection rates as measured by seroconversion. It is likely to be useful in cases of accidental lab exposure or military/adventure travel. Vaccines are available against specific serovars.
The genus Rickettsia is a member of the Rickettsiales order, which also includes Coxiella, Ehrlichia, and Bartonella. All are maintained in a cycle involving mammal reservoirs and arthropod vectors. Rickettsia organisms are fastidious, obligate intracellular Gram-negative coccobacilli (0.3 micrometre by 1–2 micrometre). They survive only briefly outside a host (unlike Coxiella). Isolation is usually only performed in reference laboratories.
Zoonotic reservoirs are varied and include wild rodents, dogs, and livestock. Humans are incidental hosts with the exception of louse-borne typhus where humans are the main reservoir. R. rickettsii, R. typhi, R. tsutsugamushi, and R. akari can exist as vector commensals. R. prowazekii however kills its human body louse vector within 3 weeks. See Table 4.17 for geographical distribution.
Table 4.17 Overview of Rickettsial Desease
Spotted fever group
Rocky Mountain spotted fever
Ixodid ticks (Western hemisphere)
Incubation ~7 days. Fever, headache, myalgia, eschar, rash. Multisystem involvement; 20% untreated mortality
Mediterranean Spotted Fever
Ixodid ticks (Mediterranean, Africa, and India)
Incubation ~5 days. Eschar and local lymphadenopathy. Rash. Mild.
Mite (USA, Africa, Korea and CIS (Commonwealth of Independent States))
Incubation ~7 days. As for R. conorii plus vesicular rash resembling chickenpox
Body louse (S America, Africa, Asia)
Incubation ~10 days. Fever, headache, neurological and GI symptoms. Rash. 20–50% untreated mortality
Nil – recurrence years after primary attack
Similar, milder illness than epidemic typhus developing years after recovery – in west was seen in E European immigrants after World War 2. Lasts around 2 weeks
Murine (endemic) typhus
Flea (worldwide where human/rat co-exist)
Similar to epidemic typhus but much milder
Scrub typhus a
Mite (S Pacific, Asia, Australia)
Eschar common. Similar to epidemic typhus
a So-called ‘scrub’ typhus as the vector is harboured in scrub vegetation. The chigger mites stay within several metres of where they hatch and are trans-ovarially infected. Infection therefore occurs in very focused rural ‘mite islands’.
• Culture – usually only performed in reference laboratories. Blood or biopsy tissue from skin lesions should be frozen at −70°C. Organisms may be isolated in small lab animals or in embryonated eggs. They are highly infectious if aerosolized and have been responsible for lab-acquired infections (some fatal).
• Detection of antigen – direct immunofluorescence of skin lesions in cases of Rocky Mountain spotted fever (RMSF) can identify organisms at the time the rash appears (day 3 to 5 of illness). Sensitivity is around 70%, with specificity approaching 100%. Organisms are most likely to be in a blood vessel near the centre of the lesion – the biopsy should include this to increase chances. Availability is limited.
• Serology – the main means of confirming diagnosis. Antibodies first appear around day 7–10 after infection. A fourfold rise on a convalescent sample is required for diagnosis but a single titre over 1:64 is very suggestive of infection. Currently the most sensitive and specific serological assay is the micro-immunofluorescence test – it requires trained personnel and a fluorescent microscope. Latex agglutination tests are available for the diagnosis of RMSF – a single positive test is considered diagnostic – they rarely produce positive reactions in convalescence. Both complement fixation and the classic Weil–Felix test (see Box 4.11 ) are now considered to be neither sufficiently sensitive nor specific. Cross-reactions among rickettsial species occur and vary from patient to patient. They tend to be strongest between rickettsial subgroups – e.g. difficult to distinguish spotted fevers from each other.
Rickettsiae replicate within the cytoplasm of infected endothelial and smooth muscle cells of capillaries, and small arteries. They cause a necrotizing vascultitis with consequent protean manifestations. The classic triad of fever, headache, and rash with the appropriate travel and exposure history should alert to the possible diagnosis. An eschar (black, ulcerated lesion) may develop at the bite site. Severity varies greatly with species – any organ can be involved.
Rocky Mountain spotted fever
• Clinical features – the most virulent spotted fever with 20% mortality if untreated. Fever, myalgia, and headache follow a 2–14-day incubation. GI involvement may suggest an acute surgical abdomen. Maculopapular rash (90% cases – more likely to be spotless in elderly or black) appears around day 3–5, often starting at the hands and may become petechial or necrotic. Gangrene is seen in 4%. Severe multisystem involvement is common including lung (pneumonia, effusions, odema), nervous system (meningitis, focal deficits, e.g. deafness), and renal impairment. Thrombocytopenia and DIC can occur. Death at around 10 days (less in fulminant cases which are seen more frequently in black males with glucose-6-phosphate dehydrogenase (G6PD) deficiency).
• Treatment – tetracycline, chloramphenicol (preferred in pregnancy), or doxycycline given for 7 days, continuing for 2 days after the patient becomes afebrile. It is recommended that doxycycline is used even in children with suspected Rocky Mountain spotted fever given the life-threatening nature of the disease. No demonstrated benefit from steroid therapy.
Other spotted fevers
• Clinical features – Mediterranean spotted fever (also known as African tick typhus) is a much milder disease; 5–7 days after inoculation patients develop an eschar with local tender lymphadenopathy and a generalized maculopapular rash. Mortality is very rare, although severe disease can occur in patients with diabetes, cardiac disease, G6PD deficiency, and in the elderly. Rickettsialpox is similar in presentation with the addition of a vesicular rash that resembles chickenpox.
• Treatment – as above. A single dose of 200 mg doxycycline has been proposed and seems effective.
• Clinical features – unusual in that humans are the reservoir and outbreaks are thus commonest in conditions of crowding – especially winter and war. The louse feeds on an infected person, bites and defaecates on the next, and infected faeces are scratched into the bite. After 1 week incubation abrupt onset of headache and fever is followed by maculopapular rash at day 5. This involves the entire body within a few days. Neurological features are common as is multisystem involvement. Mortality is 20–50% untreated and is low in children, high in the elderly.
• Treatment – as for Rocky Mountain spotted fever. Early therapy nearly eliminates fatal illness.
• Clinical features – longer incubation (up to 2 weeks) and patients rarely recall flea exposure. Fever, headache, and myalgia are followed by rash in 50%. Some may develop multi-system features but this is less common than with epidemic louse-borne typhus. Mortality is less than 1%.
• Treatment – as for Rocky Mountain spotted fever.
• Clinical features – not as severe as epidemic typhus. An individual is inoculated by the bite of the chigger mite and develops abrupt fever and headache 6–18 days later. Usually tender lymph nodes and an eschar at the inoculation point. Severity varies widely – neurological features can occur. Untreated mortality varies – up to 30%. Many serotypes (unlike the other organisms) so people may become infected again.
• Treatment – as for Rocky Mountain spotted fever, but resistance to doxycycline and chloramphenicol has been seen in Northern Thailand. Treatment may need to be prolonged to avoid relapse (2 weeks).
Coxiella burnetii (Q fever)
Coxiella burnetii is distinct from other rickettsiae. It is a significantly hardier organism and may be transmitted by aersosol or infected milk. It can form spores and is able to survive outside a host for some time – over 40 months in skimmed milk at room temperature! It grows in the phagosomes of infected cells rather than the cytoplasm (as other rickettsia) – appreciating the more acidic environment the phagosome affords.
Found around the world, and a zoonosis, the organism is usually acquired from occupational exposure to cattle or sheep but can be caught from many different animals – exposure to parturient cats is an important risk factor! Acquisition from unpasteurized dairy products has occurred and person-to-person spread is possible but unusual. It exists in ticks but this is thought to be an insignificant route of human infection – it is likely they maintain the organism and infect those animals from which man may acquire it. Infected ungulates are usually asymptomatic although abortion/stillbirth may result. Organisms from a heavily infected placenta may be found in the soil for 6 months, and the air for 2 weeks after parturition.
Humans are infected by inhalation (occasionally ingestion). Organisms proliferate in the lungs and bacteraemia follows. Presentation is 2–5 weeks after infection and ranges from a self-limited febrile illness (commonest) to pneumonia. The pneumonia may be an incidental finding as part of a fever of unknown origin (PUO) or a severe atypical pneumonia with dry cough, fever, fatigue, pleuritic chest pain, pleural effusion, and diarrhoea. It may be rapidly progressive, resembling legionnaire’s disease. Hepatomegaly and rashes are common. Acute Q fever can be complicated by behavioural disturbances, Guillain–Barré syndrome, myocarditis, arthritis, glomerulonephritis, among others. Autoantibodies are often found (anti-mitochondria, smooth muscle). Mortality is around 1% and associated with myocarditis.
Chronic Q fever can occur. The commonest manifestation is culture-negative endocarditis which may be accompanied by many extracardiac manifestations. Hepatitis, osteomyelitis, vascular graft infection, and neurological infection are also recognized. Immunocompromised and pregnant hosts (who have increased risk of miscarriage) are at higher risk.
• Culture – difficult and hazardous for lab staff – a category 3 organism
• Serology – the organism has two biological phases. Antibodies to phase II are produced first. Phase I antibodies appear weeks later. If antibodies to both phases are present simultaneously, chronic infection (specifically endocarditis) should be considered. Cross-reactions occur with Bartonella infection. The complement fixation test is most widely used. Fourfold rise between acute and convalescent titres is considered diagnostic of Q fever
• Molecular – PCR tests exist but are not in widespread use
• Pneumonia – infection is nearly always self-limited. Even without therapy people begin to recover at around 2 weeks. However, treatment is indicated in all cases to reduce chance of chronic disease: tetracycline, doxycycline, or chloramphenicol for 2–3 weeks.
• Endocarditis – combination antibiotic therapy e.g. tetracycline with rifampicin for a prolonged period (a year, even lifelong has been mooted in some cases). Valve replacement may be required.
Bartonellae are Gram-negative intracellular organisms belonging to the genus Bartonella. There are 14 species, four of which have been found to cause human disease.
• Oroya fever (B. bacilliformis) – develops 3–12 weeks after inoculation by the fly vector and may be mild or abrupt and severe (high fever, sweats, headache, confusion, anaemia due to erythrocyte invasion). Complications: abdominal pain, thrombocytopenia, seizures, dyspnoea, hepatic and GI dysfunction, and angina can occur. High mortality rate. Survivors have a high incidence of opportunistic infections (e.g. salmonella, toxoplasmosis). Asymptomatic bacteraemia with B. bacilliformis occurs in 15% of survivors.
• Veruga peruana (B. bacilliformis) – a late-stage manifestation characterized by crops of skin lesions weeks to months after untreated acute infection. Initially of a miliary appearance they become nodular, then ‘mulaire’ – erythematous round lesions, 5 mm diameter. Lesions occur on mucosal surfaces and internally. Histology demonstrates neovascular proliferation with occasional organisms.
• Cat scratch disease (B. henselae) – the commonest cause of lymphadenopathy in children and young adolescents; 3–10 days after inoculation a papule or pustule may be visible at the site. Most present at 2–3 weeks with the onset of regional lympadenopathy and low-grade fever. Rarer features: headache, sore throat, and skin rash. Lymphadenopathy settles over 2–4 months, even without treatment. Complications (commoner in the immunocompromised): encephalopathy, retinitis, bone and skin involvement, granulomatous hepatitis. Conjunctival exposure may present as the Parinaud’s oculoglandular syndrome (ocular granuloma or conjunctivitis, preauricular lymphadenopathy). Other atypical presentations: fever of unknown origin (PUO), osteomyelitis, hepatic and splenic granulomas. Diagnosis is based on history (cat exposure) but biopsy may be necessary to exclude lymphoma. Organisms may be visible on Warthin–Starry silver stain. Culture is possible from blood and tissue and should be attempted in cases of PUO, neuro-retinitis, or encephalitis after cat exposure, especially in the immunocompromised.
• Bacillary angiomatosis (BA) – unusual vascular proliferation caused by B. henselae or B. quintana infection. Usually occurs in the immunocompromised, mostly HIV patients with CD4 <100/mm3, but also transplant patients and those on chemotherapy. Lesions begin as small papules that grow to form round red to purple nodules which can ulcerate. They can also appear as flat hyperpigmented plaques. Occur on skin, liver, spleen, bone, mucosal surfaces, heart, CNS and bone marrow. Pathogenesis: the organism’s outer membrane adhesin binds to endothelial cells, and induces endothelial proliferation and new vessel formation. Numerous organisms are visible in lesions stained with Warthin–Starry silver stain. Diagnosis: lesion biopsy. All patients should be treated – 6–8 weeks erythromycin or doxycycline for cutaneous disease, longer if recurrence occurs. Skin lesions may be excised. Without therapy, systemic infection (fever, abdominal pain and anorexia) can occur.
• Bacterial peliosis (BP) – this is characterized by blood-filled cystic lesions scattered throughout a visceral organ. Cases involving the liver (peliosis hepatic) and spleen present with weight loss, diarrhoea, abdominal pain, nausea, fever, hepatosplenomegaly, and elevated liver enzymes. Caused by B. henselae or quintana (less common, affecting bone) infection. Most patients also have bacillary angiomatosis and previous cat exposure.
• Fever, bacteraemia, endocarditis (B. quintana) – acquired by scratching infected louse faeces into skin lesions. Epidemics have occurred across the world, usually in conditions of overcrowding and poor sanitation (e.g. soldiers in World War I – ‘trench fever’). Bacteraemia has also been described in homeless alcoholics. Incubation is 3–40 days, followed by a relapsing fever with headache, rash, and splenomegaly. It is a recognized cause of culture-negative endocarditis in those with HIV, and immunocompetent alcoholics. B. henselae bacteraemia occurs in the immunocompetent as well as those with HIV. Diagnosis is by culture. PCR and serology are available where culture negative. Treatment is by macrolides and tetracyclines – for 4–6 weeks. Endocarditis may need surgery and very prolonged antibiotic courses.
• Direct examination – Giemsa-stained blood films may be used to detect B. bacilliformis in areas of endemic Oroya fever due to the large number of organisms present. This is not feasible in the detection of B. henselae or B. quintana due to the low level of blood-borne organisms – they may be detected by silver staining of lesions in BA (bacillary angiomatosis or BP and in lymph nodes in the early stages of cat scratch disease.
• Culture –B. henselae grows on chocolate agar with characteristic white, dry, cauliflower- like colonies. These become visible 5–14 days after incubation at 37°C in 5% CO2. On Gram stain they are small, curved bacilli 2 micrometre by 0.5 micrometre, and display twitching motility when mounted in a saline drop. They are non-reactive for many standard biochemical tests.
Colonies with the appropriate morphological characteristics can have their identity confirmed by cellular fatty acid analysis, immunofluorescent antibody, or using commercial enzymatic substrate kits.
• Molecular – PCR or DNA-hybridization techniques can be used to speciate isolates. Direct detection of Bartonella organisms in pus or tissue is possible using PCR with wide-ranging sensitivity, depending on technique and sample.
• Serology – enzyme immunoassay or immunofluorescence kits may be used to demonstrate anti-Bartonella antibodies in culture-negative endocarditis, those with cat scratch disease, or HIV-associated aseptic meningitis etc. There is substantial cross-reactivity between B. henselae and B. quintana as well as with certain Chlamydia species.
Small (0.2 micrometre diameter). They lack a rigid cell wall and are bound only by a trilaminar membrane. They have a small genome with consequent limited biosynthetic capabilities, and require enriched media for growth. They can be distinguished by their differing phenotypic characteristics. Found in a wide range of animals and plants. Most found in humans are in the genus Mycoplasma with one (U. urealyticum) in the genus Ureaplasma. Whereas M. pneumoniae has a clear role in disease, most of the other organisms can be isolated in asymptomatic individuals. Several species have specialized structures at the cell ends which act as adhesins.
One of the commonest causes of respiratory tract infections and a cause of atypical pneumonia (3–10%). Droplet transmission. Infections occur all year round (peaks in winter) and affect all ages but most significant clinical disease (e.g. pneumonia) is seen from age 5 years to young adulthood. Once exposed, the organism attaches to respiratory tract epithelial cells and multiplies locally. Incubation is for 2–3 weeks and presentation is usually insidious with flu-like symptoms. Pneumonic symptoms follow – purulent sputum, haemoptysis. Multiple lobes may be involved but without consolidation and CXR infection is usually more dramatic than clinical presentation suggests. Pleural effusions in 20%. Disease usually self-limited with resolution over 3–10 days without antibiotics – CXR abnormalities may take 6 weeks to clear. Antibiotic therapy (e.g. erythromycin) speeds resolution but rarely eradicactes the organisms from the respiratory tract – recurrences can occur. Rare complications include: pleuritis, pneumothorax, lung abscess, haemolytic anaemia (secondary to cold agglutinins), thrombocytopenia, arthritis, rashes (e.g. erythema nodosum and multiforme), Guillain–Barré syndrome. Most complications are immune mediated with the exception of neurological complications such as meningoencephalitis which are thought to be due to direct invasion. Humoral and cellular immunodeficiency predisposes to more severe disease.
U. urealyticum and M. hominis
May be part of the commensal flora in male and female urogenital tracts. Sexually transimitted – the rate is related to sexual activity and rates are much lower among women using barrier contraception. They are implicated in endometritis and chorioamnionitis, are statistically linked with prematurity, low birth weight, and infertility, and are frequently recovered in culture along with other genital tract pathogens. Both may be isolated from blood cultures in women with post-partum fever (10% of cases). In men they are causes of non-gonococcal urethritis and a rare cause of epididymitis. In neonates, they are a cause of neonatal meningitis and there is an association between neonatal U. urealyticum colonization of the respiratory tract and the development of chronic lung disease of the newborn. Both may cause septic arthritis and subcutaneous abscesses in those with immunodeficiency. Sternal wound infections with M. hominis have occurred in heart and lung transplant patients.
M. genitalium is a cause of non-gonococcal urethritis and may also have a role in respiratory tract disease. M. fermentans, M. penetrans, and M. pirum have been isolated in those with HIV and are unusual in their ability to actively invade cells. Certain organisms found in animal hosts have caused human disease in cases of sufficient exposure and significant predisposing comorbidity (e.g. M. arginini – many animals, M. canis – in, you guessed it, dogs).
• Direct detection – indirect fluorescent antibody tests to detect M. hominis in genital samples have been developed (not widely used).
• Culture – M. pneumoniae can be recovered from respiratory tract specimens. Genital mycoplasmas can be isolated from many specimens. Fastidious organisms, they should be inoculated to culture media as soon as possible. Several media are used for culture of mycoplasma – most are diphasic (media with agar overlayed by media without agar). All species grow at 35–37°C but differ in their optimal pH and atmospheric conditions as well as their substrate utilization.
• M. pneumoniae isolates should be kept for at least 4 weeks, initially in selective broth. Growth is indicated by change in pH. Positives are subcultured to agar. Colonies should be visible by 1 week and identity confirmed by serological methods, or enzyme substrate tests (e.g. tetrazolium reduction).
• Genital mycoplasma samples are inoculated into broth and onto agar and should be kept for 8 days (although M. genitalium and M. fermentans can take longer and are not routinely looked for). Broths that exhibit a change in colour are plated to the appropriate agar. Plates are examined by microscope each day for colonies. Selective plates and colonial morphology are usually sufficient to allow identification. Anti-sera are available to confirm M. hominis.
• Molecular – PCR and DNA probe techniques have been developed but are not in wide use.
• Serology – the time required to culture these organisms means diagnosis is often made by serology. M. pneumoniae complement fixation tests detect mostly the early IgM – a fourfold rise between acute and convalescent (7–10 days) samples is diagnostic of recent infection. Enzyme immunoassays are available and detect IgM and IgG.
• M. pneumoniae is sensitive to a wide range of agents – tetracyclines, quinolones or macrolides.
• M. hominis is usually resistant to erythromycin. Some genital mycoplasma isolates have been found to be tetracycline resistant and carry the tetM resistance determinant (also found in other genital tract organisms e.g. group B streptococci).
• U. urealyticum – usually resistant to clindamycin and less susceptible to quinolones. Erythromycin effective.
Small, obligately intracellular (they are unable to produce ATP themselves), Gram-negative organisms. Outside a host cell they are tiny (300 nm diameter), inactive ‘elementary’ bodies. They infect cells primarily by receptor-mediated endocytosis. They are able to inhibit lysosome fusion and reside in a membrane-protected ‘inclusion’ body where they activate and increase to a diameter of around 800 nm. Three species produce human disease: Chlamydia trachomatis, C. psittaci, and C. pneumoniae. They differ antigenically, in host cell preference and in antibiotic susceptibility.
The family Chlamydiaceae was reorganised in 1999 on the basis of generic similarities. C. trachomatis remains in the genus Chlamydia, but psittaci and pneumoniae were moved to a new genus, Chlamydiophila.
There are 15 different serovars causing distinctive clinical syndromes. Natural infection confers only short-lived protection against re-infection.
• Lymphogranuloma venereum – serovar L1, L2, L3 – endemic in Africa, India, SE Asia, S America, and the Caribbean. Sexually transmitted – the organism enters through skin abrasions (it cannot infect squamous epithelial cells) and causes a small papule or ulcer (primary lesion) on genital mucosa or nearby skin 3–30 days after infection. It heals rapidly, and perhaps weeks later the patient develops secondary symptoms: lymphadenopathy (usually inguinal or femoral), fever, headache, myalgias, proctitis (can resemble inflammatory bowel disease), and occasionally meningitis. Nodes may coalesce forming abscesses and buboes.
• Trachoma – serovar A, B1, B2, C – chronic follicular keratoconjunctivitis which leads to corneal scarring and is the commonest cause of preventable blindness in the developing world (around 9 million blind, and 500 million affected). First infections usually acquired in childhood, resolves spontaneously but multiple re-infections and the consequent host immune response result in conjunctival scarring and corneal damage. The inner surface of the eyelid scars, and inturning eye lashes further abrade the cornea. There is a WHO grading of trachoma (Box 4.12 ).
• Inclusion conjunctivitis – serovars D to K – sexually transmitted eye infection of adults (of whom slightly over half have concurrent genital tract infection) and a cause of neonatal conjunctivitis (probably from mother’s genital tract but can occur even if delivered by Caesarean section – 5 days to 6 weeks after delivery). No corneal scarring.
• Neonatal pneumonia – serovars D to K – most acquired from the mother’s genital tract. Seen in 10–20% of infants born to infected mothers. Usually symptomatic by 8 weeks with nasal congestion, cough, etc. and only moderately ill.
• Sexually transmitted infections – serovars D to K – epididymitis (along with N. gonorrhoeae the common cause in the under-35s), urethritis, salpingitis, cervicitis with consequent pelvic inflammatory disease, and infertility. Reactive arthritis and Reither’s syndrome may follow.
Trachoma may be diagnosed on clinical grounds. Other clinical presentations require lab identification for a definitive diagnosis.
• Direct detection – microscopy of certain Giemsa-stained clinical specimens (particularly neonatal conjunctivitis) may allow direct visualization if there are sufficient bacterial inclusion bodies in the cytoplasm. Monoclonal antibodies and immunofluorescence increase sensitivity (in one study comparing Giemsa staining and immunofluorescence in detecting C. trachomatis in a series of infants with neonatal conjunctivitis, sensitivity was 42% and 100% respectively). Enzyme immunoassay can demonstrate chlamydial antigen in respiratory or genital secretions.
• Culture – being obligate intracellular organisms, the techniques for culturing are similar to those used in virus cuture. The infective elementary bodies must first be extracted by centrifugation before infecting a cell line. Inclusion bodies containing the organism can be seen at 48–72 h, and species-specific monoclonal antibodies used to confirm identity.
• Serology – most useful for epidemiological studies. Chlamydial complement fixation tests do not distinguish between species; microimmunofluorescence does.
• Molecular tests for chlamydial DNA are the preferred initial investigation for the diagnosis of C. trachomatis in all specimen types. PCR- and ligase chain reaction (LCR)-based tests are available, and sensitivity is a high as 99% for some of these but varies with specimen type and quality. The notable advantage is it allows the diagnosis of urethritis on non-invasively acquired specimens.
• Trachoma – transmission is by flies or eye-to-hand in endemic areas, thus hygiene is important for control (rates fall quickly with socioeconomic improvement). Systemic therapy (erythromycin or doxycycline) is effective in areas of low transmission (where re-infection is less frequent). Mass treatment at village level has been shown to be effective. Eyelid surgery can prevent further mechanical damage.
• Lymphogranuloma venereum – buboes should be aspirated. Doxycycline for 3 weeks.
• Genital and ocular infections in adults – single-dose azithromycin, or doxycyline for 7 days. Longer courses of amoxicillin may be effective in pregnant women.
• Neonatal infections – topical eye therapy is not recommended as it does not eliminate carriage. Erythromycin orally for 14 days for both conjunctivitis and pneumonia. Prenatal screening of mothers and treating those infected with Chlamydia is 90% effective in preventing infants from acquiring infection.
C. psittaci can infect many kinds of birds. The classic term for infection caused by this bacteria, ‘psittacosis’ (derived from the Greek word for parrot) is therefore not so accurate a description as ‘ornithosis’. It is an occupational disease of zoo workers, petshop workers, and poultry farmers. Human-to-human transmission occurs but is very rare. Infection is primarily acquired by inhalation of organisms from aerosolized avian excreta or respiratory secretions from sick birds (mouth-to-beak resuscitation has been implicated in acquisition). Transient exposure is sufficient (e.g. petshop customers). The disease is found worldwide. Incubation is 5–14 days and presentation is with fever, chills, malaise, cough, headache, breathlessness, mild pharyngitis, and epistaxis. Less commonly, nausea, vomiting, and jaundice may be seen. Examination may demonstrate the features of an atypical pneumonia. Other features are bradycardia, peri/myocarditis culture-negative endocarditis, splenomegaly, meningitis, encephalitis, Guillain-Barré syndrome, rashes, acute glomerulonephritis, severe respiratory failure, sepsis, and shock. Relapses can occur. Diagnosis is by serology: the demonstration of a fourfold rise in CF antibody titre is considered diagnostic. Antibodies may cross-react with other chlamydial species. Culture is possible but avoided due to the risks to lab staff. Other tests: ELISA, PCR available. Treatment is with tetracycline or doxycycline for 2–3 weeks (reduces the risk of relapse). Erythromycin may be used in children and the pregnant.
The cause of 3–10% of community-acquired pneumonia cases among adults. Adolescents tend to experience a mild pneumonia/bronchitis, whereas older adults can experience more severe disease and repeated infections. Fifty per cent of young adults have serological evidence of previous infection. Unlike C. psittaci, human-to-human transmission by respiratory secretions is the norm. In most populations infection is more common in males and this may reflect cigarette use. Incubation is 3–4 weeks and symptoms of a URTI are followed by bronchitis or pneumonia 1–4 weeks later. Most infections are asymptomatic or cause only mild symptoms. Other features: hoarse voice, non-productive cough, headache. Fever is often absent. Symptoms can be very prolonged even with appropriate treatment. Diagnosis is by serology (preferably microimmunofluorescence as CF tests cross-react with other chlamydial species). A definite case requires a four-fold rise in titre – single elevated IgG titres may be seen in the uninfected elderly as a consequence of repeated infections. Antibody tests may be negative in the early weeks after infection – it can take as long as 8 weeks for a significant IgG response to develop after primary infection. Other tests: PCR and cell culture tests are available. Treatment: doxycycline or erythromycin.
The Mycobacterium tuberculosis complex comprises five species: M. tuberculosis, M. bovis, M. africanum, M. microti, and M. ulcerans. The term tuberculosis (TB) describes a broad range of clinical diseases caused by M. tuberculosis (and less commonly M. bovis).
M. tuberculosis infects one-third of the world’s population and is the most-frequent infectious cause of death worldwide, accounting for 3 million deaths per year. Most cases occur in the developing world, with 13 countries accounting for 75% of cases. In the developed world, despite a general downward trend, there has been an increase in incidence in certain groups, e.g. immigrants from high-prevalence countries and HIV-infected patients.
• Transmission – infection is acquired by inhalation of infectious droplet nuclei. Occasionally due to skin inoculation (e.g. pathologists, laboratory personnel) or sexual transmission – determined by closeness of contact with infectious (sputum smear positive) source. Ninety per cent of primary infections are asymptomatic (person clinically well with positive tuberculin skin test).
• HIV infection increases the risk of developing all forms of TB. The risk of disease progression is highest at the extremes of age and in the immunocompromised. Immunocompetent people have a 5–10% lifetime risk of progression; this increases to 7–10% per year in HIV-infected patients.
Tuberculosis is the prototype of infection that elicits a cellular immune response. CD4+ T lymphocytes recognize mycobacterial antigens in association with major histocompatibility (MHC) class II molecules, presented by macrophages. The T cells becomes activated and proliferate, resulting in the production of cytokines (interferon-γ, migration inhibition factor) which attract and activate more macrophages at the site of infection. Macrophages also secrete a number of cytokines (TNF-α, platelet-derived growth factor, transforming growth factor-β, fibroblast growth factor). The interplay of these factors determines the nature of the pathological and clinical features. The Langhan’s giant cell consists of epithelioid cells (activated macrophages) oriented around mycobacterial antigens, and is a characteristic feature of tuberculous granulomas. When the population of activated lymphocytes reaches a certain size, a cutaneous delayed hypersensitivity reaction to tuberculin occurs.
Primary infection results from inhalation of infectious droplet nuclei which lodge in the alveoli and multiply, resulting in the formation of a Ghon focus. Involvement of the regional lymph nodes produces the Ghon complex. Depending on the host’s immune response the infection will either become quiescent or progress and/or disseminate. Reactivation of disease may occur in later life, particularly in the immunosuppressed.
Pulmonary tuberculosis is the most common presentation. Tuberculosis may also disseminate (military TB) or affect almost any other organ (extra-pulmonary TB): pleural cavity, pericardium, lymph nodes, GI tract, and peritoneum, GU (genitourinary) tract, skin, bones and joints, and CNS.
• Diagnosis is based on a combination of compatible clinical syndrome, supportive radiological investigations, and detection of acid-fast bacilli or culture of M. tuberculosis from clinical specimens. The gold standard for diagnosis is culture. Patients are often treated on the basis of a presumptive diagnosis.
• M. tuberculosis is an aerobic, non-sporing, non-motile, weakly Gram-positive bacillus with a thick cell wall containing mycolic acid, which renders it acid-fast.
• Samples of sputum or tissue are liquefied, decontaminated, neutralized, centrifuged, and the deposit inoculated into solid or liquid media. Normally sterile samples, e.g. CSF, need not be decontaminated, as loss of mycobacterial viability may occur.
• Acid-fast stains:
• Auramine stain (fluorochrome phenolic auramine or auramine-rhodamine stain, acid-alcohol decolourization, potassium permanganate counterstain).
• Ziehl–Neelsen (ZN) stain (carbol fuschin stain, decolourize with acid-alcohol, counterstain with methylene blue)
• Kinyoun stain (ZN stain modified to make heating unnecessary)
• Molecular methods:
• PCR-based tests may be used to detect M. tuberculosis in clinical pecimens, e.g. Amplicor test (Roche), amplified M. tuberculosis Direct Tests (MDT, GenProbe), strand displacement amplification (BD ProbeTec-SDA), ligase chain reaction (Lcx, Abbott systems). Although specificity is high, sensitivity is lower: 90–100% and 60–70% for smear-positive and -negative specimens
• identification of mycobacterial species e.g. high-pressure liquid chromatography (HPLC) of mycolic acids, DNA sequencing of 16S rRNA, PCR restriction enzyme assay (PRA), DNA probe hybridization (LiPA MYCOBACTERIA, Innogenetics)
• identify drug resistance mutations e.g. rpoB mutations (InnoLiPA Rif.TB assay, Innogenetics)
• typing for epidemiological studies e.g. IS6110 RFLP, MIRU, spoligotyping.
• Culture methods:
• solid media e.g. Lowenstein–Jensen media, Middlebrook agar
• liquid culture e.g. Kirschner broth, BACTEC 460 system, MB/BacT (Organon Teknika), Mycobacterial Growth Indicator Tube (MGIT, Becton Dickinson), MODS
• lysis-centrifugation method
• solid media, e.g. Lowenstein–Jensen media, Middlebrook agar
• liquid culture, e.g. Kirschner broth, BACTEC 460 system, MB/BacT (Organon Teknika), Mycobacterial Growth Indicator Tube (MGIT, Becton Dickinson), MODS
• Identification and susceptibility testing is usually done at reference lab level. For definitions of drug resistance see Box 4.13 .
• Tuberculin skin test – purified protein derivative (PPD) is a standardized protein precipitate of tuberculin. The Mantoux test is a quantitative tuberculin test which is performed by intracutaneous injection of five tuberculin units (TU) of PPD in 0.1 mL of solution. The reaction is usually read after 48–72 h. A positive result is defined as >10 mm of induration. Reactions of 5–10 mm may be due to BCG (bacilli Calmette–Guérin) vaccination, but are also suspicious of TB in low-prevalence areas. False-positive results can occur with non-tuberculos mycobacteris (NTM) infections. False-negative reactions can occur in up to 20% of patients with TB and in HIV-infected patients. Delayed reactivity (>10mm induration after 6 days) may occur in certain populations e.g. Indochinese immigrants.
• Interferon gamma-release assays (IGRA) – this is a relatively new method of detecting T cells specific for M. tuberculosis antigens. It has a sensitivity >80% and is more specific than the tuberculin skin test. Sensitivity remains high in children <3 years and in HIV co-infection, and is not confounded by BCG vaccination or infection with NTM. The main limitation of the test is that it cannot distinguish active from latent infection.
Treatment is with combination chemotherapy for several months. For most types of tuberculosis, the usual regimen is a 2-month intensive phase with three or four drugs, followed by a 4-month continuation phase. The choice of drugs and duration of treatment depend on the likelihood of drug resistance, the site of infection and the patient’s HIV status. For details see Antituberculous agents—1st line, p. [link] and British Thoracic Society (BTS) or CDC guidelines (Box 4.14 ).
BCG vaccine is a live attenuated vaccine derived from M. bovis. It is given to infants and children in high-prevalence areas and results in a 60–80% reduction in the incidence of TB. It should only be given to infants <12 weeks or children who are tuberculin skin test (TST)-negative. Although it does not prevent infection, BCG vaccination reduces the risk of disseminated disease in children. BCG is contraindicated in HIV-infected individuals. Vaccination can occasionally cause disseminated BCG infection, usually in immunosuppressed patients. Intravesical BCG (used to treat bladder cancer) can cause liver or lung granulomas, psoas abscess, or osteomyelitis. Newer vaccines include MVA (modified vaccinia Ankara DNA vaccines which use a prime-boost strategy to induce M. tuberculosis specific immune responses. These vaccines look promising in phase II clinical trials.
Chemoprophylaxis is given to individuals at increased risk of tuberculosis, e.g. contacts of active cases, recent TST conversion, abnormal CXR, HIV infected or certain medical conditions, e.g. renal transplant myeloproliferative or haematological malignancies. For detailed indications see BTS and CDC guidelines (Box 4.14 ). The usual regimen is isoniazid 300 mg for 6–12 months. The risk of isoniazid hepatotoxicity increases with age and daily alcohol consumption.
Leprosy or Hansen’s disease is caused by infection with Mycobacterium leprae, an obligate intracellular parasite whose only natural hosts are humans and armadillos. Experimental infections can be induced in the mouse footpad. The clinical manifestations of leprosy include skin lesions, deformities, and peripheral neuropathy, making it one of the most socially stigmatizing diseases. Leprosy exhibits a spectrum of clinical features ranging from lepromatous (multibacillary) to tuberculoid (paucibacillary) forms.
Worldwide, there are an estimated 6 million people living with leprosy, 3 million of whom are untreated. Although Africa has the highest prevalence, Asia has the greatest number of cases. Leprosy is associated with poverty and rural residence but not HIV infection. Distribution in endemic countries is non-homogeneous, suggesting that genetic factors may play a role in disease expression. The mode of transmission remains uncertain, but is thought to be human-to-human, via nasal droplet infection. Leprosy may also be acquired by direct inoculation into the skin. The incubation period is long, with an average of 5–7 years (range 2–40 years) and the peak onset is in young adults.
M. leprae has a dense, mainly lipid, capsule outside the cell wall, which is rich in M. leprae-specific phenolic glycolipid 1 (PGL-1). This has been implicated as a scavenger for free radicals, allowing intracellular survival and limiting antibiotic penetration. PGL-1 and lipoarabinomannan have been implicated as causing immunological hyporesponsiveness of both lymphocytes and macrophages in the anergic, highly bacillary lepromatous form of leprosy.
Clinical manifestations of leprosy are largely confined to the skin, upper respiratory tract, and and peripheral nerves. Most of the serious sequelae are a result of peripheral nerve damage resulting in deformities (e.g. ulnar, median, and peroneal nerve palsies), loss of peripheral parts of digits, and plantar ulceration.
• Lepromatous leprosy – characterized by symmetric skin nodules, plaques, and a thickened dermis that typically occur in cool areas of the body, e.g. earlobes and feet. This condition is multibacilliary (infectious) and associated with poor cell-mediated immunity. Involvement of the nasal mucosa results in congestion, epistaxis and, rarely, septal collapse (‘saddle nose’ deformity). May also cause loss of eyebrows and eyelashes, trichiasis, corneal scarring, uveitis, lagophthalmos, testicular dysfunction, and amyloidosis.
• Tuberculoid leprosy – characterized by hypopigmented, anaesthetic skin plaques, and asymmetric peripheral nerve involvement. It is typically paucibacillary (non-infectious) and associated with a good cell-mediated immune response.
• Borderline leprosy – the majority of patients have manifestations intermediate between the two polar forms, a condition termed borderline leprosy.
• Reversal reactions – an abrupt increase in inflammation within previously quiescent skin lesions, as well as new skin lesions, neuritis, and low-grade fever may develop in borderline leprosy patients either before (downgrading reaction) or after (reversal reacion) the initiation of therapy. If the neuritis is not treated promptly, irreversible nerve damage may occur.
• Erythema nodosum leprosum – affects >50% of lepromatous and borderline leprosy patients after initiation of therapy. Clinical features include painful nodules (usually on extensor surfaces, may pustulate or ulcerate), neuritis, fever, malaise, anorexia, uveitis, lymphadenitis, oorchitis, anaemia, leucocytosis, glomerulonephritis.
A firm diagnosis of leprosy requires the presence of a characteristic peripheral nerve abnormality or the demonstration of acid-fast bacilli in skin biopsies or split skin smears. In atypical cases two of the following three criteria are required: a clinically compatible skin lesion, dermal granuloma on skin biopsy, hypoaesthesia within the lesion. Skin biopsies should be taken from skin plaques or nodules in lepromatous patients and from the periphery of lesions in tuberculoid patients. Nerve biopsies may result in loss of function and should only be performed if there is sufficient diagnostic uncertainty to warrant it.
M. leprae is a Gram-variable, acid-fast bacillus that is best visualized by a modified Fite stain (as it may be decolourized by the Ziehl–Neelsen stain). Viable bacilli stain brightly, whereas dead bacilli stain irregularly.
M. leprae grows best at temperatures <37° C in humans and armadillos.
Unique properties of M. leprae include loss of acid-fastness by pyridine extraction, presence of dopa oxidase activity, multiplication in the mouse footpad with a doubling time of 12–14 days.
Experimental infection of the mouse footpad can be used to assess antimicrobial susceptibility.
Treatment requires combination therapy with two or more agents, e.g. dapsone, clofazimine, and rifampicin. Ethionamide, prothionamide, and certain aminoglycosides have also been used. Newer agents such as minocycline, clarithromycin fluoroquinolones look promising. For treatment regimens see Antileprotics, p. [link] .
This group of organisms comprises about 50 species of mycobacteria, excluding those in the M. tuberculosis complex and M. leprae. Other names for non-tuberculous mycobacteria (NTM) include atypical mycobacteria, opportunistic mycobacteria or mycobacteria other than tuberculosis (MOTT).
NTM were previously classified according to growth rate, colonial morphology, and pigmentation (Runyon classification). This has been superseded by molecular methods but nonetheless remains useful to separate NTM into three groups:
• rapidly growing mycobacteria (≤7 days incubation) e.g. M. fortuitum complex, M. chelonae/abscessus group, M. mucogenicum, and M. smegmatis
• slow-growing mycobacteria (>7 days incubation) e.g. M. avium complex, M. kansasii, M. xenopi, M. simiae, M. szulgai, M. scrofulaceum, M. malmoense, M. terrae/nonchromogenicum complex, M. malmoense, M. haemophilum, and M. genavense
• intermediately growing mycobacteria (7–10 days), e.g. M. marinum, M. gordonae.
The NTM can cause a wide spectrum of diseases (Table 4.18 ).
Table 4.18 Clinical syndromes caused by NTM.
Most common causes
Chronic bronchopulmonary disease (adults, CF patients)
M. avium complex, M. kansasii, M. abscessus
Cervical lymphadenitis (children)
M. avium complex
Skin and soft tissue infections
M. fortuitum group, M. chelonae, M. abscessus, M. marinum, M. ulcerans
Bone and joint infections
M. marinum, M. avium complex, M. kansasii, M. fortuitum group, M. abscessus, M. chelonae
Disseminated infection (HIV positive)
M. avium, M. kansasii
Disseminated infection (HIV negative)
M. abscessus, M. chelonae
M. fortuitum, M. abscessus, M. chelonae
Because the signs and symptoms of NTM lung disease are often variable and non-specific, diagnosis requires multiple positive respiratory cultures. Diagnosis of NTM infections at other sites requires positive cultures from pus, tissue biopsies or blood cultures.
• Microscopy – the acid-fast stains used for identifying M. tuberculosis (p. [link] ) also work well for identifying NTM.
• Culture – appropriate culture media include Middlebrook 7H10 or 7H11 agar or BACTEC broth. Samples from skin and soft tissue infections need to be plated at 28–30°C as well 35–37°C, as some species only grow at low temperatures, e.g. M. chelonae, M. haemophilum, and M. marinum. M. xenopi grows best at 42°C. Other species have special growth requirements, e.g. M. genavense (BACTEC broth for 6–8 weeks) and M. haemophilum (iron supplementation).
• Identification – although traditional biochemical and other standard tests may be performed, identification of NTM increasingly uses rapid molecular methods:
• HPLC of mycolic acids
• PCR-RFLP analysis of the heat-shock protein gene
• genetic probes for mycobacterial RNA
• 16S ribosomal DNA sequencing
• Drug susceptibility testing – various methods are used:
• agar disk elution
• broth microdilution
• BACTEC radiometric detection
• Strain comparison – for epidemiological studies, standard biochemical identification and susceptibility testing have been superseded by molecular methods.
Treatment of NTM infections may be medical, surgical, or a combination of the two. The choice of drugs and duration of treatment depend on the causative organism, site of infection and patient’s HIV status:
Table 4.19 Summary of viruses and common clinical syndromes
Consider in the differential of…
Adenovirus (p. [link] )
HSV-1 and -2 (p. [link] )
Genital, eye, dissemination
EBV (p. [link] )
VZV (p. [link] )
CMV (p. [link] )
HHV-6 and -7 (p. [link] –9)
HHV-8 (p. [link] )
Poxviridae (also smallpox) (p. [link] )
Parvovirus B19 (p. [link] )
Arthropathy, aplastic anaemia, congenital
HPV (p. [link] )
Warts, epithelialtumours of skin
Polyomavirus (p. [link] )
Hepatitis B (p. [link] )
Progressive focal neuro deficits
Influenza (p. [link] )
Parainfluenza (p. [link] )
Mumps (p. [link] )
Parotitis, epididymo-orchitis, GBS
Measles (p. [link] )
RSV/metapneumovirus (p. [link] )
Coronavirus (p. [link] )
Poliovirus (p. [link] )
Non-polio enteroviruses (p. [link] )
Hepatitis A (p. [link] )
Rhinovirus (p. [link] )
HTLV-1 and -2 (p. [link] )
Spastic paraparesis, leukaemia/lymphoma
HIV-1 and -2 (p. [link] )
Togaviridae (p. [link] )
Rubella (p. [link] )
Alphaviruses (p. [link] )
Yellow fever (p. [link] )
Dengue (p. [link] )
Hepatitis C (p. [link] )
Japanese encephalitis (p. [link] )
Bunyaviridae (also Rift Valley fever)
Hantavirus (p. [link] )
Haemorrhage, renal failure
CCHF (p. [link] )
Californian encephalitis (p. [link] )
Lassa (p. [link] )
LCMV (p. [link] )
Marburg, Ebola (p. [link] )
Rabies (p. [link] )
Astrovirus and calcivirus (p. [link] )
••• very frequently seen, •• commonly seen, • occasionally seen.
‘Congenital’ indicates viruses with the potential to cause significant sequelae if infecting the developing fetus.CCHF: Congo–Crimean haemorrhagic fever; CMV: cytomegalovirus; EBV: Epstein–Barr virus; GBS: Guillain–Barré syndrome; HHV: human herpesvirus HIV: human immunodeficiency virus; HPV: human papillomavirus; HSV: herpes simplex virus; HTLV: human T-cell lymphotrophic virus; LCMV: lymphocytic choriomeningitis virus; RSV: respiratory syncytial virus; SSPE: subacute sclerosing panencephalitis; VZV: varicella zoster virus.
a HIV may of course present in a multitude of ways - however the commoner presentations of primary HIV infection (as opposed to a subsequent opportunistic infection) are rash and encephalitis
One of the commonest infectious diseases of man, primarily causing epidemics of upper respiratory tract infection. Global pandemics may follow dramatic antigenic changes – 21 million died in the 1918–1919 pandemic.
Members of family Orthomyxoviridae. Negative sense single-strand (ss)RNA viruses.
The three distinct influenza viruses:
• influenza A causes the typical influenza syndrome and can precipitate pandemics
• influenza B is similar clinically but does not cause pandemics
• influenza C causes an afebrile common cold-like syndrome and does not occur in epidemics.
All have host cell-derived envelopes embedded with glycoproteins important to viral entry and exit. These have haemagglutinin (HA) or neuraminidase (NA) activities, are key antigenic components and may alter gradually by mutation (antigenic drift – see Box 4.15 ).
At least 16 HA and 9 NA variants have been identified in influenza A viruses. In the UK in 2008 three subtypes of influenza A were circulating: H1N1, H1N2 and H3N2. Electron microscopy (EM) appearance of the viral particles is variable: spherical to filamentous. Viruses are named by their type, place of initial isolation, strain, year, and antigenic subtype, e.g. A/Victoria/3/75/H3N2.
• Outbreaks are associated with excess rates of pneumonia and influenza-related illness and mortality, peaking in the winter and varying with the viral type responsible. Not all influenza-related deaths present as pneumonia. Sporadic cases of severe disease have occured in some countries as a result of human acquisition of viral strains adapted to birds - see Box 4.16 .
• Attack rates are highest in the young, mortality highest in the elderly, both are increased in those with pre-exisiting medical problems, e.g. cardiovascular, pulmonary, and renal impairment or immunodeficiency.
• Person-to-person transmission occurs by dispersion in small-particle aerosols. Virus is present in large quantities in the secretions of infected people. One individual can infect a large number of others contributing to the explosive nature of outbreaks. Outbreaks can occur in an epidemic or pandemic fashion:
• epidemics – confined to a single location, e.g. a town or country, and occur almost exclusively in winter. They start abruptly with cases seen initially in children and then adults, peak within 3 weeks and last around 6 weeks. Different strains may circulate simultaneously
• pandemics – severe outbreaks that spread to all parts of the world. Caused only by type A viruses. Associated with the emergence of a new virus to which the population has no significant immunity, and characterized by rapid transmission across the world, often out of the usual patterns of seasonality and with high levels of mortality among healthy young adults. Influenza virus’ capacity to continue to cause human disease on such a scale is a function of frequent antigenic changes (see Box 4.15 ). In the early years after a pandemic, disease is clinically severe, becoming milder as herd immunity improves. At present (winter 2008), late in the inter-pandemic cycle, severe disease is rare.
• Virus enters respiratory epithelial cells, replicates and progeny are released – the cell dies. Viral shedding may start within 24 h of infection – illness follows 24 h later.
• There is diffuse inflammation of the trachea and bronchi with an ulcerative, necrotizing tracheobronchitis in severe cases. Primary viral pneumonia is uncommon but is severe when it occurs. Bacterial superinfection is common, facilitated by damage to the mucociliary escalator, and virus-induced defects in lymphocyte and leucocyte function. Viral levels fall rapidly after 48 h of illness, becoming undetectable by 5–10 days.
• Uncomplicated disease – 1–2-day incubation is followed by an abrupt onset of symptoms. Fever, chills, headache, malaise, myalgia, eye pain, anorexia, dry cough, sore throat, and nasal discharge. After around day 3, respiratory features dominate as fever and other systemic features settle. Convalescence may take two or more weeks. Elderly patients may present with fever and confusion and few respiratory features. Attack rates are highest in children, who may present with croup.
• Respiratory complications – outside of a pandemic these complications are more common in the elderly. Primary viral pneumonia occurs more commonly in those with pre-existing cardiac and lung disorders and presents with worsening cough, breathlessness, and cyanosis shortly after disease onset (resembles acute respiratory distress syndrome). Mortality is high. Secondary bacterial pneumonia develops shortly after an initial period of improvement following the influenza syndrome. Pathogens: Strep. pneumoniae, H. influenzae and less commonly S. aureus. Other respiratory complications include croup, COPD, and cystic fibrosis exacerbations.
• Immunocompromised patients – more severe disease may occur in some groups of HIV-infected patients, but this has not been widely observed. HIV-infected children with low CD4 counts may shed virus for prolonged periods. Severe disease does occur in immunosuppressed children with cancer, bone marrow transplant recipients, and those with leukaemia. Viral shedding may be prolonged in these groups.
• Non-pulmonary complications – myositis, pericarditis, myocarditis, toxic shock syndrome, encephalitis, GBS.
In the context of a community outbreak, the diagnosis of influenza can be made with some confidence on clinical criteria alone – 85% accuracy in some studies. Outside of outbreaks or in the institutional setting, laboratory diagnosis is required.
• Viral culture – virus is readily isolated from sputum, throat, or nasal swabs. It is cultured in cell lines and detected within 3–5 days by its cytopathic effect.
• Viral antigen detection – rapid detection within 1–2 days is possible with immunofluoresence or ELISA. Sensitivity approaches that of culture. Reverse transcription (RT)-PCR techniques are in increasing use.
• Serology – acute and convalescent (10–20 days apart) samples showing a fourfold rise in antibody titre can be considered diagnostic but are not useful in clinical decision making.
• The cornerstone of influenza management is an effective vaccination strategy (see below). With this understanding, guidance is produced in the UK by the National Institute for Health and Clinical Excellence (NICE) on the use of antiviral agents in treatment and prevention (Box 4.17 ). Amantadine (see p. [link] ) is licensed but no longer recommended for the treatment or prophylaxis of influenza. A. Zanamivir (see p. [link] ) is an inhaled neuraminidase inhibitor and used for both prophylaxis and treatment of types A and B. NB - A study 1 of influenza A isolates collected in late 2007 and early 2008 demonstrated that the global prevalance of oseltamivir resistance among H1N1 viruses was 6.4% - a large increase from the previous season. By January of 2009 the overwhelming majority of UK H1 isolates had become resistant. H3 isolates were mostly sensitive and both remained susceptible to Zanamivir.
• General measures – adequate hydration, antipyretics (not aspirin in children), and decongestants.
• Severe disease – there is little information available on the effectiveness of antiviral agents in severe disease. The rapidly progressive nature of secondary bacterial infections argues for early presumptive use of antibiotics where suggested by the clinical scenario.
• Inactivated vaccines are the main control measure and the Department of Health (DH) recommends annual vaccination for certain risk groups. These are: those with diabetes, immunocompromise, cardiac, lung or renal disease, all people over 65 years of age, and healthcare workers.
• They are prepared each year and are usually trivalent, containing two type A, and one type B strain. Strains are collected continuously across the world, and those to be included in the year’s vaccine chosen by educated guess. It takes 6–9 months to produce a vaccine once its components are decided.
• Two doses are required in children under 9 years who have not been previously vaccinated. Otherwise a single dose is sufficient, usually given in October. The main contraindication to vaccination is hypersensitivity to hens’ eggs
• Protection is around 70% and lasts for 1 year. Diminished responses are seen in organ transplant recipients receiving immunosuppressive therapy. Protection is reduced in the elderly.
• See NICE guidance (Box 4.17 ).
National Institute for Health and Clinical Excellence. www.nice.org.uk (accessed 4 August 2008).
A group of five viruses causing a spectrum of respiratory illnesses, from upper respiratory tract symptoms in healthy children to severe pneumonia in the immunosuppressed.
• Paramyxoviridae and members of either genus Paramyxovirus (parainfluenza types 1 and 3) or Rubulavirus (types 2, 4A and 4B).
• Negative sense ssRNA viruses, spherical in appearance with a host cell derived envelope.
• Sixty per cent of childhood croup cases in which virus is isolated are due to parainfluenza. Second only to RSV as a cause of childhood respiratory hospitalization; 10% of adult acute respiratory illness can be attributed to parainfluenza. Nosocomial and residential care outbreaks have occurred.
• Parainfluenza type 3 is the most frequently isolated member, and like RSV is commonly seen in the first 6 months of life. It is endemic and may be isolated throughout the year, peaking in Spring.
• Types 1 and 2 occur in autumn, often alternating each year.
• Type 4 viruses are rarely isolated.
• Transmission is by droplet spread. Replication occurs in cells of the respiratory epithelium. Clinically, illness most frequently involves larger airways of the lower respiratory tract, causing croup.
• Re-infection may occur and tends to cause milder upper airway disease, probably representing waning of immunity – antigenic variation is not progressive (unlike influenza virus). Mucosal immunity is most important for resisting infection. CD8 T cells are important in viral clearance.
• Healthy individuals – children: upper respiratory tract illness (those under 5 years), otitis media, croup, bronchiolitis (infants under 6 months); adults: upper respiratory tract infection.
• Immunocompromised – severe disease in recipients of BMT or lung transplants in all age groups. May cause pneumonia with high rates of mortality. Other immunodeficiencies are associated with prolonged viral shedding.
• Viral isolation by tissue culture and immunofluorescence is the standard.
• Multiplexed RT-PCR-based tests are faster and can distinguish viral type.
• Paired serology can confirm a diagnosis but is unhelpful.
RSV is a major cause of lower respiratory tract infection (LRTI) in young children.
• A member of the Paramyxoviridae family. An enveloped (bilipid layer derived from the host cell) ssRNA virus which may survives up to 24 h in patient secretions depositing on non-porous surfaces, and around an hour on porous surfaces (tissues, fabric, skin).
• RSV isolates fall into two antigen groups (A and B), which differ in envelope proteins and non-structural protein-1. Several strains from both groups may circulate in the same outbreak.
• RSV infection has been found worldwide. In temperate parts, outbreaks are annual, occurring in the winter months. Mild outbreaks may be followed by a more-severe one the next year.
• The major cause of childhood pneumonia/bronchiolitis (90% of children admitted with a LRTI in the peak of an epidemic); 95% of children are seropositive by age 2 years. Naturally acquired immunity to RSV is incomplete but subsequent infections rarely produce severe illness.
• Boys and those under 2 years experience the most-severe illness – severity is also affected by socioeconomic factors.
• An important nosocomial infection – virus may spread from an infected infant (secretions, staff) or an infected adult with mild symptoms.
• Incubation between 2 and 8 days. Inoculation is by nose and eye, with infection confined to the respiratory tract. Infants often have evidence of pneumonia as well as bronchiolitis.
• Lymphocytic infiltration of the areas around the bronchioles with wall and tissue oedema is followed by proliferation and necrosis of the bronchiolar epithelium: bronchiolitis. Sloughed epithelium and mucus blocks small airway lumens leading to air trapping and hyperinflation. Air absorbed distal to obstructed airways leads to multiple areas of atelectasis.
• Disease is due to the vulnerability of the small airways of the very young to inflammation and obstruction (resistance to air flow being inversely related to the cube of the radius), as well as immunopathology, perhaps immune complex formation. The severest forms are experienced by infants when maternally derived specific antibody is at a high level, and severe disease was seen in children vaccinated with a trial inactivated vaccine in the 1960s.
• Young children – pneumonia and bronchiolitis (see p. [link] ) are the commonest manifestations of RSV infection in infants. Tracheobronchitis and croup are less common. All may occur in association with fever and otitis media (RSV is present in 75% of middle ear effusions from children with respiratory RSV infection). Rarely asymptomatic. Those with LRTI may have a preceding URTI with nasal congestion and pharyngitis. Cough and fever are common in young children Clinical findings include wheeze and crepitations. It is difficult to differentiate pneumonia from bronchiolitis, and many infants have both. Minimal CXR changes regardless of severity. Hypoxia may be profound. Duration of illness is 7–21 days. Acute complications: apnoea, secondary bacterial infection. RSV has been shown to be a contributing factor to sudden infant death syndrome. Long-term studies suggest those hospitalized with RSV LRTI may have a higher rate of later reactive airway disease.
• Older children and adults – a severe ‘common cold’ with nasal congestion, cough, fever, earache (in children); <50% of infected older people develop pneumonia (particularly those in residential homes); 2–6% of hospitalized adults with pneumonia have RSV. Secondary infections cause URTI or tracheobronchitis.
• Severe disease – young infants, the premature and those with underlying disease (congenital and cardiopulmonary disease, e.g. cystic fibrosis) are at risk of severe RSV; <66% of deaths occur in those with underlying disease. Prematurity is a risk into the third year of life. Immunodeficient patients (including those with transplants and on chemotherapy) have extensive pulmonary infiltration and prolonged viral shedding.
• Clinical diagnosis can be made with some confidence in children during an outbreak. Serology is only useful epidemiologically.
• Cell culture – NPA provides the best sample with a high rate of virus isolation. It should be inoculated into cell lines as soon as possible. Infection is characterized by the typical syncytial appearance, and the cytopathic effect is visible at around day 3–7. The major advantage of culture techniques is they can identify other pathogens.
• Rapid tests – immunofluorescence antibody test (IFAT), PCR, and enzyme immunoassays are all available.
• Supportive care – oxygen to maintain saturation at 92% or above. Other therapies of potential benefit: heliox, inhaled nitric oxide. No specific benefit shown with bronchdilators, steroid or antibiotics.
• Ribavirin – indicated for RSV LRTI in hospitalized infants considered at high risk of complicated or severe disease (underlying cardiac, pulmonary, or immunosuppressive conditions). Given as an aerosol for 8–20 h each day for 2–5 days. Follow-up over a year shows better pulmonary function and a reduced incidence of reactive airway disease at 1 year in those treated with ribavirin.
• Immunotherapy – active immunization is not available. Palivizumab (RSV monoclonal antibody) reduces morbidity in infants at risk of severe RSV. Administered to those at risk once a month during outbreaks, it significantly reduces disease severity and hospital admissions for respiratory illness. It has a role in prophylaxis to the high-risk exposed in hospital.
• Infection control – vital in hospitalized cases. Handwashing, eye–nose goggles, and glove use reduce nosocomial inections. Infected patients should be isolated or cohorted, especially on wards with high-risk patients.
Other respiratory viruses
• Enveloped viruses of the family Coronaviridae. Large positive-sense single-stranded RNA genome. They are found worldwide.
• The name derives from Latin ‘corona’ (crown) reflecting the EM appearance of the viral spike protein that populates the surface of the virus and determines its host tropism.
• Human respiratory strains cause colds in adults, have been isolated from infants with pneumonia, are associated with bouts of wheeze in children with asthma or recurrent bronchitis, and may be a contributor to exacerbations of COPD. Enteric viruses may cause gastroenteritis in infants and have been associated with outbreaks of necrotizing enterocolitis.
• Severe acute respiratory syndrome (SARS) was recognized in China in November 2002 and had spread to affect 29 countries across the world by February 2003. The epidemic had died out by July 2003; 8422 cases were reported with a fatality rate of 11% (43% in those over 60 years of age). Between July 2003 and May 2004 there were four small and rapidly contained outbreaks of SARS, three of which were associated with laboratory releases and the fourth thought to be due to an animal source. The possibility of SARS re-emergence remains.
• It is caused by a novel coronavirus. Animals are thought to be the main reservoir. Transmission is by droplets and contact with contaminated surfaces – nosocomial transmission was common in the early stages of the outbreak. Virus is present in stool and may cause diarrhoea. Incubation is 2–10 days.
• Inter-epidemic case definitions proposed by the WHO and adopted by the HPA define a possible case as an individual meeting the clinical criteria, within 10 days of onset of illness with either a history of travel to an area classified by WHO as a potential zone of re-emergence of SARS (mainland China, and Hong Kong SAR) or a history of exposure to laboratories or institutes which have retained SARS virus isolates and/or diagnostic specimens from SARS patients.
• Clinical criteria: fever of ≥38°C, one or more symptoms of lower respiratory tract illness (cough, difficulty breathing, shortness of breath), radiographic evidence of pneumonia or ARDS, or autopsy findings consistent with the pathology of pneumonia or ARDS without an identifiable cause and no alternative diagnosis to fully explain the illness. Note that this definition is for public health, not diagnostic purposes.
• Confirmation is by PCR, seroconversion, or virus isolation, and details of these and further case definitions are available on the HPA website. 1
• A newly identified virus (family Paramyxoviridae) first reported in June 2001 as a cause of respiratory tract disease in Dutch children.
• Clinical features are indistinguishable from those caused by RSV and it is now known to be a cause of respiratory tract disease in both children and adults worldwide; 98–100% of people are seropositive by age 10 years, it is a significant pathogen in LRTI of children and is implicated in nosocomial spread of infection in hospital wards.
• Symptoms are identical to those of RSV in children (mild URTI to severe cough, bronchiolitis, and pneumonia) and older adults (cough, fever, respiratory distress).
• Diagnosis is by PCR of respiratory secretions or serology. There is as yet no specific treatment (experimental evidence suggests a role for ribavirin) or vaccine.
1 Health Protection Agency. Severe acute respiratory syndrome (SARS). http://www.hpa.org.uk/accessed Sept. 2008.
An acute highly infectious disease of children characterized by cough, coryza, fever, and rash.
• A member of the family Paramyxoviridae, genus Morbillivirus; ssRNA, enveloped virus; covered with short surface projections: the haemagglutinin (H) and fusion (F) glycoproteins.
• Humans are the only natural host.
• Found in every country in the world. Without vaccination, epidemics lasting 3–4 months would occur every 2–5 years.
• Airborne, spread by contact with aerosolized respiratory secretions, and one of the most communicable of the infectious diseases. Sensitive to light and drying but can remain infective in droplet form for some hours.
• Patients are most infectious during the late prodromal phase when coughing is at its peak. Immunity after infection is lifelong.
• Virus invades the respiratory epithelium and local multiplication leads to viraemia and leucocyte infection. Reticulo-endothelial cells become infected and their necrosis leads to a secondary viraemia. The major infected blood cell is the monocyte.
• Tissues that become infected include the thymus, spleen, lymph node, liver, skin, and lung. Secondary viraemia leads to infection of the entire respiratory mucosa with consequent cough and coryza. Croup, bronchiolitis, and pneumonia may also occur.
• Koplik’s spots and rash appear a few days after respiratory symptoms – may represent host hypersensitivity to the virus.
• Incubation is 2 weeks (longer in adults than children). A prodromal phase (coinciding with secondary viraemia) of malaise, fever, anorexia, conjunctivitis, and cough is followed by Koplik’s spots (blue-grey spots with a red base classically found on the buccal mucosa opposite the second molars. Severe cases may involve the entire mucosa) then rash. Patients feel most ill around day 2 of the rash. From late prodrome to resolution of fever and rash is around 7–10 days. Rash begins on the face and proceeds down involving palms and soles last. Erythematous and maculopapular and may become confluent. It lasts around 5 days and may desquamate as it heals.
• Complications – bacterial superinfection (pneumonia and otitis media); acute encephalitis – seen in 1 in 2000 and probably due to host hypersensitivity to virus. Characterized by fever recurrence, headache, seizures, and consciousness changes during convalescence; SSPE – chronic degenerative neurological condition occurring years after measles due to persistent CNS infection with measles virus despite a vigorous host immune response; spontaneous abortion and premature labour – unlike rubella there is no association with fetal malformations but the disease can be more severe in pregnancy and infants can acquire it. Infants born to mothers with active infection should be given immunoglobulin at birth.
• Prior to vaccination measles was a common cause of viral meningitis and remains so in unvaccinated populations, usually occurring with rash.
• Special conditions:
• modified measles – a very mild form of the disease seen in people with some degree of passive immunity, e.g. those receiving immunoglobulin or babies under 1 year.
• atypical measles – seen in those who received early killed measles vaccines and are later infected by wild-type virus. The rash is atypical and may resemble Henoch–Schönlein purpura (HSP), varicella, spotted fever, or a drug eruption. High fever, peripheral oedema, pulmonary infiltrates, and effusions may occur. The disease is more severe, has a longer course, and is thought to be due to hypersensitivity to virus in a partially immune host. It is rare now but those who have received only killed vaccine should be offered live.
• immunocompromised patients (including the malnourished) may experience severe disease, e.g. primary viral giant cell pneumonia, encephalitis, SSPE-like encephalitis. They may not develop rash, making diagnosis difficult. Immunocompromised people should be passively immunized following exposure even if previously vaccinated.
Diagnosis can usually be made clinically. Lab confirmation is useful in atypical cases or the immunocompromised.
• Virus isolation – possible in renal cell lines, growth slow. Useful in the immunodeficient where antibody responses may be minimal.
• Serology – a fourfold increase in measles antibody titre between acute and convalescent specimens is diagnostic. ELISA is capable of detecting specific IgM on a single sample.
• Other – immunofluoresent microscopy of cells in secretions, RT-PCR.
• Supportive therapy and treatment of bacterial superinfection.
• Vitamin A 200,000 IU given orally to children for 2 days has been shown to reduce severity but should be avoided at or after immunization when it appears to reduce seroconversion.
• Measles vaccine is given as part of measles, mumps, rubella (MMR) (12 months and preschool). It can be given earlier in at-risk populations but responses are suppressed and an additional dose should be given later.
• Passive immunization with immunoglobulin is recommended for those exposed susceptible people at risk of severe or fatal measles – it must be given within 6 days of exposure to be effective. Such groups include:
• children with defects in cell-mediated immunity
• children with malignant disease – particularly if receiving chemo- or radiotherapy
• children with HIV should be given immunoglobulin after exposure even if already vaccinated – cases have occurred in vaccinated HIV-infected children.
Mumps is an acute generalized viral infection of children and adolescents causing swelling and tenderness of the salivary glands and rarely epididymo-orchitis. More-severe manifestations are commoner in older patients. Mumps was recognized as far back as Hippocrates. The name may derive from the English verb ‘to mump’ – to be sulky.
• A member of the Paramyxoviridae family; single-stranded RNA virus, irregular spherically shaped virion (average diameter 200 nm), nucleocapsid is enclosed by a three-layer envelope.
• Nucleocapsid contains the S (soluble) antigen, antibodies to which may be detected early in infection.
• Glycoproteins on the surface have haemagglutinin, neuraminidase, and cell-fusion activity, and include the V (viral) antigen detected in late infection by complement fixation.
• Endemic throughout the world. Prior to vaccination, epidemics took place every 2–5 years with 90% of cases occurring in those under 15 years.
• In the US one-third of cases occur in those over 15 years. UK incidence has been increasing among those born between 1981 and 1989 who missed routine MMR (first introduced in 1988) and are now at university. In 2004 there was a dramatic increase of cases in England and Wales among those born before 1987, many of whom had received just one dose of MMR in the 1998 “catch-up” campaign.
• Passive immunity makes infection uncommon in children under 1 year.
• Transmitted by droplet spread or direct contact. Most infectious just before parotitis.
• During incubation the virus proliferates in the upper respiratory tract with consequent viraemia and localization to glandular and neural tissue.
• Parotid glands show interstitial oedema and serofibrinous exudate with mononuclear cell infiltration. Cases of orchitis are similar with the addition of interstitial haemorrhage, polymorphonuclear infiltration, and areas of local infarction due to vascular compromise.
• Incubation is 2–4 weeks. A 24-h non-specific prodrome of fever, headache, and anorexia is followed by earache and ipsilateral parotid tenderness. The gland swells over 2–3 days and is associated with severe pain. Swelling can lift the ear lobe up and outward. The other side follows within a couple of days in most cases – unilateral parotid involvement is seen in 25%. Patients experience difficulty in pronunciation and mastication and may develop fever. Once swelling has peaked recovery is rapid – within a week. Complications of parotitis (e.g. sialectasia) are rare. Other salivary glands may be involved.
• CNS involvement – the commonest extra-glandular manifestation in children:
• meningitis is seen in <10% of those with parotitis although <50% of cases of mumps meningitis show no evidence of glandular disease. Onset is 4 to 7 days after glandular symptoms but can occur 1 week before or 2 weeks later. Men are affected more than women. Symptoms resolve 3–10 days later and recovery is complete with no sequelae. CSF findings – typical of viral meningitis (p. [link] ) – hypoglycorrachia (CSF glucose <40 mg/100 mL) is seen in up to 30% of cases, more than other viral meningitides
• encephalitis is seen in 1 in 6000 and takes two forms: early onset which represents direct neuron damage due to viral invasion, and a larger late-onset (7–10 days) group representing a post-infectious demyelinating process. Recovery takes around 2 weeks and sequelae (e.g. psychomotor retardation) and death (around 1.4% of cases) may be seen
• other neurological manifestations include transient deafness, permanent deafness (1 in 20,000), ataxia, facial palsy, transverse myelitis, GBS.
• Other extraglandular manifestations:
• presternal pitting oedema and tongue swelling (thought to be due to lymphatic obstruction by swollen regional glands) (6%)
• epididymo-orchitis – the commonest extra-glandular manifestation in adults – 20–30% of post-pubertal males with mumps develop it (1 in 6 of those bilateral). Rare before puberty. It may be the only manifestation of mumps. Onset is abrupt with fever and a warm, swollen (up to four times normal) tender testicle with erythema of the overlying skin. Fever resolves at 5 days with gonadal symptoms following. Some degree of atrophy may be seen in 50% once recovered. Infertility is rare
• oophoritis – seen in 5% of post-pubertal women with mumps – impaired fertility and premature menopause have been reported but are rare
• other manifestations – migratory polyarthritis, pancreatitis, myocarditis, nephritis, thyroiditis, mastitis, hepatitis.
• Lab confirmation is required for epidemiological purposes or when disease is atypical
• General features – exposure history and symptoms; leucocytosis may be seen, particularly with meningitis, orchitis, or pancreatitis; serum amylase is elevated in parotitis or pancreatitis (isoenzyme analysis is required to differentiate the source).
• Serology – most reliably determined using ELISA for IgM. Haemagglutination inhibition (convalescent serum prevents the adsorption of chick red cells to mumps-infected epithelial cells) can be positive with antibodies to parainfluenza 3 (another cause of parotitis).
• Virus isolation – present in saliva from 2 days before symptom onset to 5 days after. May be present in CSF up to 6 days after onset.
• Symptom control – antipyretics and fluids if persistant vomiting
• No benefit in steroid use has been demonstrated
• Anecdotal evidence that interferon-alfa speeds resolution of orchitis
An acute mild exanthematous viral infection of children and adults resembling mild measles but with the potential to cause fetal infection and birth defects.
• A member of the Togaviridae family, in the genus Rubivirus.
• Spherical in shape with a diameter of 60 nm. Relatively unstable.
• Unlike measles, rubella is only moderately contagious. Prior to vaccination incidence was highest in the spring among children aged 5–9 years.
• Once termed ‘third disease’, measles and scarlet fever being the first and second exanthematous infections in childhood.
• After infection or vaccination most people develop lifelong protection against disease. Re-infection occurs (the majority asymptomatic) as demonstrated by rises in antibody titre in previously vaccinated people.
• There have been rare cases of congenital rubella acquired through the re-infection of a vaccinated mother.
• Spread is by droplets; patients are at their most contagious when the rash is erupting and virus may be shed from 10 days before to 2 weeks after its appearance.
• Rash may be immune mediated – it appears as immunity develops and viral titres fall.
• Primary viraemia follows infection of the respiratory epithelium; secondary viraemia occurs a few days later once the first wave of infected leucocytes release virions.
• Infants with congenital rubella shed large quantities of virus for many months.
• Incubation is 12–23 days. Postnatal rubella is a mild infection. Many cases are subclinical. Adults may experience a prodrome of malaise, fever, and anorexia. The main symptoms are lymphadenopathy (cervical and posterior auricular) and a maculopapular rash (starting on the face and moving down) which may be accompanied by coryza and conjunctivits and lasts 3–5 days. Splenomegaly can occur.
• Complications are uncommon – arthritis affecting the wrists, finger and knees and resolving over a month may be seen as the rash appears (women more than men); haemorrhagic manifestations occur in 1 in 3000 (children more than adults) and may be due to thrombocytopenia as well as vascular damage; encephalitis occurs in 1 in 5000 (adults more than children), with a mortality of up to 50%.
• Congenital rubella – can be catastrophic in early pregnancy leading to fetal death, premature delivery, and many congenital defects. Rare since the introduction of vaccination. The younger the fetus when infected, the more severe the illness. In the first 2 months of gestation there is an up to 85% chance of being affected by either multiple defects or spontaneous abortion. In the third month there is around a 30% chance of developing a single defect (e.g. deafness), dropping to 10% in the fourth month and nil after 20 weeks. Temporary defects include low birth weight, low platelets, hepatosplenomegaly, hepatitis, meningitis, and jaundice. Permanent defects include hearing loss, cardiac abnormalities, microcephaly, inguinal hernia, cataract, and glaucoma. Developmental defects may become apparent as the infant grows, e.g. myopia, mental retardation, diabetes, behavioural and language disorders.
• Its mild nature makes clinical diagnosis difficult.
• Serology – positive IgM on a single sample or a fourfold rise in IgG in paired sera is diagnostic. IgM may be positive in cases of re-infection. Serological diagnosis of congenital rubella in neonates may necessitate the analysis of several samples over time to determine whether antibody titres are falling (maternal antibody) or rising (recent infection). Detection of rubella IgM in a newborn’s serum indicates infection.
• Intrauterine diagnosis has been made by placental biopsy and by cordocentesis with detection by PCR.
• No treatment is indicated in most cases of postnatal rubella.
• Immunoglobulin used to be given to exposed susceptible pregnant women – however it does not prevent viraemia despite suppressing symptoms.
• All women of child-bearing age should be vaccinated before pregnancy.
Parvovirus B19 has a wide variety of clinical manifestations depending on the state of the host: in immunocompetent children, ‘slapped-cheek’ disease, in those with underlying haemolytic disorders, an aplastic crisis. Parvovirus B19 was found in 1974 whilst evaluating assays for hepatitis B surface antigen using panels of serum samples – sample 19 in panel B gave a ‘false-positive’ and EM revealed the guilty virus.
• A member of the family Parvoviridae, genus Erythrovirus (so called because replication occurs only in human erythrocyte precursors). B19 remains the only known human pathogenic parvovirus. They are non-enveloped and extremely resistant to physical inactivation.
• Infection common in childhood – 50% are IgG-positive by 15 years, 90% antibody positive by 90 years. Infected children pass virus on to uninfected members of their family. Patients are infectious from 24–48 h before viral prodrome until rash appearance.
• In temperate climates infection is commonest from late winter to early summer. Rates peak every 3 to 4 years. Prevalence higher in Africa.
• Infection may be passed vertically, by respiratory secretions or from blood and blood products (standard thermal treatments and solvent-detergents are not completely effective) although viraemia is rare.
• Parvovirus B19 infects erythroid progenitors and erythroblasts. The receptor it uses to infect cells – P antigen – is found on megakaryocytes, endothelial cells, fetal myocardial cells, and erythroid precursors. Those who lack P antigen on their erythrocytes are resistant to infection.
• Infected individuals may experience an acute, self-limited (4–8-day) halt in red blood cell manufacture as infected cells are destroyed. This may be unnoticed In those with normal erythroid turnover (falls in haemoglobin >1g/dL are uncommon) but in those with a high turnover (e.g. haemoglobinopathies, haemolytic anaemia) falls of 2–6 g/dL are not uncommon and aplastic anaemia may develop.
• The infected fetus may have severe manifestations due to high red cell turnover and the immature immune response: anaemia, myocarditis, and heart failure can occur. These effects are reduced by the third trimester.
• Rash and arthralgia associated with some forms of the disease are probably immune complex related.
• 20% of infections are asymptomatic.
• Erythema infectiosum (EI) – the commonest manifestation. The classic slapped cheek-appearing rash (fiery red eruption with surrounding pallor) follows a 5–7-day prodrome of fever, coryza, and mild nausea/diarrhoea. A second erythematous maculopapular rash may follow on the trunk and limbs 1–2 days later, fading to produce a lacey appearance. Adults have milder manifestations. Pruritus (especially soles of the feet) can occur.
• Arthropathy – seen in adults, especially women. Symmetric, mainly small joints of hands and feet lasting for 1–3 weeks. May persist or recur for months. May be confused with acute rheumatoid arthritis.
• Transient aplastic crisis (TAC) – the first clinical illness associated with B19 infection – the abrupt cessation of erythropoiesis with absent erythroid precursors in bone marrow. Described in a wide range of haemolytic conditions: sickle cell (nearly 90% of TAC episodes), thalassaemia, pyruvate kinase (PK) deficiency, autoimmune haemolytic anaemia. It has also been seen after haemorrhage, in iron-deficiency anaemia, and those who are otherwise well (who are likely to see deficiencies in other blood lineages: neutropenia, thrombocytopenia). Patients can be severely ill: dyspnoea, confusion, cardiac failure. It does not appear to cause true, permanent aplastic anaemia.
• Pure red cell aplasia (PRCA) – anaemia in the immunosuppressed (HIV, congenital immunodeficiency, patients undergoing transplantation). Administration of immunoglobulin may be beneficial.
• Virus-associated haemophagocytic syndrome – usually healthy patients with cytopenia – characterized by histiocytic hyperplasia and haemophagocytosis. Self-limiting.
• Fetal infection – 10–15% of non-immune hydrops foetalis. Where maternal infection occurs, fetal loss averages 9% and occurs within the first 20 weeks of pregnancy. There is no evidence of long-term abnormality in those who survive.
• Other manifestations – encephalitis, myocarditis, hepatitis, vasculitis, erythema multiforme, glomerulonephritis, idiopathic thrombocytopenia purpura, Henoch-Schönlein purpura.
• IgM detection – 90% of cases are positive by the time of the rash in EI or by day 3 of TAC. IgM remains detectable for up to 3 months. IgG is detectable by day 7 of illness and remains detectable for life (50% of the population are IgG positive). It is not useful in diagnosing acute infection or in attributing manifestations such as chronic arthropathy to B19.
• Virus detection is possible by DNA hybridization – however, in immunocompetent people viral DNA can only be detected for 2–4 days.
• Immunocompromised people with chronic infection do not mount an immune response and diagnosis relies on detecting DNA by PCR.
• Fetal infection can be confirmed by amniotic fluid sampling, and investigations should include maternal B19 serology.
• Those with aplastic anaemia will show a fall of at least 2 g/dL from baseline Hb.
• General measures – e.g. non-steroidal anti-inflammatory drugs (NSAIDS) for arthritis, transfusions for TAC.
• Immunosuppressed patients with persistent infection may benefit from temporary cessation of immunosuppression to allow them to mount an immune response. If this is not feasible, IV IgG over 5 days may help. If disease recurs they may require repeated infusions. Some HIV-infected patients will resolve chronic B19 infection with the initiation of highly active antiretroviral therapy (HAART).
• Intrauterine blood transfusions may help some cases of hydrops.
A lymphotropic virus and the single most common cause of hospital visits in infants with fever. The cause of roseola (also known as sixth disease and exanthem subitum).
• A herpesvirus, originally called B-lymphotropic virus, now shown to grow in many different cell types (T cells, macrophages etc).
• Two subtypes (A and B), which differ in their epidemiology and growth.
• Nearly all humans infected by age 2 years, probably by saliva exchange.
• Most isolates from healthy people are HHV-6B, the only variant to have been linked to specific clinical syndromes.
• Primary infection is via the oropharynx. Regional lymph nodes and mononuclear cells are subsequently infected and virus spreads throughout the body. Incubation is 5–15 days. Like other herpesviruses it causes an initial infection, a lifelong latency, and has the potential for clinical reactivation, especially in hosts who are immunocompromised (see box 4.18 ). Asymptomatic carriers may continue to excrete the virus for months.
• Specific mechanisms for host immune evasion facilitate persistent infection.
• Infantile fever – fever without rash is the commonest manifestation and may be accompanied by periorbital oedema. 10% of cases in one series of acute febrile illnesses were attributed to HHV-6. Benign febrile convulsions may occur and are more frequent than fever alone explains – perhaps due to viral replication within the CNS.
• Exanthem subitum (6th disease) – an illness of infants and young children. 3–5 days of fever and URT symptoms are followed by the development of rose-pink papules which are mildly elevated, non-pruritic, and blanche on pressure. Rash lasts around 2 days and may be associated with malaise, vomiting, diarrhoea, cough, pharyngitis, and lymphadenopathy. Most infants are asymptomatic.
• Encephalitis – can occur alone or as a complication of exanthem subitum. Virus is frequently detected in the CNS even in the absence of symptoms.
• Immunocompromised hosts – as with other herpesviruses, immune suppression permits replication with the potential for clinical illness. Viral DNA has been isolated in bone marrow transplant recipients with pneumonitis, but often in the presence of other pathogens (such as CMV) with better established pathological pedigrees. There are other anecdotal reports, including associations with certain leukaemias and lymphomas, but no definitive links have been established.