Medical Microbiology and Infection at a Glance. Stephen H. Gillespie

IgA protease that degrades host immunoglobulin in the mucosa.

       Staphylococcus aureus expresses protein A, which binds host immunoglobulin, preventing opsonization and complement activation.

       S. pneumoniae has a polysaccharide capsule, which inhibits phagocytosis by polymorphonuclear neutrophils.

       Toxoplasma gondii, Leishmania donovani and Mycobacterium tuberculosis are adapted to survive the killing mechanisms of macrophages by different mechanisms.

       The lipopolysaccharide (LPS) of Gram‐negative organisms makes them resistant to the effect of complement.

       Trypanosoma alter surface antigens to evade the adaptive humoral immune response.

Damaging the host

      Toxins

       Endotoxins

      Endotoxins exert their effect through host mechanisms by, for example, stimulating macrophages to produce cytokines such as interleukin‐1 (IL‐1) and tumour necrosis factor (TNF) that lead to fever and shock (see Chapter 55).

       Exotoxins

      Bacterial exotoxins can cause local or distant damage. They are usually proteins and may have a subunit structure.

       Cholera toxin B subunit binds to the epithelial cell and the A subunit activates adenyl cyclase, which results in sodium and chloride efflux from the cell, thus causing diarrhoea.

       Staphylococcal enterotoxins act as superantigens, causing non‐specific activation of T‐cells that result in intense cytokine production leading to fever, shock, gastrointestinal disturbance and rash (toxic shock syndrome).

       Diphtheria toxin and Pseudomonas aeruginosa exotoxin A stop protein synthesis by blocking peptide chain elongation.

       Clostridial toxins interfere with host functions, e.g. neurological or neuromuscular signalling in botulism or tetanus.Neutralizing toxin with antibody may ameliorate the damaging effects, e.g. human‐tetanus immunoglobulin (see Chapter 24).

5 Making a microbiological diagnosis of infection

Specimens

      Any tissue or body fluid can be used for microbiological investigation to identify the infecting pathogen and predict response to therapy.

      When planning infection diagnosis one needs to:

       understand when, where and in what concentration the organism is in the body throughout the natural history of infection;

       take samples aseptically as poor aseptic technique leads to contamination of sterile samples and false‐positive or confusing results;

       transport samples rapidly to the laboratory in a suitable medium as many organisms survive poorly outside the body (e.g. strict anaerobes are readily killed by atmospheric oxygen). Direct inoculation in a clinic can overcome this as in the case of Neisseria gonorrhoeae.

       Nucleic acid amplification techniques obviate the need for organisms to grow and can provide a rapid diagnosis.

Laboratory examination

      Specimens may be examined grossly, e.g. to see adult worms in faeces. Microscopy is rapid but it is insensitive and requires considerable expertise; specificity may also be a problem if commensal organisms can be mistaken for pathogens. Microscopy can also be used to define specimen quality: epithelial cells in sputum suggest excessive salivary contamination.

      Special stains can identify organisms and Giemsa staining of blood films and tissues, can demonstrate malaria and Leishmania (Chapter 49). Immunofluorescence can provide precise identification of a pathogen by using antibodies that specifically bind the target organism.

Culture

      Culture is used to amplify the number of pathogens to make identification and drug susceptibility testing possible by isolating single colonies (a clonal population).

       Most human pathogens are fastidious, requiring special media and conditions to let them grow artificially.

       An appropriate atmosphere must be provided: e.g., fastidious anaerobes require an oxygen‐free atmosphere.

       Antibiotic therapy renders samples falsely culture negative.

       Selective agents such as antibiotics or dyes can suppress unwanted organisms in specimens with a normal flora but can also reduce the number of pathogens detected.

       Most pathogenic bacteria are incubated at 37°C, but some fungi are incubated at 30°C.

Identification

      Pathogen identification is important because it can predict disease and prognosis, e.g. Vibrio cholerae causes severe watery diarrhea and is potentially fatal.

      Identification of some organisms requires prompt public health action: e.g. contact tracing for patients with meningococcal disease.

      Bacterial identification depends on colonial morphology on agar, microscopic morphology, and biochemical tests. Matrix‐assisted‐laser‐desorption/ionization‐time‐of‐flight (MALDI‐TOF) can achieve this in 20 minutes. Nucleic acid amplification tests (NAATs) and gene sequencing are used especially when organisms are slow growing (e.g. Mycobacterium tuberculosis) or impossible to grow (e.g. Trophyrema whippelii).

Susceptibility testing

      Susceptibility testing (DST) determines whether treatment is likely to be successful remembering that clinical response depends on host factors too. A susceptible organism should respond to a standard dose of an antimicrobial; an intermediate resistant strain should respond to a larger dose; and a resistant organism is likely to fail therapy with that antibiotic.

      DST can be achieved by measuring the diameter of an inhibition zone around a paper disc with incorporated antibiotics. Susceptibility is defined by a ‘breakpoint’ in growth. These methods are standardized by international bodies such EUCAST to ensure reproducibility. Automated methods can achieve this more rapidly.

      The minimum inhibitory concentration, which is the lowest dose that completely inhibits growth, is a more objective method and enables resistance levels to be related to the concentration of antibiotic that is achievable in the tissues.

      Susceptibility can be assessed rapidly by hybridization or sequence‐based methods that detect specific antibiotic‐resistance mutations.

Serology

      An infection can be diagnosed by detecting the immune response to the pathogen: for example by detection of rising or falling antibody concentrations more than a week apart, or by the presence of a specific IgM or specific pathogen antigen. Detecting the activity of specific T‐cells can provide evidence of exposure to tuberculosis. Serological techniques are used for organisms that are difficult or impossible to grow such as viruses (e.g. HIV or