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 hepatitis B).
Molecular techniques
Southern blotting and nucleic acid hybridization
A labelled DNA probe can bind to components of the pathogen and be detected by the activity of its attached label. This technique is specific and rapid, but less sensitive because there are no amplification steps.
Nucleic acid amplification tests
Nucleic acid amplification tests (NAATs) amplify specific regions of the pathogen genome (DNA or RNA) to make the diagnosis until there is sufficient for detection. Primers are designed to bind to target a specific pathogen sequence and a polymerase synthesizes new nucleic acid over multiple cycles. Automated systems and commercial kits can follow this process in real time and make these tests available in many laboratories. NAATs have the advantage that they can detect slow or difficult to grow organisms, or make a diagnosis in the patient who has taken antibiotics. PCR can also detect virulence determinants or resistance determinants creating a surrogate susceptibility result (e.g. rpoB gene mutation for rifampicin resistance in M. tuberculosis).
Whole genome sequencing
The reducing cost of sequencing the whole pathogen sequence means that this is increasingly widely available. It provides extremely detailed information on the pathogen. Using complex manipulations of the genomic data it is possible to determine the relationship between organisms and identify their transmission. It has become vital in tracking viruses like influenza, and resistant or virulent bacteria in community or hospital outbreaks. Rapid availability of data can rapidly rule in or rule out an outbreak.
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