INVESTIGATION OF INFECTION

Microbiological tests detect either a microorganism (e.g. direct detection and culture) or the host response to the organism (immunological tests).

They also provide information on responsiveness to antimicrobial therapy.

Careful and appropriate sampling increases the chance of useful results.

The results must be interpreted in light of the normal flora in the site from which the sample was obtained and the likely findings in a person without infectious disease.

Like all tests, whether a positive test result is diagnostic of disease depends on the specificity and positive predictive value (PPV) of the test, and whether a negative test result excludes disease depends on its sensitivity and negative predictive value (NPV).

Test sensitivity varies according to the time between infection and testing (there is a ‘window of opportunity’ during which sensitivity is maximal), and the PPV and NPV depend on the prevalence of the condition in the test population.

Microbiological results are therefore interpreted in light of other findings consistent with infectious disease, including the clinical scenario and results of other investigations (e.g. neutrophilia, elevated C-reactive protein (CRP)).

Given this complexity, effective two-way communication between the clinician and the microbiologist is a vital component of test interpretation.

1. Direct detection:

Direct detection methods provide rapid results, and may be applied to organisms that cannot be grown easily on artificial culture media, such as Chlamydia spp.

They do not usually provide information on antimicrobial susceptibility or the degree to which organisms are related to each other (which is important in the investigation of possible disease outbreaks) unless relevant specific nucleic acid sequences are detected by polymerase chain reaction (PCR).

a. Detection of whole organisms: Whole organisms are detected by

microscopic examination of biological fluids or tissue using a light or electron microscope.

o Bright field microscopy (in which the test sample is interposed between the light source and the objective lens) uses stains to enhance visual contrast between the organism and its background. Examples include Gram-staining of bacteria and Ziehl–Neelsen or auramine staining of acid- and alcohol-fast bacilli (AAFB) in tuberculosis. In histopathological examination of tissue samples multiple stains are used to demonstrate not only the presence of microorganisms, but also features of disease pathology.

o Dark field microscopy (in which light is scattered to make organisms appear bright on a dark background) is used, for example, to examine genital chancre fluid in suspected syphilis.

o Electron microscopy is used to examine stool and vesicle fluid to detect enteric and herpes viruses, respectively.

b. Detection of components of organisms: Components of microorganisms that are

detected for diagnostic purposes include nucleic acids (DNA and RNA), cell wall molecules, toxins and other antigens.

Examples of antigen detection include Legionella pneumophila serogroup 1 antigen in urine, HIV p24 antigen in blood and cryptococcal polysaccharide antigen in cerebrospinal fluid (CSF).

Non-immunological methods may also be used, e.g. detection of Clostridium botulinum toxin in a mouse bioassay.

In toxin-mediated disease, detection of toxin may be of greater relevance than identification of organism (e.g. detection of C. difficile toxin in stool).

oNucleic acid amplification tests (NAAT):

Specific sequences of microbial DNA and RNA are identified using a nucleic acid primer which is amplified exponentially by enzymes to generate multiple copies of the specific sequence.

The most commonly used amplification method is the polymerase chain reaction (PCR).

Reverse transcription (RT) PCR is used to detect RNA from organisms such as hepatitis C virus and HIV-1.

In modern amplification systems the use of fluorescent-labelled primers and probes allows ‘real-time’ detection of amplified DNA.

Quantification is based on the principle that the time taken to reach the detection threshold is proportional to the initial number of copies of the target nucleic acid sequence.

Determination of nucleic acid sequence is also used to assign microorganisms to specific strains according to their genotype, which may be relevant to treatment and/or prognosis (e.g. in hepatitis C infection).

Nucleic acid sequences that are relevant to pathogenicity (such as toxin genes) or antimicrobial resistance can also be detected.

For example, detection of the mecA gene is used to screen for meticillin-resistant Staphylococcus aureus (MRSA).

Nucleic acid amplification provides the most sensitive direct detection methods, but their extreme sensitivity can produce false positive results from contamination.

PCR techniques are particularly useful when a rapid diagnosis is required.

They are used widely in virology, where the possibility of false positive results from colonising or contaminating organisms is remote, and are applied to blood, respiratory samples, stool and urine.

In bacteriology, PCR is used mainly to examine samples from normally sterile sites, such as CSF, blood and, increasingly, tissue.

The role of PCR in mycology and parasitology is not yet established.

2 .Culture: Organisms in samples of tissue, swabs and

body fluids may replicate in culture, allowing their detection and characterisation.

o In vivo culture (in a living organism) is not used in routine diagnostic microbiology.

o Ex vivo culture (tissue culture) was widely used in the isolation of viruses, but is being gradually supplanted by nucleic acid amplification techniques.

o In vitro culture (in artificial culture media) of bacteria and fungi is used for definitive identification, to test for antimicrobial susceptibility and to subtype the organism for epidemiological purposes.

However, culture has its limitations. Results are not immediate, even for

organisms which are easy to grow, and negative culture rarely excludes infection completely.

Organisms such as M. tuberculosis are inherently slow-growing, typically taking 2 weeks to be detectable in the most specialised systems (liquid culture with constant monitoring) and longer when grown on solid media.

Certain organisms, such as M. leprae and Tropheryma whipplei, cannot be cultivated on artificial media, and others (e.g. Chlamydia spp. and viruses) grow only in ex vivo systems, which are slow and labour-intensive to use.

oBlood culture:

Rapid microbiological diagnosis is required for bloodstream infection (BSI).

To diagnose BSI, a liquid culture medium is inoculated with freshly drawn blood.

Bacterial growth can be detected by serial subculture or the detection of radio-labelled CO2 produced by bacteria on breakdown of radio-labelled carbon sources provided in the growth media.

Modern blood culture systems allow constant monitoring of liquid media for products of microbial respiration (mainly CO2) using fluorescence.

It is likely that constant-monitoring systems will be replaced or enhanced by nucleic acid amplification-based techniques.

3 .Immunological tests : Immunological tests detect the host response to a

specific microorganism and can be used to diagnose infection with organisms that are difficult to detect by other methods or are no longer present in the host.

The term ‘serological’ describes tests carried out on serum, and includes both antigen and antibody detection.

Immunological tests can also be performed on fluids other than blood (e.g. CSF, urine).

Immunological tests require a functional host immune system, and often provide only a retrospective diagnosis.

a. Antibody detection: Detection of antibodies specific to the antigens of

microorganisms is applied mainly to blood. Results are typically expressed as titres: that is,

the reciprocal of the highest dilution of the serum at which antibody is detectable (for example, detection at serum dilution of 1:64 gives a titre of 64).

‘Seroconversion’ is defined as either a change from negative to positive detection or a fourfold rise in titre between acute and convalescent serum samples.

An acute sample is usually taken during the first week of disease and the convalescent sample 2–4 weeks later.

Earlier diagnosis can be achieved by detection of IgM antibodies, which are produced early in infection.

Many immunological tests may be adapted to detect antigens instead of antibodies.

oEnzyme-linked immunosorbent assay (ELISA, EIA):

These assays rely on linking an antibody with an enzyme which generates a colour change on exposure to a chromogenic substrate.

Various configurations allow detection of antigens or specific subclasses of immunoglobulin (e.g. IgG, IgM, IgA).

ELISA may also be adapted to detect PCR products, using immobilised oligonucleotide hybridisation probe and various detection systems.

oImmunoblot (western blot):

Microbial proteins are separated according to molecular weight by polyacrylamide gel electrophoresis (PAGE) and transferred (blotted) on to a nitrocellulose membrane, which is incubated with patient serum.

Binding of specific antibody is detected with an enzyme-anti-immunoglobulin conjugate similar to that used in ELISA, and specificity is confirmed by its location on the membrane.

Immunoblotting is a highly specific test, which may be used to confirm the results of less specific tests, such as ELISA.

oImmunofluorescence assays (IFA):

IFAs are highly specific. In indirect immunofluorescence a serum sample

is incubated with immobilised antigen (e.g. cells known to be infected with virus on a glass slide) and antibody binding is detected using a fluorescent-labelled anti-human immunoglobulin (the ‘secondary’ antibody).

This method can also detect organisms in clinical samples (usually tissue or centrifuged cells) using a specific antibody in place of patient serum.

In direct immunofluorescence clinical samples are incubated directly with fluorescent-labelled specific antibodies to detect antigen, eliminating the need for secondary antibody.

oComplement fixation test (CFT): In a CFT, patient serum is heat-treated to

inactivate complement, and added to specific antigen.

Any specific antibody present in the serum will complex with the antigen.

Complement is then added to the reaction. If antigen–antibody complexes are present, the

complement will be ‘fixed’ (consumed).

Sheep erythrocytes, coated with an anti-erythrocyte antibody, are added.

The degree of erythrocyte lysis reflects the remaining complement and is inversely proportional to the level of the specific antigen–antibody complexes.

oAgglutination tests:

When antigens are present on the surface of particles (e.g. cells, latex particles or microorganisms) and cross-linked with antibodies, visible clumping (or ‘agglutination’) occurs.

• In direct agglutination, patient serum is added to a suspension of organisms that express the test antigen. For example, in the Weil–Felix test, host antibodies to various rickettsial species cause agglutination of Proteus bacteria because they cross-react with bacterial cell surface antigens.

• In indirect (passive) agglutination, specific antigen is attached to the surface of carrier particles which agglutinate when incubated with patient samples that contain specific antibodies.

• In reverse passive agglutination (an antigen detection test), the carrier particle is coated with antibody rather than antigen.

oOther tests: Immunodiffusion: This involves antibodies and antigen migrating

through gels, with or without the assistance of electrophoresis, and forming insoluble complexes where they meet.

The complexes are seen on staining as ‘precipitin bands’.

Immunodiffusion is used in the diagnosis of chronic and allergic bronchopulmonary aspergillosis.

Immunochromatography:

This is used for detection of antigen. The system consists of a porous test strip (e.g. a

nitrocellulose membrane), at one end of which there is target-specific antibody, complexed with coloured microparticles.

Further specific antibody is immobilised in a transverse narrow line some distance along the strip.

Test material (e.g. blood or urine) is added to the antibody-particle complexes, which then migrate along the strip by capillary action.

If these are complexed with antigen, they will be immobilised by the specific antibody and visualised as a transverse line across the strip.

If the test is negative, the antibody–particle complexes will bind to a line of immobilised anti-immunoglobulin antibody placed further along the strip, which acts as a negative control.

Immunochromatographic tests are rapid and relatively cheap to perform, and are appropriate for point-of-care testing, e.g. in HIV 1.

b. Antibody-independent immunological tests:

It is also possible to measure components of the host immune response other than antibody responses.

Many investigations in patients with infectious disease reflect the non-specific innate immune response and acute phase response, including peripheral blood neutrophil counts and CRP levels.

These are used in combination with microbiological tests to assess the likelihood of infectious disease and its severity.

A few tests assess specific cellular immunity. For example, IFN-γ release assays are used to diagnose tuberculosis, since blood lymphocytes from infected individuals produce IFN-γ in response to specific mycobacterial antigens.

4 .Antimicrobial susceptibility testing : If growth of microorganisms in culture is inhibited

by the addition of an antimicrobial agent, the organism is considered to be susceptible.

This information is often used to select antimicrobial therapy.

Bacteriostatic agents cause reversible inhibition of growth and bactericidal agents cause cell death.

The lowest concentration of antimicrobial agent at which growth is inhibited is the minimum inhibitory concentration (MIC), and the lowest concentration that causes cell death is the minimum bactericidal concentration (MBC) (the terms fungistatic/fungicidal are used for antifungal agents, and virustatic/virucidal for antiviral agents).

If the MIC is less than a predetermined threshold, the organism is considered to be susceptible, and if the MIC is greater than or equal to the threshold, the organism is resistant.

Threshold MICs are determined for each antimicrobial agent from a combination of pharmacokinetic and clinical data.

In reality, the relationship between antimicrobial susceptibility in culture and clinical response is more complex, as it depends on comorbidities, immune status, pharmacokinetic variability and antibiotic dosing, as well as MIC/MBC.

Thus, susceptibility testing does not guarantee therapeutic success, but does indicate its probability.

Susceptibility testing is most often carried out by disc diffusion:

Antibiotic-impregnated filter paper discs are placed on an agar plate containing bacteria.

The antibiotic diffuses through the agar, resulting in a concentration gradient centred on the disc.

Bacteria are unable to grow where the antibiotic concentration exceeds the MIC, and the size of the resulting zone of inhibition is inversely proportional to the MIC.

Susceptibility testing methods using serial dilutions of antimicrobial in liquid media are generally more accurate and reproducible, and are used for generating epidemiological data.

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