Sensitivity and contamination

    Commentary

    C2.1.1
    Most nucleic acid tests for microorganisms are designed to detect the presence or absence of a particular organism. Usually, the target organism should not be present at all, although viral load testing seeks information about the levels of target organisms. Examples of detection tests are those for hepatitis C RNA in blood and for herpes simplex DNA in cerebrospinal fluid. Such tests are designed to maximise sensitivity and therefore have a greater risk of false positives caused by low-level contamination. Microorganisms are often present in samples in large numbers or are cultured at high concentrations in microbiology laboratories. The small size of microorganisms compared to eukaryotic cells creates greater potential for aerosol contamination. For these reasons, more stringent conditions are applied to microbiological testing in some circumstances.

    Supplemental testing

    Commentary

    C2.1.2
    Any single nucleic acid amplification test may be part of a testing algorithm that uses supplemental testing to improve specificity. A similar method is used in serological testing in which a sensitive but nonspecific screening assay is followed by supplemental tests (eg the Western blot for human immunodeficiency virus) to improve the specificity of the result. Currently, multiple tests are most commonly used in nucleic acid amplification testing for Neisseria gonorrhoeae, for which a single test is not specific enough to report a positive result, so that combinations of tests are used to yield a reliable result.

    Standards and controls

    Commentary

    C2.1.3
    Standards and controls are a routine part of the quality systems for all laboratory testing. However, these are not readily available for nucleic acid amplification testing for many organisms, especially uncommon pathogens. Under these circumstances, laboratories should seek or manufacture standards and controls that most closely mimic clinical samples. For example, a negative sample seeded with a synthetic target may be used to mimic a positive patient sample.

    Detection of human DNA

    Commentary

    C2.1.4
    Laboratories should be aware that when a nucleic acid amplification test is undertaken on human material, especially on samples such as tissues that have large amounts of human DNA, there may be nonspecific amplification of unrecognised human DNA sequences that are similar to the target organism sequences. Appropriate validation of the test should take into account this possibility.