In-House Nucleic Acid Amplification Assays in Research: How Much Quality Control Is Needed before One Can Rely upon the Results?

Abstract

3Over the last 20 years, nucleic acid amplification tests (NAATs) have become a major tool for detection of microorganisms, for diagnostic testing, and for research purposes in the field of infectious diseases. NAATs offer significant sensitivity and speed compared to culture and do not require viable organisms. However, validated, commercially available, U.S. Food and Drug Administration-cleared assays exist for the following microorganisms: Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrhoeae, methicillin-resistant Staphylococcus aureus, group B streptococcus, Legionella pneumophila, human immunodeficiency virus, hepatitis B virus, and hepatitis C virus. Some of these tests are for very limited indications, for example, the methicillin-resistant Staphylococcus aureus assay is intended only for use with nasopharyngeal swabs as an infection control tool.There are also a number of so-called analyte-specific reagents commercially available for clinically relevant pathogens and pathogenicity factors like herpes simplex virus, EpsteinBarr virus, cytomegalovirus, Streptococcus pyogenes, and Bordetella pertussis and the genes for vanA/vanB and mecA, respectively. Next to these relatively closed and standardized kit concepts, the use of NAATs for research purposes has expanded dramatically. These assays range from those that are well validated to not validated at all, yet these assays are frequently used and cited in the literature. A review of the current literature on the association of a particular microorganism and a particular disease frequently reveals inconsistent results, even apparently when the same methods are used.Although NAATs offer the promise of exquisite sensitivity, theoretically allowing for detection of a single organism in a clinical sample, both false-negative and -positive results can and do occur. There can be problems with sensitivity, specificity, and contamination, which can be secondary to a very large number of technical issues, as listed in Tables 1 to 6. Some of the more common problems in context with NAAT-based studies of microorganisms and disease associations are described below. FALSE-POSITIVE RESULTS DUE TO CONTAMINATIONThe possible association between human herpesvirus 8 (HHV-8) and a direct role in Kaposi's sarcoma pathogenesis is very important. The presence of this potentially oncogenic virus in semen donated for use in artificial insemination would be critically important to know. However, the prevalences of HHV-8 DNA in prostate and semen as measured by NAATs range from 0 to over 90% (32). A recent study (32) assessed interlaboratory sensitivity and reproducibility in the analysis of blinded, identical experimental panels, each consisting of 48 samples composed of semen specimens from artificial insemination donors, human immunodeficiency virus-infected patients, and positive and negative controls. As no commercial, validated assay is available, each laboratory followed its standard procedures for HHV-8 PCR; thus, procedures varied from laboratory to labora...

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