Abstract
Advantages of PCR assays over more conventional culture-based diagnostics include significantly higher sensitivities and shorter turnaround times. They are particularly useful when patient treatment has already been initiated or for specimens that may contain microorganisms that are slow-growing, difficult to culture, or for which culture methods do not exist. However, due to genome variability, single target testing might lead to false-negative results. This paper focuses on examples from our own experiences and the literature to provide insight into the limitations of single target testing in molecular biology. Lessons learned from these experiences include the careful design of diagnostic assays, preferably multitargeted, the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the importance of continuous attentiveness by investigators when confronted with inconsistent results. In conclusion, multitargeted testing in microbiological molecular assays should be a rule.
Highlights
The introduction of molecular methods has had a positive impact in many areas of diagnostic microbiology
Lessons learned from these experiences include the careful design of diagnostic assays, preferably multitargeted, the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the importance of continuous attentiveness by investigators when confronted with inconsistent results
We reported a case of vertebral spondylodiscitis caused by Mycobacterium bovis, where diagnosis was complicated because of the lack of insertion sequence 6110 (IS6110) [2]
Summary
The introduction of molecular methods has had a positive impact in many areas of diagnostic microbiology These tests have been proven to be often more sensitive and specific than classical testing, and they are useful for specimens that may contain fastidious, slow-growing, or unculturable microorganisms or when patient treatment has already been initiated. The development of a commercial or an in-house molecular assay begins with a review of the current literature. This provides information concerning the choices of target genes used in previous studies, potential specificity or sensitivity problems, and additional information of clinical importance (e.g., cutoff values). This paper focuses on examples from our own experiences and the literature to provide insight into how single target testing might lead to false-negative results and the important lessons to take into account in the future
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