Towards Multitarget Testing in Molecular Microbiology

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. 1. Introduction 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 particularly useful for specimens that may contain fastidious, slow-growing, or unculturable microorganisms or when patient treatment has already been initiated. In addition, identification based on genetic traits is more objective than the interpretation of conventional phenotypic characteristics. 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). All known subtypes or other known sequence variants (mutations, insertions, deletions, etc.) of the pathogen should be included in the specificity testing if feasible [1]. Once an appropriate target is selected, primers and probes can be designed. However, due to genome variability, single target testing might lead to false-negative results. Indeed, many variants exist today, but not all variants are known, and new variants emerge constantly according to the ever present Darwin’s evolution theory. 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