Introducing New Laboratory-Developed Molecular Methods in the Clinical Microbiology Laboratories

Commentary The verification of laboratory-developed tests aims to characterize and compare the diagnostic accuracy of a new method to that of a reference one accepted by the laboratory community as the standard of care for a particular analyte or disease. The article entitled " Evaluation of a PCR assay to detect Enterococ-cus faecalis in blood and determine glycopeptides resistance genes: Van A and Van B " published in the current issue of the Iranian Journal of Medical Sciences (page 194-199), seeks to verify a laboratory-developed multiplex PCR assay. Over the past two decades, molecular methods such as polymerase chain reactions (PCR) have been in use in the areas of infectious diseases including diagnostic work-ups of bloodstream infections. 2 Clinical pathogens can be detected and identified earlier and more accurately by PCR methods. As reported, such methods with lower detection limit of three colony-forming units of bacteria/ml, could identify organisms missed by blood culture. 2 Moreover, direct detection of resistant organisms in clinical samples by PCR methods are already available in many clinical laboratories. 2 The enterococci are usually recognized as the second or third most frequent nosocomial pathogens, predominantly inhabit in the gastrointestinal tract, and act as opportunistic agents. Among the least 20 species isolated from human sources, E. faecalis and E. faecium are the most frequent ones accounting for at least 85% of the isolates. Enterococci, as already reported, have exhibited resistance to glycopeptide, vancomycin and teicoplanin, by seven types of respective genes. Van A and Van B are considered the most clinically relevant phenotypes and are usually associated with E. faecalis and E. faecium isolates. Other types of glycopeptide resistance encoded by the Van D, Van E, Van G, and Van L genes are also reported in E. faecalis. 3 In the above-mentioned study, the identifications of E. faecalis and the two glycopeptide resistance genotypes described for enterococci has been based on specific amplification of fragments intragenic to ddl E. faecalis gene and specific amplification of portions of the Van A and Van B genes, respectively. Three pairs of oligodeoxynucleotides, developed by Dutka-Malen and colleagues, 4 to prime the amplifications of these fragments, were used. They, 4 developed a PCR assay that allows simultaneous detection of four glycopeptide resistance genotypes (Van A, Van B, Van C-1, and Van C-2) and identification of the species level of clinically relevant enterococci (Enterococcus faecium, E. faecalis, E. gallinarum, and E. casseliflavus). …