A clinical laboratory performs real-time PCR for detection of MRSA in the area where specimens are plated. The supervisor discovers that the last 50 specimens tested were positive. However, companion cultures were positive for only 15 of the specimens. What is the most likely reason for this discrepancy?
Correct Answer: The PCR workstation has been contaminated with MRSA DNA
Description: In general, real-time PCR is more sensitive than culture (a), due to the ability of the assay to detect very few copies of bacterial DNA present in the specimen. Recently, many clinical laboratories have begun using real-time PCR for the mecA gene or the SCC mec cassette that carries mecA to detect the presence of MRSA in nasal specimens collected from patients upon admission to the hospital. Numerous studies have shown that the sensitivity of real-time PCR is slightly higher than that of optimally performed chromogenic culture for MRSA. However, even at 85% sensitivity, if the 50 positive samples were true positives, one would expect to have recovered 42 to 43 isolates in the companion cultures rather than 15. Real-time PCR was thought to be more specific than culture (b), but the appearance of increasing numbers of mecA gene variations has led to false-negative results due to primer mismatch and failure to amplify the gene even when present. Detecting SCC mec was thought to be the solution to this problem, only to come under fire because the same SCC mec cassette found in community-acquired MRSA is present in methicillin-resistant S. epidermidis, leading to misclassification of patients with MRSE. This finding has led to the development of multiplex PCR tests that detect genes specific for S. aureus in addition to the mecA gene. Unfortunately, many laboratories have implemented nucleic acid testing without having access to dedicated space for sample preparation and the PCR instrument, as described in the vignette. This frequently leads to contamination of the PCR workstation with MRSA DNA (d), which is then amplified in the reaction producing a false-positive result. Failure of the thermocycler, the PCR instrument, because it needs to be calibrated (c), is a possibility, but should have been addressed through required instrument maintenance and running of appropriate controls.
Category:
Microbiology
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