Development of a Real-Time PCR Protocol Requiring Minimal Handling for Detection of Vancomycin-Resistant Enterococci with the Fully Automated BD Max System

Abstract

. We evaluated two protocols: one using a liquid master mix and the other employing commercially ordered dry-down reagents. The BD Max VRE PCR was evaluated in two rounds with 86 and 61 rectal elution swab (eSwab) samples, and the results were compared to the culture results. The sensitivities of the different PCR formats were 84 to 100% for vanA and 83.7 to 100% for vanB; specificities were 96.8 to 100% for vanA and 81.8 to 97% for vanB. The use of dry-down reagents and the ExK DNA-2 kit for extraction showed that the samples were less inhibited (3.3%) than they were by the use of the liquid master mix (14.8%). Adoption of a cutoff threshold cycle of 35 for discrimination of vanB-positive samples allowed an increase of specificity to 87.9%. The performance of the BD Max VRE assay equaled that of the BD GeneOhm VanR assay, which was run in parallel. The use of dry-down reagents simplifies the assay and omits any need to handle liquid PCR reagents.

Adaptation and validation of a quantitative vanA/vanB DNA screening assay on a high-throughput PCR system

“…The vanA assay is highly specific (100%), while the vanB test resulted in an increased proportion of false positives. The vanB resistance determinant is known to be present in gram-positive anaerobes, as well as in enterococci, which likely explains the comparably lower specificity (90.7%) and positive predictive value (PPV) (34.6%) of the vanB PCR (cutoff ct35) in this study and in other published assays for VRE screening 22 , 23 , 28 , 29 . The use of enterococcus enrichment broth, which facilitates the outgrowth of enterococci over anaerobes, improved the PPV of the vanB screening to 87.2%, which is in line with results from other studies 26 .…”

“…Patient-related risk factors for infection and the dynamics of hospital spread are still a subject of research and it has been hypothesized that the respective enteric bacterial load of VRE may play a role in both infection and transmission 19 . Currently, vanA and vanB can be detected by qPCR using manual tests on a Light Cycler 20 , 21 or automated tests on the BD MAX 22 or Xpert Xpress systems 23 . Fully-automated PCR systems offer several advantages such as reproducibility, a lower risk of contamination and less hands-on time compared to manual PCR workflows.…”

Shorter Incubation Times for Detecting Multi-drug Resistant Bacteria in Patient Samples: Defining Early Imaging Time Points Using Growth Kinetics and Total Laboratory Automation

“…As early as 2001, Rubinovitch and Pittet [1] stated that successful programs for reducing hospital transmission of MRSA are based on early identification of the MRSA reservoir and prompt implementation of contact precautions. Various PCR techniques have been developed to accelerate reporting [23], with the drawbacks of high cost, the need for highly qualified personnel, and limited detection of a very narrow spectrum of resistance mechanisms. In addition, the issue of false-positive results has to be seriously considered [4].…”

Increasing Cytomegalovirus Detection Rate from Respiratory Tract Specimens by a New Laboratory-Developed Automated Molecular Diagnostic Test

“…A fully integrated, automated platform for nucleic acid extraction and real-time PCR using the Becton Dickinson (BD) MAX system enables many commercial IVD assays for infectious agents [ 20 , 21 , 22 , 23 , 24 , 25 , 26 ], with the possibility of creating user-defined protocols using open-system reagents to meet emerging diagnostic demands and address regional healthcare needs. The evaluation of laboratory developed assays using the BD MAX system for the detection of numerous microbes has recently been published, however, none of them pertain to CMV [ 27 , 28 , 29 , 30 , 31 , 32 ]. Herein, we designed a new QMT assay applied on the BD MAX system for the detection of CMV DNA in various clinical specimens, especially different respiratory tract specimens.…”