Suzane Silbert

Rapid detection of carbapenemase genes by multiplex real-time PCR

OBJECTIVESnTo develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48).nnnMETHODSnA total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA).nnnRESULTSnEach one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified.nnnCONCLUSIONSnThe new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

Evaluation of BD Max StaphSR and BD Max MRSAXT Assays Using ESwab-Collected Specimens

ABSTRACT The BD Max MRSAXT and the BD Max StaphSR assays were validated for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in ESwab samples. In addition, the BD Max StaphSR assay was evaluated for its ability to detect and differentiate S. aureus and MRSA in the same sample. A total of 255 ESwab samples collected from the anterior nares of patients were tested by each of three BD Max assays, including the BD Max MRSA first-generation assay. The results were compared to those of direct and enrichment culture. Additionally, a challenge panel comprising 14 control strains was evaluated to determine the ability of these assays to correctly identify MRSA and also appropriately differentiate S. aureus by the BD Max StaphSR assay. Out of 255 clinical samples tested, 161 were negative and 30 were positive for MRSA, and 45 were positive for S. aureus (by BD Max StaphSR) and negative for MRSA by all three PCR assays and culture. Nineteen samples had discrepant results; all of them were retested by additional laboratory testing. All strains from the challenge panel were correctly identified or excluded by the BD Max MRSAXT and BD Max StaphSR assays. The results showed that the BD Max StaphSR and the BD MRSAXT assays have excellent sensitivity (94.3%) and specificity (97.7%) for detecting MRSA. The BD Max StaphSR assay demonstrated excellent sensitivity (96.4%) and specificity (93.6%) for detecting S. aureus.

Comparison of ESwab with Traditional Swabs for Detection of Methicillin-Resistant Staphylococcus aureus Using Two Different Walk-Away Commercial Real-Time PCR Methods

ABSTRACT The ESwab system (Copan Diagnostics) was evaluated as a nasopharyngeal specimen collection device to be used for methicillin-resistant Staphylococcus aureus (MRSA) detection by the GeneXpert and BD Max MRSA assays. Different MRSA strains and dilutions of each strain were tested in triplicate. ESwabs proved to be a suitable collection system for the two assays tested.

Laboratory evaluation of the BD MAX MRSA assay.

ABSTRACT A comparison between the BD MAX MRSA and Xpert MRSA assays was performed using 239 nares samples. A 97.9% overall agreement between the two molecular assays was observed. The BD MAX MRSA assay proved to be a reliable alternative for a highly automated system to detect methicillin-resistant Staphylococcus aureus (MRSA) in patient nares samples.

Detection of Group B Streptococcus Directly from Collected ESwab Samples by Use of the BD Max GBS Assay

ABSTRACT Group B Streptococcus detection directly from Copan ESwab collected samples, using the BD Max GBS assay, was evaluated on receipt in the laboratory and after 24 h at room temperature. Results were compared to those using Lim broth enrichment PCR and culture. No significant difference was observed between 24 h ESwab and Lim broth PCRs.

Evaluation of the New FecalSwab System for Maintaining Stability of Stool Samples Submitted for Molecular Tests

Gastrointestinal disease accounts for significant morbidity and mortality worldwide and may be caused by a variety of agents, including bacterial, viral, and parasitic pathogens (1, 2, 3). Rapid molecular multiplex testing has recently been introduced into enteric diagnostics and is revolutionizing patient diagnosis and treatment for diarrheal diseases (4, 5, 6, 7). Maintaining organism viability (especially the viability of bacteria) is still important for further antimicrobial susceptibility testing, when pertinent. However, inability to obtain a stool specimen at the time of the patient visit can delay the diagnostic process and contribute to inappropriate treatment (1, 2, 3, 4). The new FecalSwab system (Copan Diagnostics, Murrieta, CA) is a convenient system for collecting rectal swab samples or for transporting fecal specimens in small instrument-ready tubes, making it easier to transport the specimen to the laboratory (8). The FecalSwab comes in a tube with 2 ml of modified Cary-Blair medium and a flocked swab. While the FecalSwab is FDA cleared for transport and culture of gastrointestinal (GI) pathogens, it is not FDA cleared for use with any molecular GI assays (9, 10). The objective of this study was to evaluate the FecalSwab for the simultaneous qualitative detection and identification of 22 GI pathogens, using a FilmArray GI Panel (Biofire Diagnostics, Salt Lake City, UT) and the FilmArray system. A total of 103 clinical stool samples were evaluated in this study. Only one sample was included from each patient. Samples were tested by the use of a FilmArray GI Panel and the FilmArray System using two different protocols: the standard of care (SC) and the FecalSwab (FS) protocols. The SC entailed suspending fresh stool sample in Cary-Blair medium, as recommended by the FilmArray GI Panel manufacturer’s protocol. Approximately 1 g of fresh clinical stool samples received in the laboratory was transferred to a screw-cap tube filled with 15 ml of Cary-Blair liquid medium (Remel, Lenexa, KS). For the FS protocol, a residual fresh stool sample was transferred to the Cary-Blair media using the provided flocked swab in accordance with the manufacturer’s recommendations (11). Briefly, a small amount of fresh stool was collected by insertion of the tip of the flocked swab into the stool sample and rotation of the swab. The swab was carefully transferred into the FecalSwab tube to ensure that the swab did not exceed the filling limit indicated on the label. The vial was shaken until the sample appeared homogeneous. Fresh stool samples were transferred at the same time to the 15-ml Cary-Blair tube (SC) and to the FecalSwab Cary-Blair tube (FS). Testing on the FilmArray platform was performed according to the manufacturer’s instructions (12) using 200 l of either Cary-Blair stool samples (SC procedure) or FecalSwab transport medium (FS procedure). Results determined for the SC and FS from the same sample were compared. Additionally, a preservation/stability analysis was performed to assess the capability of the two transport media to preserve the nucleic acid in the sample. For this analysis, FS and SC stools from the first 25 positive samples were retested by the Accepted manuscript posted online 15 March 2017 Citation Silbert S, Gostnell A, Kubasek C, Widen R. 2017. Evaluation of the new FecalSwab system for maintaining stability of stool samples submitted for molecular tests. J Clin Microbiol 55:1588 –1590. https://doi.org/10 .1128/JCM.00273-17. Editor Peter Gilligan, UNC Health Care System Copyright

Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System

ABSTRACT A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

Evaluation of a rapid immunochromatographic test for detection of distinct variants of Klebsiella pneumoniae carbapenemase (KPC) in Enterobacteriaceae

The rapid detection of KPC-producing Enterobacteriaceae by microbiology laboratories has been required for infectious control programs. Herein we evaluated the performance of a novel immunochromatographic test for detecting KPC-2-, KPC-3-, KPC-4-, KPC-6-, KPC-7-, KPC-8-, and KPC-11-producing isolates and the influence of different growth media on the test performance.

Differentiation of Klebsiella pneumoniae carbapenemase (KPC) variants by pyrosequencing.

A fast and reliable protocol using the pyrosequencing technique was developed to identify 11 different types of the KPC enzyme. A total of 65 blaKPC positive bacterial isolates were tested and characterized. In the end, the pyrosequencing proved to be a powerful tool for epidemiological studies of KPC producer isolates.

Rapid detection of four non-fermenting Gram-negative bacteria directly from cystic fibrosis patient's respiratory samples on the BD MAX™ system

The aim of this study was to develop a multiplex PCR test to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from CF patients respiratory samples using the open mode of the BD MAX™ System. A total of 402 CF respiratory samples were evaluated by culture and PCR. Specific sets of primers and probes for each target were designed in-house. Out of 402 samples tested, 196 were identified as negative and 206 as positive by culture for AX, PSA, BC and SM. Among culture positive samples, PCR detected 21/27 AX, 4/5 BC, 138/140 PSA and 29/34 SM. In addition, PCR assay identified 35 samples as positive that were initially negative by culture for those 4 targets. The CF BDM test proved to be an excellent tool to detect AX, BC, PSA and SM by real-time PCR on an automated platform.