BD MAX Research Articles

Routine detection of Clostridium difficile in Western Australia

Despite increasing infection rates, Clostridium difficile is not currently routinely tested for in all diarrhoeal faecal specimens in Australia. In July 2014, all diarrhoeal specimens submitted to a diagnostic laboratory in Western Australia were surveyed to determine the true prevalence of C. difficile. In total, 1010 diarrhoeal non-duplicate faecal specimens were received during the month. Testing for C. difficile was requested, or the criteria for a C. difficile investigation were met, for 678 specimens which were investigated by PCR for the tcdB gene using the BD MAX platform, followed by toxigenic culture on PCR-positive samples. The remaining 332 specimens, with either no C. difficile test request or the criteria for a C. difficile investigation were not met, were examined by toxigenic culture. All isolates were PCR ribotyped. C. difficile was the most commonly detected diarrhoeal pathogen among all specimens. The overall prevalence of C. difficile in all 1010 specimens was 6.4%; 7.2% in the routinely tested group, and 4.8% in the non-requested group. The proportion of non-requested positive detections among all cases was 24.6%. Community-onset infection was present in 50.8% of all cases. The median age of all CDI cases was 60.0 years and the age range in CDI patients in the routine group was 0.6–96.6 years (median 72.7 years), compared to 0.2–2.3 years (median 0.8 years) in the non-requested group. The most common ribotype (RT) found was RT 014/020 (34.1% in the routine group, 43.8% in the non-requested group), followed by RTs 002, 056, 005 and 018. While the routine testing group and the non-requested group differed markedly in age and patient classification, C. difficile was the most common cause of diarrhoea in hospitals and the community in Western Australia. The significance of finding C. difficile in the community paediatric population requires further study.

Evaluation of the BD Max Enteric Parasite Panel for Clinical Diagnostics.

We compared the performance of the BD Max enteric parasite panel to routine microscopy and an in-house PCR for the detection of Giardia intestinalis, Entamoeba histolytica, and Cryptosporidium spp. The enteric parasite panel showed good specificity for all targets and good sensitivity for E. histolytica and Cryptosporidium spp. Sensitivity for G. intestinalis with the BD Max enteric parasite panel was equivalent to that with microscopy.

O15.2 Combined detection of chlamydia, gonorrhoea and trichomonas using the bd maxtmct/gc/tv assay

Background Chlamydia trachomatis (CT) and Neisseria gonorrhoea (GC) are the two most common bacterial STIs. Screening for CT and/or GC, is recommended in many countries. The prevalence of Trichomonas vaginalis (TV) is also high and negative consequences of untreated infection may be serious. Inclusion of TV as part of a combination assay would facilitate screening of these STI. Here we evaluated the performance of the BD MAX CT/GC/TV assay compared to currently available assays for these STI. Methods Eight STD and Family Planning clinics enrolled participants for this study. Vaginal and endocervical swabs, and female and male urine specimens were obtained from 1854 women and 843 men. Female samples were used for evaluation of the BD MAX CT/GC/TV assays while male urine was used only for CT/GC evaluation. BD MAX CT/GC/TV results were compared to the Aptima Combo 2 ® CT/GC; BD ProbeTec TM CT/GC; BD Viper TM CT Q x /GC Q x ; TV microscopy and culture. Participants were classified as infected if at least one positive result from each of 2 comparator assays were obtained. Positive wet mount and/or culture results were used as evidence of TV infection for women only. Results Among women, 7.3%, 2.4% and 14.7% were infected with CT, GC and TV, respectively. Among men the rates were 22.0% and 14.6% for CT and GC. The BD MAX CT/GC/TV assay detected 92.2–99.2%, 94.9–95.1% and 92.9–96.1% of CT, GC and TV infections among women, depending on specimen type, and 96.6% and 99.1% among men. The specificity for all organisms and sample types was >98.5%. Discussion In this US multi-site study, the prevalence of TV infection was high, demonstrating the benefit of screening women for TV as well as GC and CT. The BD MAX CT/GC/TV assay performance allows combined testing for all 3 STI among women and CT/GC from male urine.­­ Financial disclosure This study was funded by BD Diagnostics.

P07.10 Evaluation of the new bd max gc real time pcr assay, analytically and as a supplementary test to the bd probetec gc qx amplified dna assay, for molecular detection ofneisseria gonorrhoeae

Introduction The BD ProbeTec GC Qx Amplified DNA assay (Becton, Dickinson and Company) is used on the BD Viper System to detect Neisseria gonorrhoeae . However, suboptimal specificity and cross-reaction with commensal Neisseria species have been described. Recently, the BD Max GC real time (rt) PCR assay was developed for the BD Max System (BD) as a supplementary test. Methods We evaluated the performance of the new BD Max GC rt PCR assay by examining clinical specimens positive in the BD ProbeTec GC Qx Amplified DNA assay during July–October 2014 among 23815 screening or clinical patients (14846 females and 8969 males) as well as samples spiked with isolates of gonococci (n = 189), non-gonococcal Neisseria species (n = 261) and other closely related bacteria (n = 10). Results Of 23815 patients tested with the BD ProbeTec GC Qx Amplified DNA assay, 85 (0.6%) females and 259 (2.9%) males were positive. Of these 344 positive specimens, 322 were tested with BD Max GC rt PCR assay. Sixty-nine (21%) of these samples were negative in BD Max GC rt PCR assay, a gonococcal dual target PCR and in the APTIMA Combo 2 (Hologic). These 69 specimens were obtained from pharynx (50% of all screening positive pharyngeal specimens), urine (33%), vagina (11.4%), rectum (4.3%), and cervix (1.4%). In the analytical evaluation of the BD Max GC rt PCR assay, all gonococcal isolates were positive and all but one ( N. cinerea ) of the non-gonococcal isolates (99.4%) were negative. The N. cinerea isolate also cross-reacted in the BD ProbeTec GC Qx Amplified DNA assay. Conclusion The BD ProbeTec GC Qx Amplified DNA assay had a suboptimal specificity for both urogenital and extragenital clinical specimens. The new BD Max GC rt PCR assay showed a high clinical and analytical sensitivity and specificity, and might also be used for initial detection of N. gonorrhoeae . Disclosure of interest statement We are grateful to BD Diagnostics for providing the BD Max GC real time PCR tests. The present work was funded by grants from the Orebro County Council Research Committee and the Foundation for Medical Research at Orebro University Hospital, Sweden.

Detection of EBV infection by real-time PCR method in immunosuppressive patients

s / Journal of Clinical Virology 70 (2015) S1–S126 S103 frozen for several weeks, the batch is validated only once without needing the inclusion of positive controls for each run. Conclusion: These preliminary results illustrate for the first time the ability of the BD MAX platform to detect RNA viruses by using TNA-3 ExK strips. Further validation including external quality panels and clinical specimens is currently ongoing for demonstrating the use of the CHIKV and DENV BD MAX assays for rapid molecular diagnosis of these 2 mosquito-borne infections. http://dx.doi.org/10.1016/j.jcv.2015.07.237

Rapid molecular detection of dengue and chikungunya viruses on the BD MAX platform

Rapid molecular detection of dengue and chikungunya viruses on the BD MAX platform

Active surveillance of Methicillin-resistant Staphylococcus aureus in an emergency department: Comparing BD MAX StaphSR kit with the routine MRSA identification method

Methods Prospective observational study performed at the ED. Nasal and throat swab specimens from all patients > 10 years who were admitted to the ED from 01.09.2013 to 30.11.2013, were examined. The swabs were, immediately after sampling, incubated in 6% NaCl broth for at least 16 hours and further cultivated on MRSA-chromeand Columbia-agar. 150 ml of the incubated broth was used as sample material for StaphSR.

Comparison of time-motion analysis of conventional stool culture and the BD MAX™ Enteric Bacterial Panel (EBP).

BackgroundConventional bacterial stool culture is one of the more time-consuming tests in a routine clinical microbiology laboratory. In addition, less than 5 % of stool cultures yield positive results. A molecular platform, the BD MAX™ System (BD Diagnostics, Sparks, MD) offers the potential for significantly more rapid results and less hands-on time. Time-motion analysis of the BD MAX Enteric Bacterial Panel (EBP) (BD Diagnostics, Quebec, Canada) on the BD MAX System was compared to conventional stool culture in the microbiology laboratory of a tertiary care pediatric hospital.MethodsThe process impact analysis of time-motion studies of conventional cultures were compared to those of EBP with 86 stool specimens. Sample flow, hands-on time, processing steps, and overall turnaround time were determined and analyzed. Data were obtained and analyzed from both standard operating procedures and direct observation. A regression analysis was performed to ensure consistency of measurements. Time and process measurements started when the specimens were logged into the accessioning area of the microbiology laboratory and were completed when actionable results were generated.ResultsWith conventional culture, negative culture results were available from 41:14:27 (hours:minutes:seconds) to 54:17:19; with EBP, positive and negative results were available from 2:28:40 to 3:33:39.ConclusionsThis study supports the suggestion that use of the EBP to detect commonly encountered stool pathogens can result in significant time savings and a shorter time-to-result for patients with acute bacterial diarrhea.

Evaluation of 3 automated real-time PCR (Xpert C. difficile assay, BD MAX Cdiff, and IMDx C. difficile for Abbott m2000 assay) for detecting Clostridium difficile toxin gene compared to toxigenic culture in stool specimens

We evaluated the performance of the 3 automated systems (Cepheid Xpert, BD MAX, and IMDx C. difficile for Abbott m2000) detecting Clostridium difficile toxin gene compared to toxigenic culture. Of the 254 stool specimens tested, 87 (48 slight, 35 moderate, and 4 heavy growth) were toxigenic culture positive. The overall sensitivities and specificities were 82.8% and 98.8% for Xpert, 81.6% and 95.8% for BD MAX, and 62.1% and 99.4% for IMDx, respectively. The specificity was significantly higher in IMDx than BD MAX (P=0.03). All stool samples underwent toxin A/B enzyme immunoassay testing, and of the 254 samples, only 29 samples were positive and 2 of them were toxigenic culture negative. Considering the rapidity and high specificity of the real-time PCR assays compared to the toxigenic culture, they can be used as the first test method for C. difficile infection/colonization.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.

Development of a real-time PCR Assay for identification of Coccidioides immitis by use of the BD Max system.

Rapid real-time PCR (RT-PCR) can be performed in a community hospital setting to identify Coccidioides species using the new Becton Dickinson molecular instrument BD Max. Following sample preparation, DNA extraction and PCR were performed on the BD Max using the BD Max extraction kit ExK-DNA-1 test strip and a master mix prepared by BioGX (Birmingham, AL). Sample preparation took 2 h, and testing on the BD Max took an additional 2 h. Method sensitivity and specificity were evaluated along with the limits of detection to confirm that this convenient method would provide medically useful information. Using serial dilutions, the lower limit of detection was determined to be 1 CFU/μl. Testing with this method was validated using samples from various body sites, including bronchial alveolar lavage (BAL) fluid; sputum and lung tissue samples; and pleural and spinal fluids. Safety protocols were established, and specimen preparation processes were developed for the various types of specimens. The range for the cycle threshold (CT) indicating adequate fluorescent signal to signify a positive result was established along with the acceptable range for the internal standard. Positive controls run with each batch were prepared by spiking a pooled BAL fluid specimen with a known dilution of Coccidioides immitis organism. Our experience with testing >330 patient samples shows that clinically relevant information can be available within 4 h using an RT-PCR method on the BD Max to identify Coccidioides spp., with sensitivity equivalent to culture.

Performance assessment of the BD MAX Cdiff assay in comparison to Xpert C. difficile assay in a setting with very low prevalence of toxigenic Clostridium difficile PCR ribotype 027

In a clinical setting with low prevalence of ‘epidemic’ PCR ribotype 027, the BD MAX Cdiff assay was found to be a suitable alternative to the Xpert C. difficile assay for the detection of toxigenic Clostridium difficile in samples which are reflex PCR tested after obtaining a discrepant immunoassay result. There was no significant difference between the sensitivities and specificities of both commercial molecular assays.

Comparison of the Verigene Clostridium difficile, Simplexa C. difficile Universal Direct, BD MAX Cdiff, and Xpert C. difficile assays for the detection of toxigenic C. difficile

We compared the Verigene Clostridium difficile test (Nanosphere, Northbrook, IL, USA), the Simplexa C. difficile Universal Direct (Focus Diagnostics, Cypress, CA, USA), the BD MAX Cdiff (Becton Dickinson, Franklin Lakes, NJ, USA), and the Xpert C. difficile (Cepheid, Sunnyvale, CA, USA) assays for the detection of toxigenic C. difficile. One hundred and ninety deidentified, remnant diarrheal specimens were included in this study. After resolution of discordant results by toxigenic culture, the Xpert C. difficile assay displayed the highest sensitivity (100%), with a specificity of 98.8%. The sensitivity and specificity were 95.2% and 99.4% and 87% and 100% for the Verigene CDF and Simplexa Universal Direct assays, respectively. Finally, the BD MAX assay showed a sensitivity of 87% and a specificity of 98.8%. Despite differences in the overall performance of these assays, these results support the routine use of these platforms for the detection of toxigenic C. difficile in the clinical laboratory.

Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform

Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform

Premarket Evaluations of the IMDx C. difficile for Abbott m 2000 Assay and the BD Max Cdiff Assay

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Detection of Carbapenemases by Real-Time PCR and Melt Curve Analysis on the BD Max System

We developed a multiplex SYBR green real-time PCR for the BD Max instrument (BD Diagnostics, Sparks, MD) to detect a panel of carbapenemases. The assay was evaluated with 152 consecutive isolates sent to the German National Reference Laboratory, and 65/65 of the carbapenemase-positive and 87/87 of the carbapenemase-negative strains were identified correctly.

Comparison of the BD MAX Enteric Bacterial Panel to Routine Culture Methods for Detection of Campylobacter, Enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella Isolates in Preserved Stool Specimens

We compared the BD MAX enteric bacterial panel (EBP) to culture for the detection of Salmonella, Shigella, Campylobacter, and Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157 in seeded stool samples. The EBP panel demonstrated superior sensitivity and reliably detected Salmonella, EHEC O157, Shigella, and Campylobacter at concentrations 1- to 2-log10 lower than those needed for culture detection.

Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform

Pneumocystis jirovecii is an opportunistic pathogen in immunocompromised and AIDS patients. Detection by quantitative PCR is faster and more sensitive than microscopic diagnosis yet requires specific infrastructure. We adapted a real-time PCR amplifying the major surface glycoprotein (MSG) target from Pneumocystis jirovecii for use on the new BD MAX platform. The assay allowed fully automated DNA extraction and multiplex real-time PCR. The BD MAX assay was evaluated against manual DNA extraction and conventional real-time PCR. The BD MAX was used in the research mode running a multiplex PCR (MSG, internal control, and sample process control). The assay had a detection limit of 10 copies of an MSG-encoding plasmid per PCR that equated to 500 copies/ml in respiratory specimens. We observed accurate quantification of MSG targets over a 7- to 8-log range. Prealiquoting and sealing of the complete PCR reagents in conical tubes allowed easy and convenient handling of the BD MAX PCR. In a retrospective analysis of 54 positive samples, the BD MAX assay showed good quantitative correlation with the reference PCR method (R(2) = 0.82). Cross-contamination was not observed. Prospectively, 278 respiratory samples were analyzed by both molecular assays. The positivity rate overall was 18.3%. The BD MAX assay identified 46 positive samples, compared to 40 by the reference PCR. The BD MAX assay required liquefaction of highly viscous samples with dithiothreitol as the only manual step, thus offering advantages for timely availability of molecular-based detection assays.

Evaluation of the new CE-IVD marked BD MAX Cdiff Assay for the detection of toxigenic Clostridium difficile harboring the tcdB gene from clinical stool samples

The evaluation of the fully automated BD MAX Cdiff assay on a panel of 100 stool samples characterized by the Xpert C. difficile assay reported a high concordance between the two molecular assays (kappa coefficient of 0.96), which makes this new assay suitable for routine detection of toxigenic Clostridium difficile.

Evaluation of the BD MAX Cdiff assay for the detection of the toxin B gene of Clostridium difficile out of faecal specimens

In this evaluation study, 180 faecal specimens were tested in parallel with the new BD MAX™ Cdiff assay, the Premier™ Toxins A&B, and toxigenic culture as reference method. BD MAX™ Cdiff presented a high sensitivity (96.0%) and specificity (99.4%) with a negative predictive value of 99.4%.