J Osiecki

Multicenter Evaluation of the LightCycler MRSA Advanced Test, the Xpert MRSA Assay, and MRSASelect Directly Plated Culture with Simulated Workflow Comparison for the Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs

Rapid detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) followed by appropriate infection control procedures reduces MRSA infection and transmission. We compared the performance and workflow of two Food and Drug Administration-approved nucleic acid amplification assays, the LightCycler MRSA Advanced Test and the Xpert MRSA test, with those of directly plated culture (MRSASelect) using 1202 nasal swabs collected at three U.S. sites. The sensitivity of the LightCycler test (95.2%; 95% CI, 89.1% to 98.4%) and Xpert assay (99%; 95% CI, 94.8% to 100%) did not differ compared with that of culture; the specificity of the two assays was identical (95.5%; 95% CI, 94.1% to 96.7%) compared with culture. However, sequencing performed on 71 samples with discordant results among the three methods confirmed the presence of MRSA in 40% of samples that were positive by both molecular methods but negative by culture. Workflow analysis from all sites including batch runs revealed average hands-on sample preparation times of 1.40, 2.35, and 1.44 minutes per sample for the LightCycler, Xpert, and MRSASelect methods, respectively. Discrete event simulation analysis of workflow efficiencies revealed that the LightCycler test used less hands-on time for the assay when greater than eight batched samples were run. The high sensitivity and specificity, low hands-on time, and efficiency gains using batching capabilities make the LightCycler test suitable for rapid batch screening of MRSA colonization.

Evaluation of the cobas Cdiff Test for Detection of Toxigenic Clostridium difficile in Stool Samples

ABSTRACT Nucleic acid amplification tests (NAATs) are reliable tools for the detection of toxigenic Clostridium difficile from unformed (liquid or soft) stool samples. The objective of this study was to evaluate performance of the cobas Cdiff test on the cobas 4800 system using prospectively collected stool specimens from patients suspected of having C. difficile infection (CDI). The performance of the cobas Cdiff test was compared to the results of combined direct and broth-enriched toxigenic culture methods in a large, multicenter clinical trial. Additional discrepancy analysis was performed by using the Xpert C. difficile Epi test. Sample storage was evaluated by using contrived and fresh samples before and after storage at −20°C. Testing was performed on samples from 683 subjects (306 males and 377 females); 113 (16.5%) of 683 subjects were positive for toxigenic C. difficile by direct toxigenic culture, and 141 of 682 subjects were positive by using the combined direct and enriched toxigenic culture method (reference method), for a prevalence rate of 20.7%. The sensitivity and specificity of the cobas Cdiff test compared to the combined direct and enriched culture method were 92.9% (131/141; 95% confidence interval [CI], 87.4% to 96.1%) and 98.7% (534/541; 95% CI, 97.4% to 99.4%), respectively. Discrepancy analysis using results for retested samples from a second NAAT (Xpert C. difficile/Epi test; Cepheid, Sunnyvale, CA) found no false-negative and 4 false-positive cobas Cdiff test results. There was no difference in positive and negative results in comparisons of fresh and stored samples. These results support the use of the cobas Cdiff test as a robust aid in the diagnosis of CDI.

Performance of the cobas MRSA/SA Test for Simultaneous Detection of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus From Nasal Swabs

Objectives Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) infections are continuing problems. Rapidly determining the MRSA colonization status of a patient facilitates practice to reduce spread of MRSA clinical disease. Sensitive detection of all SA prior to surgery, followed by decolonization, can significantly reduce postoperative infection from this pathogen. Our goal was to validate a new automated assay for this testing. Methods We compared performance of the cobas MRSA/SA Test on the cobas 4800 System to direct and enriched chromogenic culture using nasal swabs collected from patients at six United States sites. Results Compared to direct and enriched culture, the sensitivity for MRSA and SA was 93.1% and 93.9%, and the specificity was 97.5% and 94.2%, respectively. After discrepancy analysis, the sensitivity for MRSA and SA was 97.1% and 98.6%, and the specificity was 98.3% and 95.5%, respectively. Compared to direct culture, sensitivity for detecting any SA was 99.6%. Conclusions The cobas MRSA/SA Test is an effective tool to simultaneously perform surveillance testing for nasal colonization of both MRSA and MSSA.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeaewith the cobas CT/NG v2.0 test: performance compared with the BD ProbeTec CT Qx and GC Qx amplified DNA and Aptima AC2 assays

Objectives Infections due to Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most common bacterial sexually transmitted infections worldwide, most of which are asymptomatic. Detection of infection using a variety of specimen types in symptomatic and asymptomatic subjects is important to effectively combat CT/NG infections. The performance of the cobas CT/NG v2.0 test was assessed for urogenital swabs, urine and cervical cytology samples collected in PreservCyt Solution from 5266 symptomatic and asymptomatic women (including 202 who were pregnant), and urine from 738 men. Methods Sensitivity and specificity were estimated compared with a patient infected status determined using two US Food and Drug Administration–cleared nucleic acid amplification tests. Results Among 6004 participants, 487 CT (8.1%) and 159 NG (2.6%) infections were identified. Sensitivity estimates for CT for women ranged from 91.2% to 97.6% depending on specimen type, and the estimate for male urine specimens was 98.4%. Specificity for CT ranged from 99.2% to 99.7%. Sensitivity estimates for NG ranged from 95.6% to 100.0% for women, and the estimate for men was 100.0%. Specificity for NG ranged from 99.3% to 100.0%. Conclusions The cobas CT/NG v2.0 test performs well using urogenital swabs, urine and cervical samples collected in PreservCyt solution.

P2.054 Optimal Processing of Chlamydia Trachomatis Simulated Samples from Proficiency Testing Panels by Dilution with Cobas© PCR Media

Background Proficiency materials are designed to resemble true clinical samples, yet challenges exist in procuring sufficient quantity of patient material. Simulated samples are often provided for this testing. Matrix effect with simulated samples can confound molecular assessment, having negative consequences for the laboratory through failed proficiency testing. This study was conducted to evaluate simulated urine samples provided for proficiency testing which generate invalid results with the cobas© CT/NG test. Methods Simulated urine proficiency panels were acquired from a commercial proficiency testing provider. Panels were evaluated in triplicate by routine procedure at neat concentrations and processed with cobas© PCR media at the following dilutions 1:1, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, and 1:500. The samples were held at room temperature (1 complete set) for 1 hour prior to loading on the cobas© 4800 system, while the second complete set of samples were processed 24 hours later. Samples were tested using two different cobas© 4800 workflows (400ul vs. 850ul of sample). Internal control and target Ct values were assessed for each sample to determine success of amplification. Results Invalid results due to internal control failures were observed at neat concentrations of simulated urine samples. Incubation of samples for 1 hour or 24 hours in cobas© PCR media, showed no significant difference between target and IC Ct values indicating incubation period in cobas© PCR media does not impact performance. Simulated Urine Sample dilution of 1:5 in cobas© PCR media using the 400ul sample input volume produced similar IC Ct values (mean Ct = 35.5) to cobas© PCR media tested alone (mean Ct = 36), and produced a robust target signal (mean Ct = 22). Conclusions Proficiency testing materials may require optimization for use on commercially available systems. Optimal processing of simulated urine specimens can be achieved by dilution in cobas© PCR media.

P3.009 Prevalence of Chlamydia Trachomatis and Neisseria Gonorrhoea Infection in Pregnant Women Enrolled in a Large Multicenter Clinical Trial

Background Pregnant women infected with sexually transmitted diseases are at higher risk for miscarriage, pre-term delivery, low birth weight, and morbidity in the neonate associated with transmission of pathogenic agents. Treatment guidelines recommend screening pregnant women for Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG) on the first prenatal visit. This study was performed to determine the frequency of CT and NG infection observed in pregnant women enrolled in a large clinical trial study population. Methods This multicenter retrospective cohort analysis was performed with data collected during the VENUS clinical trial, a study characterising the clinical performance of the cobas® CT/NG Test on the cobas® 4800 system. Two FDA-cleared nucleic acid amplification tests (NAATs) were used as comparator assays. Obstetrics-gynaecology practises, family planning clinics, and STD clinics from diverse settings in the United States served as specimen collection sites. Patient infection status (PIS) was defined as positive when results from NAATs with different target regions generated positive results with collected samples. Results Of 5,269 enrolled participants, 281 of 4315 eligible women (6.5%) were found to be positive for CT infection and 69 of 4314 (1.6%) were positive for NG according to PIS outcomes. Alternatively, 16 of 178 eligible pregnant women (9.0%) were positive for CT, where 2 of 178 pregnant women (1.1%) were considered positive for NG by PIS. Conclusion Screening of pregnant women for CT and NG with the cobas® CT/NG Test and two additional NAATs during the VENUS clinical trial revealed the prevalence of CT and NG infections are comparable to rates observed in the general female population.

P2.036 Detection of Herpes Simplex Viruses 1 and 2 from Clinical Samples with a Fully-Automated PCR Test on the Cobas ® 4800 System

Background Identification of genital herpes can have important implications for clinical management of HIV infected patients, immunosuppressed individuals, pregnant women, and individuals with HSV seronegative partners. This study was performed to establish preliminary performance characteristics for the newly developed cobas® HSV-1/2 Test by evaluating analytical sensitivity and specificity, specimen stability, and clinical performance compared with the BD ProbeTec™ HSV-1/2 Test. Methods Analytical sensitivity was determined using viral culture spiked into a contrived background matrix at predetermined concentrations. Nine levels of viral target were evaluated using the prototype cobas® HSV-1/2 Test. These viral culture panels were also used to assess analytical sensitivity compared to the BD ProbeTec™ HSV-1/2 Test. Preliminary exclusivity of the cobas® HSV-1/2 Test was evaluated with other herpes family viruses (n = 7) and a collection of microorganisms that might be found in lesion swab specimens (n = 31). We also evaluated clinical lesion swab specimens (collected in UVT media for the BD Test and MSwab Media for the cobas® Test). Transport and storage stability of anogenital lesion swab samples collected in MSwab media was assessed by testing specimens stored at RT, 2–8C and –20C. Results The cobas® HSV-1/2 test displayed excellent analytical sensitivity of 150 vp/mL (HSV 1) and 100 vp/mL (HSV-2). When compared to the BD ProbeTec™ HSV-1/2 Test, superior sensitivity was observed for both HSV-1 and HSV-2 with the cobas® HSV-1/2 Test. Exclusivity studies showed no cross reactivity. The cobas® HSV-1/2 Test showed excellent performance with lesion swab specimens, observing a sensitivity and specificity of 100% and 100% for HSV-1 and 100% and 94% for HSV-2, respectively. Preliminary specimen stability studies for routine laboratory workflow indicate favourable performance. Conclusion The cobas® HSV-1/2 test, run on the fully automated cobas® 4800 system, exhibited excellent preliminary performance characteristics, suitable for identifying low concentration HSV-1 and HSV-2 from anogenital lesions.