Ryhana Manji

Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak.

Abstract Background In response to the novel influenza A H1N1 outbreak in the NY City area, 6090 patient samples were submitted over a 5-week period for a total of 14,114 viral diagnostic tests, including rapid antigen, direct immunofluorescence (DFA), viral culture and PCR. Little was known about the performance of the assays for the detection of novel H1N1 in the background of seasonal H1N1, H3N2 and other circulating respiratory viruses. In addition, subtyping influenza A became critical for the identification of high risk and/or hospitalized patients with novel H1N1 infection and for monitoring the spread of the outbreak. Study design This study analyzed the performances of the BinaxNOW Influenza A&B test (BinaxNOW), the 3M Rapid Detection Flu A+B test (3MA+B), direct immunofluorescence, R-Mix culture and the Luminex xTAG Respiratory Virus Panel (RVP) for the detection of seasonal influenza, novel H1N1 and other respiratory viruses. RVP was also evaluated for its ability to differentiate seasonal H1N1, H3N2 and novel H1N1. Results The sensitivities, specificities, PPVs and NPVs for the detection of novel H1N1, determined by comparing all four-test methods, were: rapid antigen: 17.8%, 93.6%, 77.4%, 47.9%; DFA: 46.7%, 94.5%, 91.3%, 58.9%; R-Mix culture: 88.9%, 100%, 100%, 87.9%; RVP: 97.8%, 100%, 100%, 97.3%. The individual sensitivities of BinaxNOW and 3MA+B as compared to R-Mix culture for the detection of novel H1N1 were 9.6% and 40%, respectively. All unsubtypeable influenza A specimens identified by RVP and tested with the CDC novel H1N1 specific RT-PCR assay were confirmed to be novel H1N1. Conclusions Rapid antigen tests, DFA, R-Mix culture and the xTAG RVP test all detected the novel H1N1 strain, but with highly varied sensitivity. The RVP test provided the best diagnostic option as RVP demonstrated superior sensitivity for the detection of all influenza strains, including the novel H1N1, provided accurate influenza A subtyping and identified a significant number of additional respiratory pathogens.

Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Multicenter Study Evaluating the Vitek MS System for Identification of Medically Important Yeasts

ABSTRACT The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time.

Clinical performance of the 3M Rapid Detection Flu A+B Test compared to R-Mix culture, DFA and BinaxNOW Influenza A&B Test

BACKGROUND The rapid diagnosis of influenza allows for prompt patient management and the initiation of appropriate infection control measures to reduce spread in healthcare settings. OBJECTIVE To evaluate the clinical performance of the 3M Rapid Detection Flu A+B Test (3MA+B) as compared to R-Mix cell culture, direct immunofluorescence assay (DFA) and the BinaxNOW A&B Influenza Test (BinaxNOW). STUDY DESIGN Five hundred fresh respiratory samples, collected from patients aged 5 days to 99 years with respiratory symptoms, were tested by R-Mix culture, DFA, 3MA+B and BinaxNOW. Analytical sensitivity of 3MA+B was compared to BinaxNOW using replicates of serially diluted clinical samples positive for influenza A or B. RESULTS Sensitivity, specificity, PPV and NPV for the detection of influenza A and B, respectively, were for R-Mix (96.9%, 100%, 100%, 99.3%; 98.1%, 100%, 100%, 99.8%), DFA (80.4%, 99.2%, 96.1%, 95.3%; 74%, 100%, 100%, 97%), 3MA+B (70.1%, 99.8%, 98.6%, 93%; 86.5%, 98.7%, 88.2%, 98.4%) and BinaxNOW (46.4%, 100%, 100%, 88.6%; 34.6%, 100%, 100%, 93%). R-Mix, DFA and 3MA+B were significantly (P<or=0.0001) more sensitive than BinaxNOW for the detection of both influenza A and B. The analytical sensitivity of 3MA+B was greater than BinaxNOW. Excessive blood in samples may cause 3MA+B false positive influenza B results. CONCLUSIONS The 3MA+B provided superior results compared to BinaxNOW. The 3MA+B Reader eliminated user misinterpretation and provided quality control and result documentation. The improved sensitivity and easy of use makes 3MA+B an effective first line triage test for emergency departments, clinics and rapid response laboratories.

Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria

The VITEK MS v2.0 MALDI-TOF mass spectrometry systems performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level.

Clinical Evaluation of NucliSENS Magnetic Extraction and NucliSENS Analyte-Specific Reagents for Real-Time Detection of Human Metapneumovirus in Pediatric Respiratory Specimens

ABSTRACT In this study, we evaluated the NucliSENS miniMAG (MM) and easyMAG (EM) nucleic acid extraction platforms (bioMérieux, Durham, NC) in combination with the NucliSENS EasyQ basic kit and analyte-specific reagents (ASRs) (bioMérieux) for the detection of human metapneumovirus (hMPV) in respiratory samples. Total nucleic acids from pediatric clinical samples (n = 653) and an hMPV-specific inhibition control (h-IC) were coextracted using the MM and/or the EM. Nucleic acid sequence-based amplification and real-time molecular beacon detection of hMPV were performed using a NucliSENS EasyQ analyzer (bioMérieux). Positive results were confirmed using an in-house-validated reverse transcriptase PCR ASR-based assay. The inclusion of the h-IC monitored the entire process, including the efficiency of nucleic acid extraction, amplification, and detection. The percentages of samples with inhibited amplification of the h-IC after initial NA extraction by EM and MM were 1.88% and 3.17%, respectively. After reprocessing of a new aliquot, the final h-IC inhibition rates were 0% (EM) and 1.06% (MM). The limit of detection of the assay was between 2 (EM extraction) and 10 (MM extraction) RNA copies/reaction, and specificity was 100% when testing viral respiratory isolates and clinical samples. hMPV was detected in 5.6% of pediatric samples tested and was also detected in three coinfections with respiratory syncytial virus (RSV). hMPV was the second most frequently detected respiratory virus in children of 0 to 2 years of age, after RSV. In summary, NucliSENS extraction and ASRs provided a sensitive and specific method for the detection of hMPV in respiratory samples.

Assessment of Reproducibility of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Bacterial and Yeast Identification

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of clinical bacterial and yeast isolates. However, data describing the reproducibility of MALDI-TOF MS for microbial identification are scarce. In this study, we show that MALDI-TOF MS-based microbial identification is highly reproducible and can tolerate numerous variables, including differences in testing environments, instruments, operators, reagent lots, and sample positioning patterns. Finally, we reveal that samples of bacterial and yeast isolates prepared for MALDI-TOF MS identification can be repeatedly analyzed without compromising organism identification.

Clinical evaluation of NucliSENS magnetic extraction and NucliSENS analytical specific reagents for the real-time detection of respiratory syncytial virus (RSV) in paediatric respiratory specimens.

Aims: To evaluate the combination of NucliSENS magnetic extraction and NucliSENS analytical specific reagents (bioMérieux, Marcy L’Etoile, France) for the detection of respiratory syncytial virus (RSV) from a variety of respiratory samples. Methods: Nucleic acids (NA) from paediatric samples (n = 603) and an RSV-specific inhibition control (R-IC) were coextracted using the miniMAG and/or the easyMAG. Nucleic-acid-sequence-based amplification (NASBA) and molecular beacon detection of RSV and R-IC were performed using NucliSENS analyte-specific reagents (NRSVA) and the NucliSENS EasyQ Analyzer. NRSVA results were compared with R-Mix culture and direct fluorescent antibody detection (DFA). Results: The NRSVA analytical specificity was 100%, and the NRSVA limit of detection was 5–20 RNA copies/reaction. The prediscordant analysis, sensitivity, specificity, PPV and NPV were, respectively, for R-Mix (64.7%, 100%, 100%, 94.5%); DFA (98.8%, 99.0%, 94.4%, 99.8%); NRSVA (94.1%, 95%, 75.5%, 99%). After discordant analysis, sensitivity, specificity, PPV and NPV were, respectively, for R-Mix (56.7%, 100%, 100%, 92.3%); DFA (87.6%, 99.2%, 95.5%, 97.7%); NRSVA (93.8%, 97%, 85.9%, 99%). RSV was detected in 17.8% of the samples and in seven coinfections. Children with proven RSV infection, compared with children without a pathogen identified, had shorter median hospitalisation stays (2 days vs 3 days, p = 0.035), used fewer antibiotics (54% vs 69%) and had shorter durations of antibiotic therapy (6.2 days vs 9.3 days, p = 0.021), respectively. Conclusions: NRSVA is sensitive and specific for RSV detection in respiratory samples. The R-IC monitored the test process, including NA extraction, target amplification and detection. The rapid detection of respiratory pathogens can foster appropriate patient management.