Comparative evaluation of the Thermo fisher TaqPath™ COVID-19 combo kit with the Cepheid Xpert® Xpress SARS-CoV-2 assay for detecting SARS-CoV-2 in nasopharyngeal specimens.

PURPOSE: With over 50 SARS-CoV-2 gene amplification assays that have been EUA cleared with minimal experimental validation performed, it is likely that not all of these assays are comparable in their ability to detect SARS-CoV-2 in clinical specimens. Thermo Fisher Scientific is a relatively new company in the molecular diagnostics field and the purpose of this study was to compare the performance of the Thermo Fisher TaqPath™ Combo Kit with an established test, the Cepheid Xpert® Xpress SARS-CoV-2 assay, for its ability to detect SARS-CoV-2 in nasopharyngeal specimens. METHODS: A total of 300 randomly selected nasopharyngeal specimens were evaluated and tested by the TaqPath and GeneXpert assays. Discordant test specimens were arbitrated by performing an alternative PCR assay and Sanger sequencing. RESULTS: The TaqPath assay had a 96.7 and 99.6% positive and negative agreement respectively when compared to the Xpert Xpress test. However, after test arbitration, the three discordant specimens were arbitrated in favor of the TaqPath assay producing a positive and negative percent agreement of 100% for the TaqPath Combo Kit while the Xpress SARS-CoV-2 assay had a positive and negative percent agreement of 98.3 and 99.2% respectively. CONCLUSIONS: The TaqPath Combo Kit is a high complexity assay that compares favorably with the Xpert Xpress test and can be reliably used for the detection of SARS-CoV-2 in nasopharyngeal specimens.

Performance comparison of the cobas Liat and Cepheid GeneXpert systems for Clostridium difficile detection.

Clostridium difficile infection (CDI) is a high burden and significant cause of healthcare-acquired infectious diarrhea in the United States (US). Timely and accurate diagnosis of CDI enables the rapid initiation of antibiotic therapy and infection control policies to minimize disease transmission. Polymerase chain reaction (PCR) assays have become a preferred modality for diagnosing CDI in the US. The cobas Liat Cdiff PCR test is a novel assay that can be performed on-demand for hospital-based testing with a rapid 20-minute turnaround time from specimen collection to result reporting. We compared the clinical performance of the cobas Liat Cdiff test to the previously introduced Xpert C. difficile/Epi test; both tests are FDA-cleared PCR assays that detect the toxin B (tcdB) gene of C. difficile. Prospectively collected and remnant stool specimens from 310 patients with suspected CDI were obtained for analysis. The cobas Liat Cdiff and Xpert PCR tests showed an overall percent agreement of 97.4% (302/310; 95% CI: 95.0-98.9). Low bacterial burdens of toxigenic C. difficile, indicated by significantly delayed PCR cycle threshold (Ct) values, explained most of the discordance. Positive and negative percent agreement of the cobas Liat Cdiff test compared to the Xpert PCR test were 94.5% (52/55) and 98.0% (250/255), respectively. The clinical performance of the cobas Liat Cdiff test, combined with its simplicity of use and rapid result reporting, provides a reliable option for clinical laboratory use.

Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures.

The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.

Multicenter Evaluation of the Accelerate PhenoTest BC Kit for Rapid Identification and Phenotypic Antimicrobial Susceptibility Testing Using Morphokinetic Cellular Analysis.

We describe results from a multicenter study evaluating the Accelerate Pheno system, a first of its kind diagnostic system that rapidly identifies common bloodstream pathogens from positive blood cultures within 90 min and determines bacterial phenotypic antimicrobial susceptibility testing (AST) results within ∼7 h. A combination of fresh clinical and seeded blood cultures were tested, and results from the Accelerate Pheno system were compared to Vitek 2 results for identification (ID) and broth microdilution or disk diffusion for AST. The Accelerate Pheno system accurately identified 14 common bacterial pathogens and two Candida spp. with sensitivities ranging from 94.6 to 100%. Of fresh positive blood cultures, 89% received a monomicrobial call with a positive predictive value of 97.3%. Six common Gram-positive cocci were evaluated for ID. Five were tested against eight antibiotics, two resistance phenotypes (methicillin-resistant Staphylococcus aureus and Staphylococcus spp. [MRSA/MRS]), and inducible clindamycin resistance (MLSb). From the 4,142 AST results, the overall essential agreement (EA) and categorical agreement (CA) were 97.6% and 97.9%, respectively. Overall very major error (VME), major error (ME), and minor error (mE) rates were 1.0%, 0.7%, and 1.3%, respectively. Eight species of Gram-negative rods were evaluated against 15 antibiotics. From the 6,331 AST results, overall EA and CA were 95.4% and 94.3%, respectively. Overall VME, ME, and mE rates were 0.5%, 0.9%, and 4.8%, respectively. The Accelerate Pheno system has the unique ability to identify and provide phenotypic MIC and categorical AST results in a few hours directly from positive blood culture bottles and support accurate antimicrobial adjustment.

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinically Important Bacteria and Yeasts.

OBJECTIVES: A report on the multicenter evaluation of the Bruker MALDI Biotyper CA System (MBT-CA; Bruker Daltonics, Billerica, MA) for the identification of clinically important bacteria and yeasts. METHODS: In total, 4,399 isolates of medically important bacteria and yeasts were assessed in the MBT-CA. These included 2,262 aerobic gram-positive (AGP) bacteria, 792 aerobic gram-negative (AGN) bacteria 530 anaerobic (AnA) bacteria, and 815 yeasts (YSTs). Three processing methods were assesed. RESULTS: Overall, 98.4% (4,329/4,399) of all bacterial and yeast isolates were correctly identified to the genus and species/species complex level, and 95.7% of isolates were identified with a high degree of confidence. The percentage correctly identified and the percentage identified correctly with a high level of confidence, respectively, were as follows: AGP bacteria (98.6%/96.5%), AGN bacteria (98.5%/96.8%), AnA bacteria (98.5%/97.4%), and YSTs (97.8%/87.6%). The extended direct transfer method was only minimally superior to the direct transfer method for bacteria (89.9% vs 86.8%, respectively) but significantly superior for yeast isolates (74.0% vs 48.9%, respectively). CONCLUSIONS: The Bruker MALDI Biotyper CA System accurately identifies most clinically important bacteria and yeasts and has optional processing methods to improve isolate characterization.

Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens.

The Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producing Escherichia coli (STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx1 and stx2), as well as the E. coli serotype O:157-specific marker rfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection of stx1 and stx2 and 95.7% sensitive and 99.3% specific for detection of E. coli serotype O:157. All specimens with false-positive results were found to contain stx1 or stx2 or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negative stx1 or stx2 results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive for stx by an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative for stx1 and stx2 following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens.

The unexpected detection of varicella-zoster virus in genital specimens using the Lyra™ Direct HSV 1+2/VZV Assay.

BACKGROUND: The Lyra™ Direct HSV 1+2/VZV Assay is a moderately complex, multiplex PCR assay that qualitatively detects the presence of HSV 1, HSV 2, and VZV DNA in cutaneous and mucocutaneous specimens with a time-to-result of less than 60min. OBJECTIVES: To report a one-year laboratory experience using Lyra assay for testing cutaneous and mucocutaneous specimens for HSV and VZV that resulted in the unexpected detection of VZV in 14 male and female genital specimens. STUDY DESIGN: Over a one-year period, 2113 cutaneous and mucocutaneous specimens from male and female patients were submitted for testing using the Lyra assay. An unexpected 14 genital specimens were positive for the presence VZV DNA. Eleven of the 14 specimens were available for confirmatory testing using two alternative molecular methods and Sanger sequencing. RESULTS: Fourteen male and female genital specimens were positive for the presence of VZV DNA. All of the 11 specimens (9 female and 2 male) that were available for confirmatory testing by the alternative molecular method and Sanger sequencing were confirmed as containing VZV DNA. CONCLUSIONS: Using of the Lyra assay over a one-year time period, VZV DNA was detected in 126 specimens of which 14 (11.1%) were from male and female genital sites. This rare and unexpected finding suggests that the appearance of zoster lesions in the genital area may not be as uncommon as previously thought and that this finding would have considerable impact on patient counseling and public health considerations.

Multicenter Evaluation of the Solana Group A Streptococcus Assay: Comparison with Culture.

We compared group A Streptococcus (GAS) culture with a rapid helicase-dependent amplification (HDA) method using 1,082 throat swab specimens. The HDA method demonstrated 98.2% sensitivity and 97.2% specificity. GAS prevalence by culture was 20.7%, and it was 22.6% using the HDA method. In 35 min, the HDA method provided rapid, sensitive GAS detection, making culture confirmation unnecessary.

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

Comparative Evaluation of AmpliVue HSV 1+2 Assay with ELVIS Culture for Detecting Herpes Simplex Virus 1 (HSV-1) and HSV-2 in Clinical Specimens.

The AmpliVue HSV 1+2 assay was compared to the ELVIS HSV ID and D(3) Typing Culture System for the qualitative detection and differentiation of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in 1,351 cutaneous and mucocutaneous specimens. Compared to ELVIS, AmpliVue had sensitivities of 95.7 and 97.6% for detecting HSV-1 and HSV-2, respectively. Following arbitration of discordant results by an independent molecular method, the AmpliVue assay had a resolved sensitivity and specificity of 99.2 and 99.7%, respectively, for both HSV-1 and HSV-2, whereas ELVIS had a resolved sensitivity of 87.1% for HSV-1 and 84.5% for HSV-2.

Preliminary Evaluation of the Research-Use-Only (RUO) iCubate iC-GPC Assay for Identification of Select Gram-Positive Bacteria and Their Resistance Determinants in Blood Culture Broths.

The iC-GPC assay (iCubate, Huntsville, AL) provides a molecular option for the rapid, on-demand analysis of positive blood cultures. A preliminary evaluation of the iC-GPC assay using 203 clinical or seeded specimens demonstrated a sensitivity of 93.8% to 100% and a specificity of 98.0% to 100% for the identification of five Gram-positive bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) and three associated genetic resistance determinants (mecA, vanA, and vanB) in positive blood culture broths.

Multicenter evaluation of the Quidel Lyra Direct C. difficile nucleic acid amplification assay.

Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods.

Multicenter clinical evaluation of VRESelect agar for identification of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium.

A chromogenic medium for identification of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, VRESelect, was compared to bile esculin azide agar with 6 µg/ml vancomycin (BEAV) for the isolation of vancomycin-resistant enterococci (VRE) from stool specimens. At 24 to 28 h, VRESelect demonstrated 98.7% (confidence interval [CI], 96.1 to 99.7%) sensitivity and 99.0% (CI, 98.0 to 99.6%) specificity versus 85.1% (CI, 79.8 to 89.5%) and 90.1% (CI, 79.8 to 89.5%) sensitivity and specificity, respectively, for BEAV.

Comparative clinical evaluation of the IsoAmp(®) HSV Assay with ELVIS(®) HSV culture/ID/typing test system for the detection of herpes simplex virus in genital and oral lesions.

BACKGROUND: The novel IsoAmp(®) HSV Assay employs isothermal helicase-dependent nucleic acid amplification and a user-friendly disposable test device to achieve rapid (<1.5h), on-demand qualitative detection of herpes simplex virus (HSV) types 1 and 2 in oral and genital lesions. OBJECTIVES: To compare performance of the IsoAmp(®) HSV Assay with the ELVIS(®) HSV ID/typing (shell-vial culture and DFA) test system for clinical specimens collected from oral and genital lesions in symptomatic patients. STUDY DESIGN: A total of 994 specimens from male and female genital and oral lesions were obtained and evaluated at five study sites in the United States. Results from the IsoAmp(®) HSV Assay were compared to those from the ELVIS(®) system. Separate reproducibility studies were performed at 3 sites using a blinded and randomized study panel. Discrepant specimens were resolved by bidirectional sequencing analysis. RESULTS: After discrepant analysis, overall agreement of IsoAmp(®) with ELVIS(®) was 98.8% with 37.0% overall prevalence (all study sites). Reproducibility rates were well within expectations. CONCLUSION: The IsoAmp(®) HSV Assay showed excellent performance for clinical use for detection of HSV in genital and oral specimens. In contrast to ELVIS(®), IsoAmp(®) HSV offers excellent sensitivity plus rapid on-demand testing and simpler specimen preparation.

Comparison of premier CAMPY enzyme immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY tests with culture for laboratory diagnosis of Campylobacter enteric infections.

Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the "gold standard" for diagnosis.