Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2.

Nucleic acid amplification for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory samples is the standard method for diagnosis. The majority of this testing is centralized and therefore has turnaround times of several days. Point-of-care (POC) testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly. The inclusivity and specificity of the Simple AMplification-Based Assay (SAMBA) II SARS-CoV-2 test were determined by both in silico analyses of the primers and probes and wet testing. The SAMBA II SARS-CoV-2 test was evaluated for performance characteristics. Clinical performance was evaluated in residual combined throat/nose swabs and compared to that of the Public Health England real-time PCR assay targeting the RdRp gene. The SAMBA II SARS-CoV-2 test has an analytical sensitivity of 250 copies/ml for detecting two regions of the genome (open reading frame 1ab [ORF1ab] and nucleocapsid protein [N]). The clinical performance was evaluated in 172 residual combined nose/throat swabs provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (CMPHL), which showed an estimated positive percent agreement of 98.9% (95% confidence interval [CI], 93.83 to 99.97) and negative percent agreement of 96.4% (95% CI, 89.92 to 99.26) compared to testing by the CMPHL. The data show that the SAMBA II SARS-CoV-2 test performs equivalently to the centralized testing methods, but with a shorter turnaround time of 86 to 101 min. Point-of-care tests such as SAMBA should enable rapid patient management and effective implementation of infection control measures.

Application of Sample6 DETECT™ HT/L Kit for the Detection of Listeria in Mixed Leafy Greens.

BACKGROUND: Early and accurate detection of Listeria in foods is vital. Current methods require 24 h enrichment for detection. OBJECTIVE: This study aimed to demonstrate enhanced early detection of Listeria in mixed leafy greens using Sample6 DETECT™ HT/L, a phage-based detection system. METHODS: A method comparison between the reference method (U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 10) for the detection of Listeria spp. and the Sample6 DETECT HT/L using a new proprietary R2 Medium was performed in mixed leafy greens. RESULTS: Using the R2 Medium enrichment with Sample6 DETECT HT/L, detection of L. innocua was possible at 12 h (with a centrifugation step), and L. monocytogenes was detected by 18 h, with equivalent performance as the 24 h enrichments using the reference method detection. The Sample6 DETECT HT/L gave an equivalent performance for L. innocua at 15 h as the reference method at 24 h. Detection was accomplished by the addition of reagents in the kit, following the package insert, and reading results in a detection chamber using a 96-well plate reader that detects a fluorescent signal. CONCLUSIONS: Results indicate the new R2 Medium and Sample6 DETECT HT/L allowed for earlier detection of Listeria spp. in mixed leafy greens. HIGHLIGHTS: Sample6 DETECT HT/L with the new R2 Medium allowed the early detection of Listeria spp. and may be applied in other food matrices and environmental samples.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Simultaneous Detection of Multiple Wine-Spoilage Organisms Using a PCR-Based DNA Dipstick Assay.

Background: The presence of microbial contaminants such as Brettanomyces in wine can lead to undesirable wine. Therefore, monitoring for the presence of these spoilage organisms is critical for winemakers to ensure the quality of their end product. Objective: To address this problem, Molecular Epidemiology, Inc. (MEI, Seattle, WA) has developed a wine-spoilage organism detection kit consisting of a multiplex PCR DNA dipstick that simultaneously detects these organisms. Methods: Wine samples obtained from local wineries that tested negative by routine microbiological culture were spiked with the target microorganisms, while samples that were designated as spoiled by the wineries were used as-is without spiking for assessing the performance characteristics of the DNA dipstick assay. Microbial enumeration was performed following standard microbiological plating methods. Samples spiked with low cell numbers (<5 cells per 100 mL) were enriched using wine enrichment media (WSE; optional component of the kit) prior to analysis using the DNA dipstick assay. Suitability of WSE medium to support the growth of wine-spoilage microorganisms was compared with standard microbiological media. Results: Testing of 92 diverse bacterial and yeast strains commonly found in winery and food operations and 50 various strains of spoilage organisms isolated from wineries indicated that the dipstick assay can exclusively detect the target wine-spoilage microorganisms. All target spoilage organisms in samples containing low cell numbers (<5 cells per 100 mL) were detected by dipstick assay 48 h postenrichment in WSE, except for a few strains of Brettanomyces bruxellensis that required longer incubation times. Conclusions: The wine-spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the wine-making process to detect multiple spoilage-causing microorganisms in a single assay, thus offering a convenient test system for winemakers interested in monitoring the quality of their product.

Rapid Detection of Listeria in Ice Cream in 13 Hours Using the Roka Listeria Detection Assay.

Background: Listeria contamination is a major concern in the ice cream industry; therefore, early and accurate detection is vital. Current detection methods require about a 24 h enrichment period for detection. Objective: Enhance the early detection of Listeria in ice cream using the highly sensitive isothermal ribosomal RNA-based Roka/Atlas Listeria Detection Assay. Methods: The R2 Medium was developed for Listeria enrichment by Molecular Epidemiology, Inc. (Seattle, WA). Comparative growth curve studies were performed on the new R2 Medium for Listeria and the currently validated media for the Roka Listeria Detection Assay. Subsequently, a method comparison between the Roka Listeria Detection Assay and the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 reference method on ice cream was carried out. Results: The R2 Medium supports the growth of L. monocytogenes better than Buffered Listeria Enrichment Broth, Demi-Fraser broth, and Modified University of Vermont Broth, as indicated by the faster growth rate of the organism. When used as an enrichment medium in a method comparison study of ice cream, the results showed that R2 Medium-enriched samples tested with the Roka Listeria Detection Assay gave an equivalent performance compared with the 24 h FDA-BAM reference method at 10 and 18 h post-enrichment for Listeria. Conclusions: The results from this study indicate that the new R2 Medium and the highly sensitive Roka Listeria Detection Assay allowed for the rapid detection of Listeria species in ice cream in 13 h. Highlights: The Roka Listeria Detection Assay, in conjunction with a new media formulation (R2 Medium), allowed for the early detection of Listeria in ice cream and may be applied in other food matrixes and environmental samples.

Performance Validation of the Microbiologique MicrofilmTM Test System for AOAC Research Institute Performance Tested MethodSMCertification.

The Microfilm™ Test System is intended for quantitative microbiology and consists of three types of Microfilms for aerobic plate count (Microfilm APC), total coliform and Escherichia coli count (Microfilm TCEc), and yeast and mold count (Microfilm YMC). This study evaluated the performance of the Microfilm Test System against International Organization for Standardization (ISO) methods on 20 food matrixes and 2 environmental surfaces. Ruggedness, robustness, and stability were also determined, while inclusivity and exclusivity studies were performed on Microfilm TCEc and YMC. An independent laboratory evaluated the performance on four food matrixes and one environmental surface. No significant differences and high correlation coefficients were observed between the Microfilm Test System and the corresponding ISO methods (ISO 4833-1:2013 for APC, ISO 4832:2006 for total coliform count, ISO 16649-2: 2001 for E. coli, and ISO 21527 Part 1 and Part 2 for YMC) in spiked food matrixes and environmental samples. These results were corroborated by the independent laboratory. Inclusivity and exclusivity studies for Microfilm TCEc showed expected results for all the E. coli strains tested (blue-violet or violet color), while the related coliforms showed the expected blue-green colonies on the Microfilm. Similarly, all 100 fungal strains tested showed typical growth on Microfilm YMC. Exclusivity testing on Microfilm TCEc and YMC showed no growth of nontarget organisms. Robustness and ruggedness studies showed no significant differences in mean difference counts at varying incubation temperatures and times. Stability studies on three lots of the Microfilm Test System showed that it is stable at 2-25°C for 12 months and at 45°C for 6 weeks.

Dipstick Assay for Rapid Detection of Beer Spoilage Organisms.

Background: Beer spoilage caused by wild yeast and bacteria is a major concern to both commercial and home brewers. Objective: To address this problem, Molecular Epidemiology Inc. (MEI, Seattle, WA) has developed a beer spoilage organism detection kit consisting of an enrichment media (BSE) and a multiplex PCR DNA dipstick that simultaneously detects these organisms within 2 h following enrichment. Methods: The kit was tested by using samples obtained from breweries located in the Greater Seattle area. Samples were spiked with the target microbes, when necessary, and used for assessing the performance characteristics of the DNA dipstick assay. Microbial enumerations were performed as per the standard microbiological plating methods. The suitability of the BSE medium to support the growth of beer spoilage microbes was compared with the industry-approved NBB-C medium (Dohler, Darmstadt, Germany). Results: Inclusivity (a panel of 50 isolates) and Exclusivity (a panel of 92 isolates) testing indicated that the dipstick assay can exclusively detect the indicated target beer spoilage microbes. When compared with the NBB-C medium (Dohler, Darmstadt, Germany) approved by the European Brewers Convention for beer spoilage organisms, the BSE medium supported faster growth of critical spoilage lactic acid bacteria such as Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Conclusions: The beer spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the brewing process, thus offering a convenient, cost effective, and faster test system for brewers interested in monitoring the quality of their product.

Performance of the SAMBA I and II HIV-1 Semi-Q Tests for viral load monitoring at the point-of-care.

Although access to antiretroviral therapy for HIV infection is increasing in resource-poor countries, viral load testing for monitoring of treatment efficacy remains limited, expensive, and confined to centralized laboratories. The SAMBA HIV-1 Semi-Q Test is a nucleic acid-based amplification assay developed for viral load monitoring performed on either the semi-automated SAMBA I system for laboratory use or the fully automated SAMBA II system for point-of care use. We have assessed the performance characteristics of the SAMBA HIV-1 Semi-Q Test on SAMBA I and SAMBA II systems according to the Common Technical Specifications of the European Community's 98/79 In Vitro Diagnostic Medical Devices Directive. The sensitivity, specificity, reproducibility, and viral subtype coverage of the test were similar on the SAMBA I and SAMBA II platforms. The clinical performance on the SAMBA I system was compared with the Roche CAP/CTM assay and evaluated in-house with 130 patient specimens from London as well as in the field with 390 specimens in Kenya and Zimbabwe. The overall concordance between the SAMBA and CAP/CTM assays was 98.1%. The clinical performance of the test on the SAMBA II platform in comparison with the Abbott HIV-1 RealTime Assay was evaluated in-house with 150 specimens from Ukraine, yielding a concordance of 98.0%. The results thus show that the SAMBA HIV-1 Semi-Q Test performs equivalently on SAMBA I and SAMBA II, and they suggest that the test is suitable for implementation at the point-of-care in resource-poor regions where viral load testing is desperately needed but often unavailable.

Performance of a new rapid test for the detection of hepatitis B surface antigen in various patient populations.

BACKGROUND: Rapid diagnostic tests (RDT) have been developed for the detection of hepatitis B surface antigen (HBsAg). They represent a promising alternative to enzyme immunoassays and a powerful tool for large-scale screening and diagnosis of HBV infection, especially in regions without easy access to serological and molecular testing. OBJECTIVES: The aims of the present study were to evaluate the characteristics and clinical performance of a new CE-marked HBsAg RDT, DRW-HBsAg v2.0 assay (Diagnostics for the Real World™, Ltd., USA), in various patient populations, including those chronically infected with HBV, patients with severe acute hepatitis of unknown origin and pregnant women with unknown HBV serological status at delivery. RESULTS: The lower limit of detection of the assay, evaluated in 21 clinical samples, ranged from 0.30 ± 0.07 to 0.97 ± 0.26 international units/mL (using Abbott Architect as a reference), depending on the HBV genotype. The assay tested positive in 100% of patients with chronic hepatitis B, 96.3% of HBsAg-positive acute hepatitis patients, and 95.2% of HBsAg-positive pregnant women. Its specificity was 98.8% in HBsAg-negative patients, 98.7% in HBsAg-negative patients with acute hepatitis of unknown origin and 97.8% in HBsAg-negative pregnant women. Amino acid substitutions in the HBsAg major hydrophilic region did not affect HBsAg detection by DRW-HBsAg v2.0. CONCLUSIONS: The new DRW-HBsAg v2.0 assay is a simple, rapid, easy-to-run and highly sensitive assay that can be used in both high- and low-risk populations for the diagnosis of HBsAg carriage. It appears to be a promising new tool for large-scale screening and diagnosis of HBV infection.