Comparison of the Simplexa GBS Direct and ARIES GBS assays for the detection of S. agalactiae in broth-enriched swab specimens.

We conducted a comparative evaluation of the FDA-cleared Simplexa GBS Direct and ARIES GBS molecular assays for the detection of Streptococcus agalactiae (Group B Streptococcus, GBS) in 386 prospectively collected, broth-enriched vaginal/rectal swab specimens. The sensitivity of each test was 96.2% and specificity was ≥98.7% when compared to a combined direct and enriched culture method using chromogenic culture medium. A total of four specimens were called positive by both molecular assays but negative by culture, likely representing specimens with a low burden of GBS in these specimens. Two specimens were reported positive by culture but negative by both molecular assays. One of these specimens demonstrated atypically colored colonies on chromogenic agar; the other yielded typically colored colonies only observed after broth enrichment. Our data demonstrate equivalent performance of Simplexa and ARIES molecular assays for the detection of GBS in clinical specimens.IMPORTANCEClinical laboratories often face decisions regarding which of the multiple available molecular platforms would best fit their needs based on cost, workflow, menu, and diagnostic performance. Therefore, objective clinical comparisons of similar molecular tests are valuable resources to aid these decisions. We provide a clinical comparison of two FDA-cleared tests to routine culture and to each other that can be used by clinical laboratories when determining which of the available molecular platforms would best fit their laboratory in terms of workflow, cost, and performance.

Rapid identification of SARS-CoV-2 variants: Validation of the simplexa SARS-CoV-2 variant direct assay.

The circulation of certain SARS-CoV-2 variants may have a great impact on the epidemiological status of a geographical area; therefore, it is important that their presence is monitored. Currently, the gold standard method used to identify newly emerged variants is sequencing of either genes or whole genomes. However, since this method is relatively expensive and has a long turnaround time, other rapid strategies should also be employed. The current study aimed to evaluate the performance of the Simplexa® SARS-CoV-2 Variants Direct assay, which is a RT-PCR that determines the variant present in a nasopharyngeal swab sample in approximately two hours. Totally, 527 positive samples for SARS-CoV-2 were analyzed from January until December 2022 and next-generation sequencing (NGS) was used as the reference method. The assay showed high sensitivity, ranging from 94.12 % to 100 %, depending on the variant. The assay also showed high specificity, reaching 100 % for Delta and BA.1 variants, and 99.74 % and 98.67 % for BA.2 and BA.4/BA.5 variants, respectively. Moreover, the assay was able to identify the correct variant category in the presence of any subvariant in the sample. We conclude that the assay can be used to facilitate faster monitoring of circulating SARS-CoV-2 variants, however sequencing cannot be completely replaced, since new variants always emerge, and constant updates are needed, so that the user is able to interpret the melting curve patterns.

Diagnostic accuracy of rapid one-step PCR assays for detection of herpes simplex virus-1 and -2 in cerebrospinal fluid: a systematic review and meta-analysis.

BACKGROUND: Rapid and accurate diagnosis of herpes simplex virus (HSV)-1 and -2 (HSV1/2) in cerebrospinal fluid (CSF) is important for patient management. OBJECTIVES: Summarize the diagnostic accuracy of commercial rapid sample-to-answer PCR assays (results in <90 minutes, without a separate nucleic acid extraction step) for HSV1/2 detection in CSF. DATA SOURCES: Four databases (MEDLINE, EMBASE, Scopus, and CENTRAL) and five conference abstract datasets from January 2012 to March 2022. STUDY ELIGIBILITY CRITERIA: Eligible diagnostic accuracy studies provided sufficient data for the construction of a standard diagnostic accuracy two-by-two table. PARTICIPANTS: Patients with suspected meningitis and/or encephalitis. TESTS: FilmArray Meningitis-Encephalitis Panel and Simplexa HSV 1&2 Direct Kit PCR. REFERENCE STANDARD: Real-time PCR assay. ASSESSMENT OF RISK OF BIAS: Two investigators independently extracted data, rated risk of bias, and assessed quality using QUADAS-2. METHODS OF DATA SYNTHESIS: Accuracy estimates were pooled using Bayesian random effects models. RESULTS: Thirty-one studies were included (27 FilmArray; 4 Simplexa), comprising 9924 samples, with 95 HSV-1 and 247 HSV-2 infections. Pooled FilmArray sensitivities were 84.3% (95% credible interval, 72.3-93.0) and 92.9% (95% credible interval (CrI), 82.0-98.5) for HSV-1 and HSV-2, respectively; specificities were 99.8% (95% CrI, 99.6-99.9) and 99.9% (95% CrI, 99.9-100). Pooled Simplexa sensitivities were 97.1% (95% CrI, 88.1-99.6) and 97.9% (95% CrI, 89.6-99.9), respectively; specificities were 98.9% (95% CrI, 96.8-99.7) and 98.9% (95% CrI, 97.1-99.7). Pooled FilmArray sensitivities favoured industry-sponsored studies by 10.0 and 13.0 percentage points for HSV-1 and HSV-2, respectively. Incomplete reporting frequently led to unclear risk of bias. Several FilmArray studies did not fully report true negative data leading to their exclusion. CONCLUSIONS: Our results suggest Simplexa is accurate for HSV1/2 detection in CSF. Moderate FilmArray sensitivity for HSV-1 suggests additional testing and/or repeat CSF sampling is required for suspected HSV encephalitis when the HSV-1 result is negative. Low prevalence of HSV-1 infections limited summary estimates' precision. Underreporting of covariates limited exploration of heterogeneity.

A Tale of Two Countries: DiaSorin Molecular's Rapid Response to the COVID-19 Pandemic.

In the summer of 2019, DiaSorin Molecular started designing a multiplex respiratory panel with pan-coronavirus detection as one of the planned targets. The R&D team in Gerenzano, Italy was already searching databases, performing alignments and assessing preliminary target regions for common coronavirus RT-PCR, including SARS and MERS-CoV. In December 2019, we were vigilant and following a cluster of pneumonia cases with undetermined etiology in Wuhan, China. As we now know, the cause of the respiratory infections was the new SARS-CoV-2 virus. DiaSorin Molecular swiftly responded in line with our heritage and company history in detecting emerging infectious diseases. Early in the pandemic and in record time, using research and development teams in both Italy and the U.S. together with the U.S. manufacturing team, we were able to develop and commercialize a new diagnostic test, Simplexa™ COVID-19 Direct, to detect SARS-CoV-2. Our unique platform allowed development of a rapid diagnostic test without the need for extraction reagents. Challenges with control materials, quarantines, clinical samples, raw materials and production were overcome and the entire company worked side by side for accelerated delivery of this assay to clinical labs in Europe, the U.S. and Canada.

A comparison of the Quidel Solana GAS assay, the Luminex Aries Group A Strep assay and the Focus Diagnostics Simplexa Group A Strep Direct assay for detection of Group A Streptococcus in throat swab specimens.

We compared three commercially available group A Streptococcus (GAS) nucleic acid amplification tests, the Quidel Solana GAS assay, the Luminex Aries Group A Strep assay and the Focus Diagnostics Simplexa Group A Strep Direct assay, with GAS bacterial culture. A true positive result was defined as one positive by culture or positive by ≥2/3 molecular methods. Two hundred eighty-seven throat swabs (207 children, 80 adults) were collected. The sensitivity of culture was 84.8% (95% CI 77.7-90.3%) with a specificity of 100% (95% CI 97.5-100%). The Solana assay sensitivity was 94.2% (95% CI 88.9-97.5%) with a specificity of 98.7% (95% CI 95.2-99.8%). Simplexa assay sensitivity was 99.3% (95% CI 96.0-99.9%) with a specificity of 95.3% (95% CI 90.6-98.1%). Aries assay sensitivity was 96.4% (95% CI 91.8-98.8%) with a specificity of 98.0% (95% CI 94.2-99.6%). In summary, all the molecular methods evaluated showed high sensitivity and specificity and were more sensitive than culture.

Implementation of a rapid influenza A/B and RSV direct molecular assay improves emergency department oseltamivir use in paediatric patients.

PURPOSE: Influenza A virus (FluA), influenza B virus (FluB) and respiratory syncytial virus (RSV) illnesses increase hospitalizations during seasonal epidemics. METHODOLOGY: To determine the utility of the Simplexa FluA/B & RSV Direct Assay (Direct Flu/RSV) and its impact on oseltamivir use, we offered this assay to emergency department (ED) patients with influenza-like illness. RESULTS: Utilization of the Direct Flu/RSV provided a turnaround time (TAT) of 2 hours. Compared to the flu season prior to implementation of the Direct Flu/RSV, clinicians were more likely to prescribe 5 days of oseltamivir therapy for Direct Flu/RSV-positive patients in comparison to those with a negative test. CONCLUSIONS: Use of Direct Flu/RSV provides results rapidly, which leads to more appropriate use of oseltamivir. The ease of use of this assay and quick TAT allows for prompt decision-making, which is essential for patient care and effective disease control during the influenza season.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.

Performance of the Simplexa™ Flu A/B & RSV Direct Kit on respiratory samples collected in saline solution.

BACKGROUND: Molecular assays for diagnosis of influenza A, influenza B, and respiratory syncytial virus (RSV) with short turnaround time are of considerable clinical importance. We have evaluated the diagnostic performance of the Simplexa(™) Flu A/B & RSV Direct Kit, which has a run time of 60 min, using different types of respiratory samples collected from patients with a suspected respiratory tract infection, including materials not previously evaluated on this kit. METHODS: In total, 210 clinical respiratory samples were analyzed using both the Simplexa direct assay and a laboratory-developed assay (LDA). The 210 clinical samples included 99 nasopharyngeal aspirates collected in 0.9% saline, 91 nasopharyngeal swabs in Σ-Virocult transport medium, 9 tracheal secretions, 8 bronchoalveolar lavages (BAL), and 3 other respiratory sample materials. RESULTS: The specificity of the Simplexa assay, using the LDA as gold standard and excluding secondary viral findings, was 100% for all three viruses, whereas the sensitivity was 94.0% for influenza A (47/50), 90.7% for influenza B (49/54), and 90.1% for RSV (46/51), respectively. Discordant results were only observed for samples with cycle threshold values (Ct) > 31 in the LDA. The Simplexa assay generated higher Ct values than the LDA for all three viruses and performed equally well on nasopharyngeal swabs and aspirates. CONCLUSIONS: The short run time of the Simplexa direct assay, in combination with high specificity and good sensitivity regarding the sample materials used in this study, make it an interesting option for rapid detection of these three important viral respiratory pathogens in a variety of clinical sample materials.

Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

Influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using FOCUS direct disc assay is substantially equivalent to the traditional methods and the FOCUS nucleic acid extraction-dependent RT-PCR assay.

In this study, we evaluated FOCUS diagnostic's Flu A/B & RSV direct kit (Direct Disc assay), designed to detect influenza (FLU) and respiratory syncytial viruses (RSV) directly in clinical specimens without nucleic acid extraction. This novel 'sample-to-answer', nucleic acid extraction-independent assay uses a unique disc to process, amplify, and detect viral targets in up to 8 specimens at a time. The performance of this assay for detecting FLU and RSV viruses was compared to the traditional methods (culture and/or direct florescent antibody testing) using 945 nasopharyngeal swab specimens. In addition, a total of 150 consecutive clinical specimens positive for FLU (FLU A=50, FLU B=50) or RSV (n=50) were tested in parallel using the novel Direct Disc assay and FOCUS diagnostic's nucleic acid extraction-dependent assay to assess their relative performance. Compared to the traditional methods, the overall (prospective+retrospective) positive/negative percent agreement was determined to be 96.6%/98.1% for FLU A, 98.4%/99.9% for FLU B, and 99.3%/98.8% for RSV. Compared to the nucleic acid extraction-dependent assay, the positive percent agreement was 90% (n=45/50) for FLU A, 92% (n=46/50) for FLU B, and 98% (n=49/50) for RSV. Overall, the Direct Disc assay showed good agreement with both traditional methods and nucleic acid extraction-dependent assay. Although we encountered some failures compared to the nucleic acid extraction-dependent assay, these limitations must be balanced against the substantial advantages of the extraction-free nature of this assay and rapid turnaround time.