Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure.

SARS-CoV-2 nucleic acid detection tests enable rapid virus detection; however, it is challenging to identify genotypes to comprehend the local epidemiology and infection routes in real-time qRT-PCR. At the end of June 2022, our hospital experienced an in-hospital cluster of COVID-19. When examined using the GeneXpert® System, the cycle threshold (Ct) value of the N2 region of the nucleocapsid gene of SARS-CoV-2 was approximately 10 cycles higher than that of the envelope gene. Sanger sequencing revealed a G29179T mutation in the primer and probe binding sites. A review of past test results revealed differences in Ct values in 21 of 345 SARS-CoV-2-positive patients, of which 17 cases were cluster-related and 4 were not. Including these 21 cases, 36 cases in total were selected for whole-genome sequencing (WGS). The viral genomes in the cluster-related cases were identified as BA.2.10, and those in the non-cluster cases were closely related and classified as being downstream of BA.2.10 and other lineages. Although WGS can provide comprehensive information, its use is limited in various laboratory settings. A measurement platform reporting and comparing Ct values of different target genes can improve test accuracy, enhance our understanding of infection spread, and be applied to the quality control of reagents.

Diagnostic Efficacy of RealStar SARS-CoV-2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) in Comparison to GeneXpert System for the Detection of COVID-19.

Background and objective The coronavirus disease 2019 (COVID-19) pandemic has become a major health concern due to the rapid transmission of the virus that causes it: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address the growing demand on healthcare systems to control this pandemic, more effective diagnostic methods need to be applied. In this study, we aimed to compare the efficacy of RealStar® SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) versus the GeneXpert® system. Methods A retrospective cross-sectional study was conducted in the central lab of King Abdulaziz Medical City (KAMC) in Riyadh, Saudi Arabia. Data from all nasopharyngeal swabs (NPS) (150,000) submitted for SARS-CoV-2 analysis from July 2020 to July 2021 were reviewed retrospectively. Furthermore, all NPS (n=384) that were analyzed on both the RealStar® SARS-CoV-2 RT-PCR and GeneXpert® systems for confirmatory purposes were included in the study. Acute respiratory illness (ARI) screening forms of the selected samples were reviewed from the electronic database (BestCare system), and they were analyzed and compared at one point in time; therefore, a cross-sectional study was found to be the best suitable study design. Using the statistical analysis software, the receiver operating characteristic (ROC) curve was obtained to compare the sensitivity (Sn), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV). The test was considered significant if the area under the curve (AUC) value was >0.5. Results The diagnostic performance of the RealStar® and GeneXpert® assays in detecting SARS-CoV-2 was evaluated using ROC curve analysis, which showed AUCs of 0.597 and 0.637, respectively. In addition, 35% of the total results fell into a substantial agreement of 0.76 (95% CI: 0.6626-0.8732). The majority of the NPS were reported negative by both RealStar® (246, 80.66%) and GeneXpert® (226, 74.10%). Most samples (210, 68.85%) were obtained from asymptomatic patients, scoring less than 4 (ARI <4) based on the ARI screening form. Conclusion Based on the AUC of ROC, there is no significant difference in the performance characteristics between the RealStar® RT-PCR and GeneXpert® in detecting COVID-19.

Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test.

The GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test (PLUS assay) received Health Canada approval in January 2022. The PLUS assay is similar to the SARS-CoV-2/Flu/RSV combination test, with modifications to improve assay robustness against circulating and emerging variants. The performance characteristics of the PLUS assay were assessed at the Lakeridge Health Oshawa Hospital Centre and the National Microbiology Laboratory of Canada. The PLUS assay was directly compared to the SARS-CoV-2/Flu/RSV combination test using SARS-CoV-2 culture from five variants and remnant clinical specimens collected across the coronavirus disease 2019 pandemic. This included 50 clinical specimens negative for all pathogens, 110 clinical specimens positive for SARS-CoV-2, influenza A, influenza B, RSVA, and(or) RSVB and an additional 11 mixed samples to screen for target interactions. The PLUS assay showed a high % agreement with the widely used SARS-CoV-2/Flu/RSV combination test. Based on these findings, the PLUS assay and the Xpert SARS-CoV-2/Flu/RSV combination test results are largely consistent with no observed difference in sensitivity, specificity, or time to result when challenged with various SARS-CoV-2 variants. The reported cycle threshold (Ct) values provided by the new PLUS assay were also unchanged, with the exception of a possible 1-2 decrease reported in Ct for RSVA across a limited sample size.

Towards Accurate Point-of-Care Tests for Tuberculosis in Children.

In childhood tuberculosis (TB), with an estimated 69% of missed cases in children under 5 years of age, the case detection gap is larger than in other age groups, mainly due to its paucibacillary nature and children's difficulties in delivering sputum specimens. Accurate and accessible point-of-care tests (POCTs) are needed to detect TB disease in children and, in turn, reduce TB-related morbidity and mortality in this vulnerable population. In recent years, several POCTs for TB have been developed. These include new tools to improve the detection of TB in respiratory and gastric samples, such as molecular detection of Mycobacterium tuberculosis using loop-mediated isothermal amplification (LAMP) and portable polymerase chain reaction (PCR)-based GeneXpert. In addition, the urine-based detection of lipoarabinomannan (LAM), as well as imaging modalities through point-of-care ultrasonography (POCUS), are currently the POCTs in use. Further to this, artificial intelligence-based interpretation of ultrasound imaging and radiography is now integrated into computer-aided detection products. In the future, portable radiography may become more widely available, and robotics-supported ultrasound imaging is currently being trialed. Finally, novel blood-based tests evaluating the immune response using "omic-"techniques are underway. This approach, including transcriptomics, metabolomic, proteomics, lipidomics and genomics, is still distant from being translated into POCT formats, but the digital development may rapidly enhance innovation in this field. Despite these significant advances, TB-POCT development and implementation remains challenged by the lack of standard ways to access non-sputum-based samples, the need to differentiate TB infection from disease and to gain acceptance for novel testing strategies specific to the conditions and settings of use.

Tuberculosis

Tuberculosis (TB) is a communicable, airborne infectious disease caused by the bacterium Mycobacterium tuberculosis (MTB). A quarter of the world's population is infected with TB, affecting all age groups. Infection with MTB results in latent or active disease. Latent infection is associated with a 10% lifetime risk of developing active disease, but this is much higher in those with concurrent immunosuppression. Despite being both preventable and curable, TB remains the leading cause of global death from a single infectious agent. Active disease most commonly affects the lungs but can spread to cause extrapulmonary disease anywhere in the body. Over half of individuals in the UK now present with features of extrapulmonary TB, those with HIV being at particular risk. In all cases, obtaining samples for TB culture is absolutely vital. Standard treatment is with quadruple therapy for 6 months, extended in TB meningitis and often TB bone infection. Adjunctive corticosteroids have proven benefit in TB meningitis and TB pericarditis, and can be considered in other circumstances, such as paradoxical reactions to starting treatment in miliary TB. Despite recent gains in diagnosing and treating TB cases worldwide, the global COVID-19 pandemic is likely to have significantly affected recent progress.

A Sensitive and Rapid Wastewater Test for SARS-COV-2 and Its Use for the Early Detection of a Cluster of Cases in a Remote Community.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been used to monitor trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence in the community. A major challenge in establishing wastewater surveillance programs, especially in remote areas, is the need for a well-equipped laboratory for sample analysis. Currently, no options exist for rapid, sensitive, mobile, and easy-to-use wastewater tests for SARS-CoV-2. The performance of the GeneXpert system, which offers cartridge-based, rapid molecular clinical testing for SARS-CoV-2 in a portable platform, was evaluated using wastewater as the input. The GeneXpert demonstrated a SARS-CoV-2 limit of detection in wastewater below 32 copies/mL with a sample processing time of less than an hour. Using wastewater samples collected from multiple sites across Canada during February and March 2021, a high overall agreement (97.8%) was observed between the GeneXpert assay and laboratory-developed tests regarding the presence or absence of SARS-CoV-2. Additionally, with the use of centrifugal filters, the detection threshold of the GeneXpert system was improved to <10 copies/mL in wastewater. Finally, to support on-site wastewater surveillance, GeneXpert testing was implemented in Yellowknife, a remote community in Northern Canada, where its use successfully alerted public health authorities to undetected transmission of COVID-19. The identification of SARS-CoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest that GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories. IMPORTANCE Wastewater-based surveillance is a powerful tool that provides an unbiased measure of COVID-19 prevalence in a community. This work describes a sensitive wastewater rapid test for SARS-CoV-2 based on a widely distributed technology, the GeneXpert. The advantages of an easy-to-use wastewater test for SARS-CoV-2 are clear: it supports surveillance in remote communities, improves access to testing, and provides faster results allowing for an immediate public health response. The application of wastewater rapid testing in a remote community facilitated the detection of a COVID-19 cluster and triggered public health action, clearly demonstrating the utility of this technology. Wastewater surveillance will become increasingly important in the postvaccination pandemic landscape as individuals with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be tested.

A Rare Case of Sternoclavicular Tuberculosis Diagnosed Using Microchip-Based Polymerase Chain Reaction in a Diabetic Female.

Osteoarticular tuberculosis (TB) accounts for almost 10% of all extrapulmonary TB cases. In the majority of cases, the spine, knee, hip, and large bones are involved; other sites like the sternoclavicular joint, elbow, wrist, and smaller joints are infrequently involved. Uncommon locations of extrapulmonary TB pose a challenge in diagnosis due to lack of clinical suspicion, non-availability of samples, and unavailability of suitable diagnostic modalities. Here we report a case of uncommon location of osteoarticular TB diagnosed through microchip-based real-time polymerase chain reaction (PCR).

TB positive cases go up in ongoing COVID-19 pandemic despite lower testing of TB: An observational study from a hospital from Northern India.

The whole world is wrestling against SARS-CoV-2 infection (COVID-19). COVID-19-TB co-infection is also reported but there are limited number of studies which analyze the impact of COVID-19 pandemic in TB diagnosis and management. In this retrospective study, we observed that the TB diagnosis was reduced in pandemic time. Before COVID-19 pandemic (March-December 2019), there were 644 TB tests out of which 127 were TB positive. In ongoing COVID-19 pandemic (January-October 2020), 484 TB tests were performed and 146 patients were TB positive. Male accounted for 64%/57% of TB cases in 2019/2020 whereas female patients were 35%/42% in 2019/2020. Increase in female TB positive cases was a noticeable feature. The newly diagnosed with TB cases in 2019/2020 were 112/130 respectively. Though, we have seen only 7 COVID-TB co-infection cases, we could not establish the causal relationship in COVID-TB co-infection. The increase in the number of TB positive cases during COVID-19 pandemic clearly showed how adversely COVID-19 has affected TB diagnosis and management. Anticipating the increase in TB cases in future, we emphasize the need to ensure continuous TB testing and treatment despite the pandemic burden. Further study on the COVID-TB co-infection in high TB-burden countries like India, is required to enable analyses of interactions, risk factors in COVID-19-TB co-infection.

Validation of the GeneXpert Xpress SARS-CoV-2 PCR assay using saliva as biological specimen.

In the pandemic, rapid and accurate detection of SARS-CoV-2 is crucial in controlling the outbreak. Recent studies have shown a high detection rate using saliva/oral fluids as specimens for laboratory detection of the virus. We intended to evaluate the test performance of the Xpert Xpress SARS-CoV-2 cartridge assay in comparison to a conventional qRT-PCR testing, using saliva as biological specimen. Forty saliva samples from symptomatic participants were collected. Conventional qRT-PCR was performed for amplification of E and RdRp genes and the Xpert Xpress SARS-CoV-2 assay amplified E and N2 genes. In the conventional assay, the median cycle threshold value of the E gene was 34.9, and of the RdRp gene was 38.3. In the Xpert Xpress assay, the median cycle threshold value of the E gene was 29.7, and of the N2 gene was 31.6. These results can allow a broaden use of molecular tests for management of COVID-19 pandemic, especially in resources-limited settings.

Sample pooling on the Cepheid Xpert® Xpress SARS-CoV-2 assay.

The COVID-19 pandemic has placed unprecedented global demand on laboratory supplies required for testing. Sample pooling has been investigated by laboratories as a strategy to preserve testing capacity. We evaluate the performance of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay for testing samples in pools of 4 and 6. Clinical samples containing SARS-CoV-2, and confirmed negative clinical samples were used to create sample pools. Clinical samples had 'neat' Xpert® E gene cycle threshold values ranging between 20 and 28 and all were detected qualitatively when contained in pools of 4 or 6 samples. For these samples, pooling had a median change in cycle threshold value of 2.0 in pools of 4, and of 2.9 in pools of 6. With the use of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay, pooling of 4 or 6 samples may be an effective strategy to increase testing capacity.

Comparison of a Point-of-Care Assay and a High-Complexity Assay for Detection of SARS-CoV-2 RNA.

BACKGROUND: Numerous nucleic acid amplification assays utilizing different target genes of the SARS-CoV-2 genome have received emergency use authorization (EUA) by the United States Food and Drug Administration (FDA). Limited data are available comparing the test performance characteristics of these assays. METHODS: A diagnostic comparison study was performed to evaluate the performance of the Cepheid Xpert Xpress SARS-CoV-2 assay compared to the Hologic Panther Fusion SARS-CoV-2 assay using clinical nasopharyngeal specimens. Agreement between the two assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient. RESULTS: A total of 104 (54 positive and 50 negative) clinical nasopharyngeal samples were tested by both assays. Using the Panther Fusion as a reference standard, the Xpert demonstrated an overall agreement of 99.0% [95% confidence interval (CI): 94.8-100], positive percent agreement of 98.1% (95% CI: 90.1-100), and a negative percent agreement of 100% (95% CI: 94.2-100). The kappa coefficient was 0.98 (95% CI: 0.94-1.0). One sample positive by the Panther Fusion with a cycle threshold (Ct) of 38.6 was found to be reproducibly negative by the Xpert assay. CONCLUSIONS: The Cepheid Xpert Xpress SARS-CoV-2 assay provides test performance comparable to the Hologic Panther Fusion SARS-CoV-2 assay while offering laboratories rapid, on-demand testing capacity.

Brief validation of the novel GeneXpert Xpress SARS-CoV-2 PCR assay.

The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses.