Performances of rapid and connected salivary RT-LAMP diagnostic test for SARS-CoV-2 infection in ambulatory screening.

In the context of social events reopening and economic relaunch, sanitary surveillance of SARS-CoV-2 infection is still required. Here, we evaluated the diagnostic performances of a rapid, extraction-free and connected reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay on saliva. Nasopharyngeal (NP) swabs and saliva from 443 outpatients were collected simultaneously and tested by reverse-transcription quantitative PCR (RT-qPCR) as reference standard test. Seventy-one individuals (16.0%) were positive by NP and/or salivary RT-qPCR. Sensitivity and specificity of salivary RT-LAMP were 85.9% (95%CI 77.8-94.0%) and 99.5% (98.7-100%), respectively. Performances were similar for symptomatic and asymptomatic participants. Moreover, SARS-CoV-2 genetic variants were analyzed and no dominant mutation in RT-LAMP primer region was observed during the period of the study. We demonstrated that this RT-LAMP test on self-collected saliva is reliable for SARS-CoV-2 detection. This simple connected test with optional automatic results transfer to health authorities is unique and opens the way to secure professional and social events in actual context of economics restart.

One-step and sequential SARSCOV-2 polymerase chain reaction tests would not work every time.

INTRODUCTION: RT-PCR is widely used as a diagnostic test for the detection of SARS-CoV-2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID-19. METHOD: We collected multiple nasopharyngeal swab samples from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization. RESULT: Among 68 patients with initial negative test results, at the end of follow-up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. CONCLUSION: Based on this study, serial RT-PCR testing is unlikely to yield additional information.

Cost analysis of reflexive versus selective molecular testing for indeterminate thyroid nodules.

BACKGROUND: Molecular testing is now commonly used to refine the diagnosis of indeterminate thyroid nodules. The purpose of this study is to compare the costs of a reflexive molecular testing strategy to a selective testing strategy for indeterminate thyroid nodules. METHODS: A Markov model was constructed to estimate the annual cost of diagnosis and treatment of a real-world cohort of patients with cytologically indeterminate thyroid nodules, comparing a reflexive testing strategy to a selective testing strategy. Model variables were abstracted from institutional clinical trial data, literature review, and the Medicare physician fee schedule. RESULTS: The average cost per patient in the reflexive testing strategy was $8,045, compared with $6,090 in the selective testing strategy. In 10,000 Monte Carlo simulations, diagnostic thyroid lobectomy for benign nodules was performed in 2,440 patients in the reflexive testing arm, compared with 3,389 patients in the selective testing arm, and unintentional observation for malignant nodules occurred in 479 patients in the reflexive testing arm, compared with 772 patients in the selective testing arm. The cost of molecular testing had the greatest impact on overall costs, with $1,050 representing the cost below which the reflexive testing strategy was cost saving compared with the selective testing strategy. CONCLUSION: In this cost-modeling study, reflexive molecular testing for indeterminate thyroid nodules enabled patients to avoid unnecessary thyroid lobectomy at an estimated cost of $20,600 per surgery avoided.

Socioeconomic Determinants of the Use of Molecular Testing in Stage IV Colorectal Cancer.

OBJECTIVES: Treatment with epidermal growth factor receptor monoclonal antibodies extends life for patients with advanced colorectal cancers (CRCs) whose tumors exhibit wild-type KRAS, but KRAS testing may be underused. We studied the role of socioeconomic factors in the application of KRAS testing. MATERIALS AND METHODS: We identified subjects with stage IV colorectal adenocarcinoma diagnosed 2010-2015 in the Surveillance, Epidemiology, and End Results (SEER) database. We used multivariable logistic regression models to evaluate associations between clinical/demographic factors and the rate of KRAS testing. We used multivariable-adjusted Cox proportional hazards models to assess survival. RESULTS: We identified 37,676 patients with stage IV CRC, 31.1% of whom were tested for KRAS mutations, of those who had documented KRAS testing, 44% were KRAS mutant. Patients were more likely to be tested if they were younger (odds ratio [OR]=5.10 for age 20 to 29 vs. 80+, 95% confidence interval [CI]: 3.99-6.54, P<0.01), diagnosed more recently (OR=1.92 for 2015 vs. 2010, 95% CI: 1.77-2.08, P<0.01), or lived in an area of high median household income (OR=1.24 for median household income of >$69,311 vs. <$49,265, 95% CI: 1.14-1.35, P<0.01). Patients were less likely to be tested if they had Medicaid (OR=0.83, 95% CI: 0.77-0.88, P<0.01) or were unmarried (OR=0.78, 95% CI: 0.75-0.82, P<0.0001). The risk of death was decreased in patients who received KRAS testing (hazard ratio=0.77, 95% CI: 0.75-0.80, P<0.01). CONCLUSIONS: We found a low rate of KRAS testing in CRC patients with those living in low-income areas less likely to be tested, even after controlling for Medicaid insurance. Our study suggests that socioeconomic disparities persist despite Medicaid insurance.

Diagnostic and prognostic predictive values of circulating sTREM-1 in sepsis: A meta-analysis.

BACKGROUND: With the increasing studies regarding the diagnostic value of soluble triggering receptor expressed on myeloid cells (sTREM)-1 in sepsis in recent years, it is essential to make an updated meta-analysis to explore the sepsis differentiation value of circulating sTREM-1 from systemic inflammatory response syndrome (SIRS). Recently, no meta-analysis was made to explore the prognostic predictive value of circulating sTREM-1 in sepsis. Thus, the present aimed to make meta-analyses to explore the diagnostic and prognostic predictive values of circulating sTREM-1 in sepsis. METHODS: Articles published before March 2021 were searched in databases: PubMed, Web of Science, EMBASE, Medline and Google Scholar. After a summary of sensitivity, specificity, positive likelihood ratios (PLR), negative likelihood ratios (NLR), and diagnostic odds ratio (DOR), the receive-operating characteristics (SROC) curve were performed to summarize true positive (TP) and false positive (FP) rates. Q test and I2 were used to explore heterogeneity between studies. RESULTS: Circulating sTREM-1 showed a high sensitivity (0.85 (95% confidence interval (CI): 0.76-0.91)) and moderate specificity (0.79 (95% CI: 0.70-0.86)) to differentiate sepsis from SIRS. The study showed a high sensitivity (0.80 (95% CI: 0.66-0.89)) and moderate specificity (0.75 (95% CI: 0.69-0.81)) to predict 28-day mortality in sepsis. CONCLUSION: In conclusion, the present study suggested that circulating sTREM-1 showed diagnostic and prognostic predictive values in sepsis.

Performance of rapid diagnostic tests, microscopy, loop-mediated isothermal amplification (LAMP) and PCR for malaria diagnosis in Ethiopia: a systematic review and meta-analysis.

BACKGROUND: Rapid accurate diagnosis followed by effective treatment is very important for malaria control. Light microscopy remains the "golden standard" method for malaria diagnosis. Diagnostic test method must have sufficient level of accuracy for detecting malaria parasites. Therefore, this study aimed to investigate the diagnostic accuracy of rapid diagnostic tests (RDTs), microscopy, loop-mediated isothermal amplification (LAMP) and/or polymerase chain reaction (PCR) for the malaria diagnosis in Ethiopia. METHODS: Data bases such as PubMed, PubMed central, Science direct databases, Google scholar, and Scopus were searched from September to October, 2020 for studies assessing the diagnostic accuracy of RDTs, microscopy, LAMP and PCR methods for malaria diagnosis. RESULTS: A total of 29 studies published between 2001 and 2020 were analysed using review manager, Midas (Stata) and Meta-disc. The sensitivity and specificity of studies comparing RDT with microscopy varies from 79%-100% to 80%-100%, respectively. The sensitivity of LAMP (731 tests) was 100% and its specificity was varies from 85 to 99% when compared with microscopy and PCR. Considerable heterogeneity was observed between studies included in this meta-analysis. Meta-regression showed that blinding status and target antigens were the major sources of heterogeneity (P < 0.05). RDT had an excellent diagnostic accuracy (Area under the ROC Curve = 0.99) when compared with microscopy. Its specificity was quite good (93%-100%) except for one outlier (28%), but lower "sensitivity" was observed when PCR is a reference test. This indicates RDT had a good diagnostic accuracy (AUC = 0.83). Microscopy showed a very good diagnostic accuracy when compared with PCR. CONCLUSIONS: The present study showed that microscopy and RDTs had high efficiency for diagnosing febrile malaria patients. The diagnostic accuracy of RDT was excellent when compared with microscopy. This indicates RDTs have acceptable sensitivities and specificities to be used in resource poor settings as an alternative for microscopy. In this study, LAMP showed an excellent sensitivities and specificities. Furthermore, the need of minimum equipment and relatively short time for obtaining results can made LAMP one of the best alternatives especially for accurate diagnosis of asymptomatic malaria.

Improved outcomes and staging in non-small-cell lung cancer guided by a molecular assay.

There remains a critical need for improved staging of non-small-cell lung cancer, as recurrence and mortality due to undetectable metastases at the time of surgery remain high even after complete resection of tumors currently categorized as 'early stage.' A 14-gene quantitative PCR-based expression profile has been extensively validated to better identify patients at high-risk of 5-year mortality after surgical resection than conventional staging - mortality that almost always results from previously undetectable metastases. Furthermore, prospective studies now suggest a predictive benefit in disease-free survival when the assay is used to guide adjuvant chemotherapy decisions in early-stage non-small-cell lung cancer patients.

Lay abstract There is a need for improvement in the way early-stage non-small-cell lung cancers are staged and treated because many patients with 'early-stage' disease suffer high rates of cancer recurrence after surgery. In recent years, a specialized test has been developed to allow better characterization of a tumor's risk of recurrence based on the genes being expressed by tumor cells. Use of this test, in conjunction with standard staging methods, is better able to identify patients at high risk of cancer recurrence after surgery. Evidence suggests that giving chemotherapy to patients at high risk of recurrence after surgery reduces recurrence rates and improves long-term patient survival.

Estimate of the contemporary live-birth prevalence of recurrent 22q11.2 deletions: a cross-sectional analysis from population-based newborn screening.

BACKGROUND: Although pathogenic 22q11.2 deletions are an important cause of developmental delays and lifelong disease burden, their variable and complex clinical expression contributes to under-recognition, delayed molecular diagnosis and uncertainty about prevalence. We sought to estimate the contemporary live-birth prevalence of typical 22q11.2 deletions using a population-based newborn screening sample and to examine data available for associated clinical features. METHODS: Using DNA available from an unbiased sample of about 12% of all dried blood spots collected for newborn screening in Ontario between January 2017 and September 2018, we prospectively screened for 22q11.2 deletions using multiplex quantitative polymerase chain reaction assays and conducted independent confirmatory studies. We used cross-sectional analyses to compare available clinical and T-cell receptor excision circle (TREC, used in newborn screening for severe combined immunodeficiency) data between samples with and without 22q11.2 deletions. RESULTS: The estimated minimum prevalence of 22q11.2 deletions was 1 in 2148 (4.7 per 10 000) live births (95% confidence interval [CI] 2.5 to 7.8 per 10 000), based on a total of 30 074 samples screened, with 14 having confirmed 22q11.2 deletions. Of term singletons, samples with 22q11.2 deletions had significantly younger median maternal age (25.5 v. 32.0 yr, difference -6.5 yr, 95% CI -7 to -2 yr), a greater proportion with small birth weight for gestational age (odds ratio 7.00, 95% CI 2.36 to 23.18) and lower median TREC levels (108.9 v. 602.5 copies/3 µL, p < 0.001). INTERPRETATION: These results indicate that the 22q11.2 deletion syndrome is one of the most common of rare genetic conditions and may be associated with relatively younger maternal ages and with prenatal growth abnormalities. The findings support the public health importance of early - prenatal and neonatal - diagnosis that would enable prompt screening for and management of well-known actionable features associated with 22q11.2 deletions.

The Dual/Global Value of SARS-CoV-2 Genome Surveillance on Migrants Arriving to Europe via the Mediterranean Routes.

Despite the pandemic, 34,154 migrants, refugees or asylum-seekers landed in Sicily (Italy) in 2020, representing the main point of entry by sea into Europe. The SARS-CoV-2 surveillance program among migrants arriving to Sicily via the Mediterranean Sea, made by the combination of clinical examination and molecular testing, has been integrated by full-genome sequencing strains using the NGS technology from the last week of February. To date, more than one hundred full-genome strains have been sequenced and 8 different lineages have been identified mostly belonging to the lineages B.1.1.7 and B.1.525. As global access to COVID-19 vaccines should be ensured, the need to provide more detailed information to inform policies and to drive the possible re-engineering of vaccines needed to deal with the challenge of new and future variants should be highlighted.

Effect of Rapid Respiratory Virus Testing on Antibiotic Prescribing Among Children Presenting to the Emergency Department With Acute Respiratory Illness: A Randomized Clinical Trial.

Importance: There is high usage of antibiotics in the emergency department (ED) for children with acute respiratory illnesses. Studies have reported decreased antibiotic use among inpatients with rapid respiratory pathogen (RRP) testing. Objective: To determine whether RRP testing leads to decreased antibiotic use and health care use among children with influenzalike illness (ILI) in an ED. Design, Setting, and Participants: A randomized clinical trial among children aged 1 month to 18 years presenting to an ED with ILI from December 1, 2018, to November 30, 2019, was conducted. Data were analyzed March 23, 2020, to April 2, 2021. All children received a nasopharyngeal swab for RRP testing and were randomized 1:1 to the intervention group or control group (results not given, routine clinical care). Results were available in 45 minutes. Intention-to-treat analyses and modified intention-to-treat (clinician knows results) analyses were conducted using multivariable Poisson regression. Interventions: Rapid respiratory pathogen test results given to clinicians. Main Outcomes and Measures: Antibiotic prescribing was the primary outcome; influenza antiviral prescribing, ED length of stay, hospital admission, and recurrent health care visits were the secondary outcomes. Results: Among 931 ED visits (intervention group, 452 children group and control group, 456 children after exclusion of those not meeting criteria or protocol violations), a total of 795 RRP test results (85%) were positive. The median age of the children was 2.1 years (interquartile range, 0.9-5.6 years); 509 (56%) were boys. Most children (478 [53%]) were Hispanic, 688 children (76%) received government insurance, and 314 (35%) had a high-risk medical condition. In the intention-to-treat intervention group, children were more likely to receive antibiotics (relative risk [RR], 1.3; 95% CI, 1.0-1.7), with no significant differences in antiviral prescribing, medical visits, and hospitalization. In inverse propensity-weighted modified intention-to-treat analyses, children with test results known were more likely to receive antivirals (RR, 2.6; 95% CI, 1.6-4.5) and be hospitalized (RR, 1.8; 95% CI, 1.4-2.5); there was no significant difference in antibiotic prescribing (RR, 1.1; 95% CI, 0.9-1.4). Conclusions and Relevance: The use of RRP testing in the ED for ILI did not decrease antibiotic prescribing in this randomized clinical trial. There is a limited role for RRP pathogen testing in children in this setting. Trial Registration: ClinicalTrials.gov Identifier: NCT03756753.

Impact of molecular testing in advanced melanoma on outcomes in a tertiary cancer center and as reported in a publicly available database.

BACKGROUND: In patients with advanced melanoma (MM), genomic profiling may guide treatment decisions in the frontline setting and beyond as specific tumor mutations can be treated with targeted therapy (TT). The range of panel sizes used to identify targetable mutations (TM) can range from a few dozen to whole exome sequencing (WES). AIM: We investigated the impact of panel size and mutation status on first-line treatment selection and outcomes in MM. METHODS AND RESULTS: We analyzed data for 1109 MM patients from three cohorts: 169 patients at NYULH and profiled with the 50 gene Ion Torrent panel (IT), 195 patients at MSKCC, profiled with the 400-gene MSK-IMPACT panel (MSK-I) and 745 patients at seven different sites profiled with WES. Data for cohorts 2 and 3 were extrapolated from the publicly available cBioPortal. Treatment information was available for 100%, 25%, and 0% of patients in cohort 1, 2, and 3, respectively. BRAF and NRAS were among the top five most commonly mutated genes in the IT and MSK-I, whereas for WES only BRAF was a top five mutation. There was no significant difference in OS for BRAF MUT patients treated with immune checkpoint inhibitors (ICI) vs TT in cohort 1 (P = .19), nor for BRAF MUT patients from cohort 1 treated with ICI vs those from cohort 2 treated with TT (P = .762). CONCLUSION: Public datasets provide population-level data; however, the heterogeneity of reported clinical information limits their value and calls for data standardization. Without evidence of clear clinical benefit of a larger panel size, there is a rationale for adopting smaller, more cost effective panels in MM.

Prenatal molecular testing and diagnosis of Beckwith-Wiedemann syndrome.

OBJECTIVE: The objective of this study was to describe molecular findings and phenotypic features among individuals referred for prenatal Beckwith-Wiedemann syndrome (BWS) testing. METHODS: Molecular diagnostic testing was performed using a sensitive quantitative real-time PCR-based assay capable of detecting mosaic methylation to the level of 3% at IC1 and IC2. Sanger sequencing of CDKN1C was performed in cases with normal methylation. RESULTS: Of the 94 patients tested, a molecular diagnosis was identified for 25.5% of cases; 70.9% of diagnosed cases had loss of methylation at IC2, 4.2% had gain of methylation at IC1, 12.5% had paternal uniparental isodisomy, and 12.5% had CDKN1C loss-of-function variants. Methylation level changes in prenatal cases were significantly greater than changes identified in cases tested after birth. Cases with a prenatal molecular diagnosis had a significantly greater number of BWS-associated phenotypic features. The presence of either macroglossia or placentomegaly was most predictive of a BWS diagnosis. CONCLUSION: Our results support the consensus statement advocating BWS molecular testing for all patients with one or more BWS-associated prenatal features and suggest that low-level mosaic methylation changes may be uncommon among prenatal BWS diagnoses.

Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.

A model based on the quantification of complement C4c, CYFRA 21-1 and CRP exhibits high specificity for the early diagnosis of lung cancer.

Lung cancer screening detects early-stage cancers, but also a large number of benign nodules. Molecular markers can help in the lung cancer screening process by refining inclusion criteria or guiding the management of indeterminate pulmonary nodules. In this study, we developed a diagnostic model based on the quantification in plasma of complement-derived fragment C4c, cytokeratin fragment 21-1 (CYFRA 21-1) and C-reactive protein (CRP). The model was first validated in two independent cohorts, and showed a good diagnostic performance across a range of lung tumor types, emphasizing its high specificity and positive predictive value. We next tested its utility in two clinically relevant contexts: assessment of lung cancer risk and nodule malignancy. The scores derived from the model were associated with a significantly higher risk of having lung cancer in asymptomatic individuals enrolled in a computed tomography (CT)-screening program (OR = 1.89; 95% CI = 1.20-2.97). Our model also served to discriminate between benign and malignant pulmonary nodules (AUC: 0.86; 95% CI = 0.80-0.92) with very good specificity (92%). Moreover, the model performed better in combination with clinical factors, and may be used to reclassify patients with intermediate-risk indeterminate pulmonary nodules into patients who require a more aggressive work-up. In conclusion, we propose a new diagnostic biomarker panel that may dictate which incidental or screening-detected pulmonary nodules require a more active work-up.

Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification.

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.

Molecular profiling of the colon cancer in South-Eastern Romania: Results from the MERCUR study.

ABSTRACT: Colorectal cancer is a heterogeneous disease with multiple epigenetic alterations and different molecular features. The molecular classification is based on 2 major distinct pathways: microsatellite stable pathway and the microsatellite instability pathway. Molecular profiling of colorectal cancer provides important information regarding treatment and prognosis. Aim of the study was to assess the frequency of microsatellite instability in colon cancer and the clinicopathological characteristics of the tumors with high level of microsatellite instability (MSI-H) in our region. The secondary outcome was to assess the frequency of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in colon cancer.The study included 129 patients with colon cancer fit for surgery. Demographic data, clinical and pathological data, immunohistochemistry staining pattern (4 mismatch repair proteins were investigated), and BRAF gene mutations were assessed. According to microsatellite instability status by polymerase chain reaction, patients were divided into 3 groups: microsatellite stable (MSS) = 108 patients, high level of microsatellite instability (MSI-H) = 15 patients and low level of microsatellite instability (MSI-L) = 6 patients. Different clinicopathological comparisons between MSS and MSI-H patients, and between MSS and MSI-L patients were performed.Microsatellite instability was found in 16.3% patients: 11.6% had MSI-H and 4.7% had MSI-L. Significantly more patients in the MSI-H group than in the MSS group were female (P = .01) and had a family history of colon cancer (P < .001). MSI-H and MSI-L groups were associated with the ascending colon location of the tumors, were mostly type G3, T2, and stage I whereas MSS tumors were mostly G2, pT3, and stage III. Overall, BRAF mutations were identified in 18/129 patients (13.9%). BRAF mutant tumors were predominantly associated with MSI-H and MSI-L tumors. Immunohistochemistry had a sensitivity of 76% and a specificity of 89% in detecting MSI tumors and an accuracy of 87.6%.The frequency of microsatellite instability in our study was 16.3%. MSI-H is a distinct molecular phenotype of colon cancer with particular features: female gender, family history of colorectal cancer, a predilection for the ascending colon, poorly differentiated, predominantly T2, and stage I. The frequency of BRAF mutations was 13.9% and mutations were more often present in the MSI tumors.

False negative rate of COVID-19 PCR testing: a discordant testing analysis.

BACKGROUND: COVID-19 is diagnosed via detection of SARS-CoV-2 RNA using real time reverse-transcriptase polymerase chain reaction (rtRT-PCR). Performance of many SARS-CoV-2 rtRT-PCR assays is not entirely known due to the lack of a gold standard. We sought to evaluate the false negative rate (FNR) and sensitivity of our laboratory-developed SARS-CoV-2 rtRT-PCR targeting the envelope (E) and RNA-dependent RNA-polymerase (RdRp) genes. METHODS: SARS-CoV-2 rtRT-PCR results at the Public Health Laboratory (Alberta, Canada) from January 21 to April 18, 2020 were reviewed to identify patients with an initial negative rtRT-PCR followed by a positive result on repeat testing within 14 days (defined as discordant results). Negative samples from these discordant specimens were re-tested using three alternate rtRT-PCR assays (targeting the E gene and N1/N2 regions of the nucleocapsid genes) to assess for false negative (FN) results. RESULTS: During the time period specified, 95,919 patients (100,001 samples) were tested for SARS-CoV-2. Of these, 49 patients were found to have discordant results including 49 positive and 52 negative swabs. Repeat testing of 52 negative swabs found five FNs (from five separate patients). Assuming 100% specificity of the diagnostic assay, the FNR and sensitivity in this group of patients with discordant testing was 9.3% (95% CI 1.5-17.0%) and 90.7% (95% CI 82.6-98.9%) respectively. CONCLUSIONS: Studies to understand the FNR of routinely used assays are important to confirm adequate clinical performance. In this study, most FN results were due to low amounts of SARS-CoV-2 virus concentrations in patients with multiple specimens collected during different stages of infection. Post-test clinical evaluation of each patient is advised to ensure that rtRT-PCR results are not the only factor in excluding COVID-19.

Integrating Xpert MTB/RIF for TB diagnosis in the private sector: evidence from large-scale pilots in Patna and Mumbai, India.

BACKGROUND: Xpert MTB/RIF (Xpert) has been recommended by WHO as the initial diagnostic test for TB and rifampicin-resistance detection. Existing evidence regarding its uptake is limited to public health systems and corresponding resource and infrastructure challenges. It cannot be readily extended to private providers, who treat more than half of India's TB cases and demonstrate complex diagnostic behavior. METHODS: We used routine program data collected from November 2014 to April 2017 from large-scale private sector engagement pilots in Mumbai and Patna. It included diagnostic vouchers issued to approximately 150,000 patients by about 1400 providers, aggregated to 18,890 provider-month observations. We constructed three metrics to capture provider behavior with regards to adoption of Xpert and studied their longitudinal variation: (i) Uptake (ordering of test), (ii) Utilization for TB diagnosis, and (iii) Non-adherence to negative results. We estimated multivariate linear regression models to assess heterogeneity in provider behavior based on providers' prior experience and Xpert testing volumes. RESULTS: Uptake of Xpert increased considerably in both Mumbai (from 36 to 60.4%) and Patna (from 12.2 to 45.1%). However, utilization of Xpert for TB diagnosis and non-adherence to negative Xpert results did not show systematic trends over time. In regression models, cumulative number of Xpert tests ordered was significantly associated with Xpert uptake in Patna and utilization for diagnosis in Mumbai (p-value< 0.01). Uptake of Xpert and its utilization for diagnosis was predicted to be higher in high-volume providers compared to low-volume providers and this gap was predicted to widen over time. CONCLUSIONS: Private sector engagement led to substantial increase in uptake of Xpert, especially among high-volume providers, but did not show strong evidence of Xpert results being integrated with TB diagnosis. Increasing availability and affordability of a technically superior diagnostic tool may not be sufficient to fundamentally change diagnosis and treatment of TB in the private sector. Behavioral interventions, specifically aimed at, integrating Xpert results into clinical decision making of private providers may be required to impact patient-level outcomes.

Rapid Detection of mrp, epf, and sly Genes by Loop-Mediated Isothermal Amplification in Streptococcus suis.

Streptococcus suis remains a serious threat to the worldwide swine industry and human health. In this study, rapid assays for the detection of three common virulence-related factors (mrp, epf, and sly) were developed, evaluated, and applied. Loop-mediated isothermal amplification (LAMP) primers were designed using Primer Explorer V5 software. The sensitivity and specificity of the LAMP assays were determined based on sample turbidity. For all three genes, LAMP assays were performed at 62°C with a reaction time of 60 min. The detection limit of conventional polymerase chain reaction (PCR) was 1 ng/µL, 10 pg/µL, and 100 fg/µL for the epf, sly, and mrp genes, respectively. For the LAMP assays, the detection limits were 10 pg/µL, 10 fg/µL, and 100 fg/µL for epf, sly, and mrp, respectively, representing sensitivities 100-1000 times higher than those of the PCR assay. Furthermore, when the LAMP assays were applied to clinical strains, the results were consistent with those of the PCR assay, confirming the LAMP assays as rapid and reliable detection techniques. In conclusion, the LAMP assays described in this study have the potential to become standard methods to detect the virulence factors mrp, epf, and sly. To the best of our knowledge, this is the first study to report the application of LAMP to detect the mrp, epf, and sly genes.

Implementation of Early Next-Generation Sequencing for Inborn Errors of Immunity: A Prospective Observational Cohort Study of Diagnostic Yield and Clinical Implications in Dutch Genome Diagnostic Centers.

Objective: Inborn errors of immunity (IEI) are a heterogeneous group of disorders, affecting different components of the immune system. Over 450 IEI related genes have been identified, with new genes continually being recognized. This makes the early application of next-generation sequencing (NGS) as a diagnostic method in the evaluation of IEI a promising development. We aimed to provide an overview of the diagnostic yield and time to diagnosis in a cohort of patients suspected of IEI and evaluated by an NGS based IEI panel early in the diagnostic trajectory in a multicenter setting in the Netherlands. Study Design: We performed a prospective observational cohort study. We collected data of 165 patients with a clinical suspicion of IEI without prior NGS based panel evaluation that were referred for early NGS using a uniform IEI gene panel. The diagnostic yield was assessed in terms of definitive genetic diagnoses, inconclusive diagnoses and patients without abnormalities in the IEI gene panel. We also assessed time to diagnosis and clinical implications. Results: For children, the median time from first consultation to diagnosis was 119 days versus 124 days for adult patients (U=2323; p=0.644). The median turn-around time (TAT) of genetic testing was 56 days in pediatric patients and 60 days in adult patients (U=1892; p=0.191). A definitive molecular diagnosis was made in 25/65 (24.6%) of pediatric patients and 9/100 (9%) of adults. Most diagnosed disorders were identified in the categories of immune dysregulation (n=10/25; 40%), antibody deficiencies (n=5/25; 20%), and phagocyte diseases (n=5/25; 20%). Inconclusive outcomes were found in 76/165 (46.1%) patients. Within the patient group with a genetic diagnosis, a change in disease management occurred in 76% of patients. Conclusion: In this cohort, the highest yields of NGS based evaluation for IEI early in the diagnostic trajectory were found in pediatric patients, and in the disease categories immune dysregulation and phagocyte diseases. In cases where a definitive diagnosis was made, this led to important disease management implications in a large majority of patients. More research is needed to establish a uniform diagnostic pathway for cases with inconclusive diagnoses, including variants of unknown significance.