Tapestry: A Single-Round Smart Pooling Technique for COVID-19 Testing

AbstractThe COVID-19 pandemic has strained testing capabilities worldwide. There is an urgent need to find economical and scalable ways to test more people. We present Tapestry, a novel quantitative nonadaptive pooling scheme to test many samples using only a few tests. The underlying molecular diagnostic test is any real-time RT-PCR diagnostic panel approved for the detection of the SARS-CoV-2 virus. In cases where most samples are negative for the virus, Tapestry accurately identifies the status of each individual sample with a single round of testing in fewer tests than simple two-round pooling. We also present a companion Android application BYOM Smart Testing which guides users through the pipetting steps required to perform the combinatorial pooling. The results of the pooled tests can be fed into the application to recover the status and estimated viral load for each individual sample.NOTEThis protocol has been validated with in vitro experiments that used synthetic RNA and DNA fragments and additionally, its expected behavior has been confirmed using computer simulations. Validation with clinical samples is ongoing. We are looking for clinical collaborators with access to patient samples. Please contact the corresponding author if you wish to validate this protocol on clinical samples.

Download Full-text

High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

10.1101/2020.05.14.097295 ◽

2020 ◽

Cited By ~ 2

Author(s):

Rania Francis ◽

Marion Le Bideau ◽

Priscilla Jardot ◽

Clio Grimaldier ◽

Didier Raoult ◽

...

Keyword(s):

High Speed ◽

Large Scale ◽

Vaccine Development ◽

Work Load ◽

Drug Susceptibility Testing ◽

High Content Screening ◽

Clinical Samples ◽

Rt Pcr ◽

Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

Download Full-text

Comparison of commercial RT-PCR diagnostic kits for COVID-19

10.1101/2020.04.22.056747 ◽

2020 ◽

Cited By ~ 3

Author(s):

Puck B. van Kasteren ◽

Bas van der Veer ◽

Sharon van den Brink ◽

Lisa Wijsman ◽

Jørgen de Jonge ◽

...

Keyword(s):

Limit Of Detection ◽

Cross Reactivity ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Specific Method ◽

Preliminary Evaluation ◽

Rt Pcr ◽

Accurate Identification ◽

Clinical Sensitivity ◽

Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

Download Full-text

Increasing Cytomegalovirus Detection Rate from Respiratory Tract Specimens by a New Laboratory-Developed Automated Molecular Diagnostic Test

Microorganisms ◽

10.3390/microorganisms8071063 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1063

Author(s):

Huey-Pin Tsai ◽

Chun-Sheng Yeh ◽

I-Ting Lin ◽

Wen-Chien Ko ◽

Jen-Ren Wang

Keyword(s):

Respiratory Tract ◽

Detection Rate ◽

Turnaround Time ◽

The Other ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Clinical Specimens ◽

Automated Assay ◽

Blood Specimens ◽

Molecular Diagnostic Test

Lots of automated molecular methods for detecting cytomegalovirus (CMV) DNA in the blood are available, but seldom for various clinical specimens. This study was designed to establish a highly sensitive automated assay to detect CMV DNA in non-blood specimens. We designed a new QMT assay using QIAGEN artus CMV RG polymerase chain reaction (Q-CMV PCR) kit applied on the BD MAX system and compared with the other assays, including an RGQ assay (LabTurbo auto-extraction combined Q-CMV PCR kit on Rotor-Gene-Q instrument), and in-house PCR assay. A total of 1067 various clinical samples, including 426 plasma, 293 respiratory tract specimens (RTS), 127 stool, 101 cerebral spinal fluid, 90 vitreous humours were analysed. Examining CMV DNA in simultaneous specimens of the same immunocompromised patient with respiratory symptoms, the detection rate of RTS (93.6%, 88/94) was significant higher than plasma (65.9%, 62/94). The positive rates for plasma samples with a low CMV viral load (<137 IU/mL) and diagnostic sensitivity of QMT, RGQ, and in-house assays were 65% and 99.1%, 45% and 100%, 5% and 65.5%, respectively. The QMT assay performs better, with shorter operational and turnaround time than the other assays, enabling the effective and early detection of CMV infection in various clinical specimens, particularly for RTS.

Download Full-text

Comparison of SARS-CoV-2 RT-PCR on a high-throughput molecular diagnostic platform and the cobas SARS-CoV-2 test for the diagnostic of COVID-19 on various clinical samples

Pathogens and Disease ◽

10.1093/femspd/ftaa061 ◽

2020 ◽

Vol 78 (8) ◽

Cited By ~ 1

Author(s):

Onya Opota ◽

René Brouillet ◽

Gilbert Greub ◽

Katia Jaton

Keyword(s):

High Throughput ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Test Results ◽

Rt Pcr ◽

Clinical Specimens ◽

Rapid Spread ◽

Bronchial Aspirate ◽

Very High

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 > 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.

Download Full-text

OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution

Biomedicines ◽

10.3390/biomedicines9101314 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1314

Author(s):

Eduarda Carvalho-Correia ◽

Carla Calçada ◽

Fernando Branca ◽

Nuria Estévez-Gómez ◽

Loretta De Chiara ◽

...

Keyword(s):

Cross Reactivity ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Analytical Sensitivity ◽

S Gene ◽

Genomic Knowledge ◽

Gene Assay ◽

One Step

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide–sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

Download Full-text

Laboratory Diagnostic Options and Challenges for Chikungunya Viruses

Pulse ◽

10.3329/pulse.v10i1.38607 ◽

2018 ◽

Vol 10 (1) ◽

pp. 18-24

Author(s):

MM Rahman

Keyword(s):

Viral Disease ◽

Medical Attention ◽

Molecular Diagnostic ◽

Diagnostic Tools ◽

Clinical Samples ◽

Rt Pcr ◽

Specific Pcr ◽

Highly Sensitive

Background: Chikungunya infection is a Aedes mosquito transmitted viral disease caused by Chikungunya virus (CHIKV), a member of the Alphavirus genus. It is an important human pathogen that causes a syndrome characterized by fever, chills, headache and severe joint pain usually of the smaller joints, with or without swelling. Though the disease is almost self-limiting, during the recent outbreak CHIKV was also found to cause long-term arthralgia, neurological disease and even few fatalities. Despite the fact that CHIKV is associated with epidemics of unprecedented magnitude, only a few specific serological and molecular diagnostic tools are available.Objective: CHIKV diagnosis is essentially based on virus isolation, reverse transcription (RT)-PCR and ELISA assays. The gold standard of CHIKV diagnosis is culture, however, required facilities and skills are not available in any routine laboratory in the country. Highly sensitive and specific PCR assays for CHIKV have been developed and commercially available.Conclusion: Although the reagents and equipment are costly for widespread use RT-PCR is the method of choice for the early detection and confirmation of virus in clinical samples as most acutely infected patients seek medical attention within the first few days of illness when role of serology based diagnosis is minimum.Pulse Vol.10 January-December 2017 p.18-24

Download Full-text

An Observational Laboratory-Based Assessment of SARS-CoV-2 Molecular Diagnostics in Benin, Western Africa

mSphere ◽

10.1128/msphere.00979-20 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Anna-Lena Sander ◽

Anges Yadouleton ◽

Andres Moreira-Soto ◽

Carine Tchibozo ◽

Gildas Hounkanrin ◽

...

Keyword(s):

Genomic Diversity ◽

Clinical Samples ◽

Central Laboratory ◽

Rt Pcr ◽

Target Domain ◽

African Countries ◽

Protein Variant ◽

Clinical Sensitivity ◽

Western Africa

ABSTRACT Information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread in Africa is limited by insufficient diagnostic capacity. Here, we assessed the coronavirus disease (COVID-19)-related diagnostic workload during the onset of the pandemic in the central laboratory of Benin, Western Africa; characterized 12 SARS-CoV-2 genomes from returning travelers; and validated the Da An RT-PCR-based diagnostic kit that is widely used across Africa. We found a 15-fold increase in the monthly laboratory workload due to COVID-19, dealt with at the cost of routine activities. Genomic surveillance showed near-simultaneous introduction of distinct SARS-CoV-2 lineages termed A.4 and B.1, including the D614G spike protein variant potentially associated with higher transmissibility from travelers from six different European and African countries during March-April 2020. We decoded the target regions within the ORF1ab and N genes of the Da An dual-target kit by MinION-based amplicon sequencing. Despite relatively high similarity between SARS-CoV-2 and endemic human coronaviruses (HCoVs) within the ORF1ab target domain, no cross-detection of high-titered cell culture supernatants of HCoVs was observed, suggesting high analytical specificity. The Da An kit was highly sensitive, detecting 3.2 to 9.0 copies of target-specific in vitro transcripts/reaction. Although discrepant test results were observed in low-titered clinical samples, clinical sensitivity of the Da An kit was at least comparable to that of commercial kits from affluent settings. In sum, virologic diagnostics are achievable in a resource-limited setting, but unprecedented pressure resulting from COVID-19-related diagnostics requires rapid and sustainable support of national and supranational stakeholders addressing limited laboratory capacity. IMPORTANCE Months after the start of the COVID-19 pandemic, case numbers from Africa are surprisingly low, potentially because the number of SARS-CoV-2 tests performed in Africa is lower than in other regions. Here, we show an overload of COVID-19-related diagnostics in the central laboratory of Benin, Western Africa, with a stagnating average number of positive samples irrespective of daily sample counts. SARS-CoV-2 genomic surveillance confirmed a high genomic diversity in Benin introduced by travelers returning from Europe and other African countries, including early circulation of the D614G spike mutation associated with potentially higher transmissibility. We validated a widely used RT-PCR kit donated by the Chinese Jack Ma Foundation and confirmed high analytical specificity and clinical sensitivity equivalent to tests used in affluent settings. Our assessment shows that although achievable in an African setting, the burden from COVID-19-related diagnostics on national reference laboratories is very high.

Download Full-text

TRIM32 triggers β-catenin signaling through ubiquitylation of AXIN1 to promote inflammatory factor-induced apoptosis of rat nucleus pulposus cells

AJP Cell Physiology ◽

10.1152/ajpcell.00386.2019 ◽

2020 ◽

Vol 318 (3) ◽

pp. C695-C703

Author(s):

Fei Chen ◽

Qunfeng Guo ◽

Qunxiang Chen ◽

Zhao Han ◽

Xin Zhou ◽

...

Keyword(s):

Nucleus Pulposus ◽

Ubiquitin Ligase ◽

Clinical Samples ◽

Induction Effect ◽

Inflammatory Factor ◽

Rt Pcr ◽

Nucleus Pulposus Cells ◽

Tnf Α ◽

Induced Apoptosis

The dysregulation of ubiquitin ligase is the cause of many human diseases. Tripartite motif protein 32 (TRIM32) is an E3 ubiquitin ligase whose role in nucleus pulposus (NP) cell apoptosis is unclear. The expression of TRIM family protein and β-catenin in 40 NP tissue samples was detected by RT-PCR. Interleukin (IL)-1β or tumor necrosis factor (TNF)-α was used to treat rat NP cells. Knockdown and overexpression of Trim32 were achieved using specific siRNA and recombinant plasmids. Western blotting, RT-PCR, and flow cytometry were used to assess the expression of TRIM32/β-catenin and the apoptosis rate of NP cells. Coimmunoprecipitation was adopted to analyze the possible interactions between AXIN1 and TRIM32. In clinical samples, TRIM32 expression was of positive relevance with the expression of CTNNB1 (β-catenin). In vitro, apoptosis of IL-1β- or TNF-α-treated rat NP cells was induced through upregulated Trim32 expression and activated β-catenin signaling, whereas Trim32 siRNA and inhibition of β-catenin reversed the induction effect of cytokines. Further studies indicated that TRIM32 activated the β-catenin signaling pathway through ubiquitination of AXIN1, thereby regulating apoptosis. Collectively, this study reveals that TRIM32 promotes inflammatory factor-induced apoptosis of rat NP cells, in part by direct degradation of AXIN1 to trigger β-catenin signaling.

Download Full-text

Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00743-20 ◽

2020 ◽

Vol 58 (8) ◽

Cited By ~ 43

Author(s):

Wei Zhen ◽

Ryhana Manji ◽

Elizabeth Smith ◽

Gregory J. Berry

Keyword(s):

New York ◽

Respiratory Infections ◽

Clinical Performance ◽

Control Measures ◽

Molecular Diagnostic ◽

Analytical Sensitivity ◽

Diagnostic Assays ◽

Infection Control Measures ◽

Diagnostic Panel

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel human coronavirus that causes coronavirus disease 2019 (COVID-19), was first discovered in December 2019 as the cause of an outbreak of pneumonia in the city of Wuhan, Hubei province, China. The clinical presentation of COVID-19 is fairly nonspecific, and symptoms overlap those of other seasonal respiratory infections concurrently circulating in the population. Furthermore, it is estimated that up to 80% of infected individuals experience mild symptoms or are asymptomatic, confounding efforts to reliably diagnose COVID-19 empirically. To support infection control measures, there is an urgent need for rapid and accurate molecular diagnostics to identify COVID-19-positive patients. In the present study, we evaluated the analytical sensitivity and clinical performance of the following four SARS-CoV-2 molecular diagnostic assays granted emergency use authorization by the FDA using nasopharyngeal swabs from symptomatic patients: the New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel (modified CDC) assay, the Simplexa COVID-19 Direct (Diasorin Molecular) assay, GenMark ePlex SARS-CoV-2 (GenMark) assay, and the Hologic Panther Fusion SARS-CoV-2 (Hologic) assay. This information is crucial for both laboratories and clinical teams as decisions on which testing platform to implement are made.

Download Full-text