SARS-CoV-2 detection in multi-sample pools in a real pandemic scenario: a screening strategy of choice for active surveillance

Background The current COVID-19 pandemic has overloaded the diagnostic capacity of laboratories by the gold standard method rRT-PCR. This disease has a high spread rate and almost a quarter of infected individuals never develop symptoms. In this scenario, active surveillance is crucial to stop the virus propagation. Methods Between July 2020 and April 2021, 11580 oropharyngeal swab samples collected in closed and semi-closed institutions were processed for SARS-CoV-2 detection in pools, implementing this strategy for the first time in Cordoba, Argentina. Five-sample pools were constituted before nucleic acid extraction and amplification by rRT-PCR. Comparative analysis of cycle threshold (Ct) values from positive pools and individual samples along with a cost-benefit report of the whole performance of the results was performed. Results From 2314 5-sample pools tested, 158 were classified as positive (6.8%), 2024 as negative (87.5%), and 132 were categorized as indeterminate (5.7%). The Ct value shift due to sample dilution showed an increase in Ct of 2.6 ±1.53 cycles for N gene and 2.6 ±1.78 for ORF1ab gene. Overall, 290 pools were disassembled and 1450 swabs were analyzed individually. This strategy allowed correctly identifying 99.8% of the samples as positive (7.6%) or negative (92.2%), avoiding the execution of 7,806 rRT-PCR reactions which represents a cost saving of 67.5%. Conclusion This study demonstrates the feasibility of pooling samples to increase the number of tests performed, helping to maximize molecular diagnostic resources and reducing the work overload of specialized personnel during active surveillance of the COVID-19 pandemic.

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SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification

Micromachines ◽

10.3390/mi12020197 ◽

2021 ◽

Vol 12 (2) ◽

pp. 197

Author(s):

Meiqing Liu ◽

Haoran Li ◽

Yanwei Jia ◽

Pui-In Mak ◽

Rui P. Martins

Keyword(s):

Signal Amplification ◽

Incubation Temperature ◽

Dna Probes ◽

Detection Sensitivity ◽

N Gene ◽

Rna Detection ◽

Gold Standard Method ◽

Complementary Method ◽

Amplification Method ◽

Virus Rna

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.

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The Arizona Healthcare, Emergency Response, and Other Essential Workers Study (AZ HEROES): Protocol (Preprint)

10.2196/preprints.28925 ◽

2021 ◽

Author(s):

Karen Lutrick ◽

Katherine D. Ellingson ◽

Zoe Baccam ◽

Patrick Rivers ◽

Shawn Beitel ◽

...

Keyword(s):

Active Surveillance ◽

Emergency Response ◽

Natural Infection ◽

Diagnostic Testing ◽

Vaccine Dose ◽

Demographic Characteristics ◽

Healthcare Personnel ◽

Molecular Diagnostic ◽

Vaccination History ◽

Baseline Information

UNSTRUCTURED Background: The Arizona Healthcare, Emergency Response, and Other Essential workers Study (AZ HEROES) aims to examine the epidemiology of SARS-CoV-2 infection and COVID-19 illness among adults with high occupational exposure risk. Methods: Eligible participants include Arizona residents aged 18–85 years who work at least 20 hours per week in an occupation involving regular direct contact (within three feet) with others. Recruitment goals are stratified by demographic characteristics (50% aged 40 or older, 50% women, and 50% Hispanic or American Indian), by occupation (40% healthcare personnel, 30% first responders, and 30% other essential workers), and by prior SARS-CoV-2 infection (with up to 50% seropositive at baseline). Information on sociodemographics, health and medical history, vaccination status, exposures to individuals with suspected or confirmed SARS-CoV-2 infection, use of personal protective equipment, and perceived risks are collected at enrollment and updated through quarterly surveys. Every week, participants complete active surveillance for COVID-19–like illness (CLI) and self-collect nasal swabs. Additional self-collected nasal swab and saliva specimens are collected in the event of CLI onset. Respiratory specimens are sent to Marshfield Laboratories and tested for SARS-CoV-2 by real-time reverse transcription polymerase chain reaction (rRT-PCR) assay. CLI symptoms and impact on work and productivity are followed through illness resolution. Serum specimens are collected every 3 months and additional sera are collected following incident rRT-PCR positivity and after each COVID-19 vaccine dose. Incidence of SARS-CoV-2 infections will be calculated by person-weeks at risk and compared by occupation and demographic characteristics and by seropositivity status and infection and vaccination history. Discussion: AZ HEROES is unique in aiming to recruit a diverse sample of essential workers and prospectively following strata of SARS-CoV-2 seronegative and seropositive adults. Survey results combined with active surveillance data on exposure, CLI, weekly molecular diagnostic testing, and periodic serology will be used to estimate the incidence of symptomatic and asymptomatic SARS-CoV-2 infection, assess the intensity and durability of immune responses to natural infection and COVID-19 vaccination, and contribute to the evaluation of COVID-19 vaccine effectiveness.

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An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

10.1101/2022.01.06.22268835 ◽

2022 ◽

Author(s):

Bongkot Ngamsom ◽

Alexander Iles ◽

Moses Kamita ◽

Racheal Kimani ◽

Pablo Rodriguez-Mateos ◽

...

Keyword(s):

Saliva Sample ◽

Rna Extraction ◽

Lab On A Chip ◽

Cross Reactivity ◽

Contact Tracing ◽

Molecular Diagnostic ◽

N Gene ◽

Sample Collection ◽

Middle Income ◽

Specificity And Sensitivity

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

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SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a widely used commercial multiplex PCR assay

10.1101/2021.03.23.21254171 ◽

2021 ◽

Author(s):

Paul Wollschlaeger ◽

Nadja Gerlitz ◽

Daniel Todt ◽

Stephanie Pfaender ◽

Thomas Bollinger ◽

...

Keyword(s):

Rdrp Gene ◽

N Gene ◽

Pcr Assay ◽

Roc Curve Analysis ◽

Ct Value ◽

Specific Pcr ◽

Multiplex Pcr Assay ◽

Gene Coding ◽

Value Shift ◽

Rapid Tool

Objectives. Increased importance in detection and surveillance of SARS-CoV-2 has been demonstrated due to the emergence of variants of concern (VOCs). In this study we evaluated if a commercially available real-time SARS-CoV-2 PCR assay can identify B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared to the S or RdRP gene. Methods. Patients samples with confirmed B.1.1.7 variant by whole-genome sequencing and variant-specific PCR (n=48) and non-B.1.1.7 samples (n=53) were tested by the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay for presence of S, RdRP and N gene of SARS CoV-2. The N gene coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) were cloned into pCR-TOPO vectors and Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay was performed. Results. All studied B.1.1.7 patient samples showed significantly higher Ct values (delta 6-10, N-gene dropout on Ct values >29) in the N gene compared to the respective values of S and RdRP gene. Receiver operating characteristic (ROC) curve analysis resulted in 100% sensitivity and specificity for delta Ct N/RdRP and delta Ct N/S. As a result of the reversed genetic experiments we found also the shift in Ct values for the 3L variant N-gene. Conclusions. N gene dropout or Ct value shift is specific for B.1.1.7 positive samples using the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

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Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples

10.1101/2020.10.09.20203430 ◽

2020 ◽

Author(s):

Vijay J. Gadkar ◽

David M. Goldfarb ◽

Virginia Young ◽

Nicole Watson ◽

Linda Hoang ◽

...

Keyword(s):

Nucleic Acid ◽

Standard Method ◽

Molecular Diagnostic ◽

Heat Inactivation ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Sample Type ◽

Mouth Rinse ◽

Nucleic Acid Extraction ◽

Single Tube

ABSTRACTBackgroundSaline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction.MethodsClinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay.ResultsA total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (CT) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔCT [ΔCT=CT(Direct PCR)-CT(Extracted RNA)], for each of the gene targets were: S (ΔCT= +4.24), ORF8 (ΔCT=+2.63), N1 (ΔCT=+2.74) and N2 (ΔCT=+5.74). The ΔCT for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method.ConclusionOur Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.

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Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong

Journal of Clinical Microbiology ◽

10.1128/jcm.00936-20 ◽

2020 ◽

Vol 58 (8) ◽

Author(s):

Jonathan Hon-Kwan Chen ◽

Cyril Chik-Yan Yip ◽

Jasper Fuk-Woo Chan ◽

Rosana Wing-Shan Poon ◽

Kelvin Kai-Wang To ◽

...

Keyword(s):

Hong Kong ◽

High Throughput ◽

Clinical Performance ◽

Diagnostic System ◽

Molecular Diagnostic ◽

N Gene ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Gene Target ◽

E Gene

ABSTRACT In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

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Optimizing SARS-CoV-2 Molecular Diagnostic Using N Gene Target

SSRN Electronic Journal ◽

10.2139/ssrn.3727304 ◽

2020 ◽

Author(s):

Raphael Contelli Klein ◽

Mary Hellen Fabres-Klein ◽

Larissa Gomes Barbosa ◽

Lívia Vasconcelos Gonzaga Knnup ◽

Larissa Paola Rodrigues Venâncio ◽

...

Keyword(s):

Molecular Diagnostic ◽

N Gene ◽

Gene Target

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A cost-benefit analysis for sonographic screening for renal tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.6_suppl.665 ◽

2018 ◽

Vol 36 (6_suppl) ◽

pp. 665-665

Author(s):

Shmuel Roizman ◽

Moshe Leshno ◽

Miki Haifler ◽

Yishai Hode Rappaport ◽

Amnon Zisman

Keyword(s):

Targeted Therapy ◽

Cost Effectiveness ◽

Cost Benefit ◽

Cost Effective ◽

Screening Strategy ◽

Renal Tumors ◽

Stage Migration ◽

Mathematical Framework ◽

Abdominal Ultrasonography ◽

The Cost

665 Background: In the last 2 decades, the rates of metastatic Renal Cell Carcinoma (RCC) at diagnosis declined from 33% to 17%This fact is attributed to massive penetration of cross sectional imaging leading to a marked stage migration. The cost of targeted therapy for metastatic RCC patients is very high. These trends led us to hypothesize that screening for RCC with ultrasound may be cost effective. Objective: To assess the cost effectiveness of screening with ultrasound for renal tumors in the general population over 60 years of age. Methods: Using the Markov model, a mathematical framework was set up describing the course of disease with and without screening for RCC using abdominal ultrasonography. Quality Adjusted Life Year (QALY) and financial costs were the outputs of the model. Results: Average costs for the screening strategy was 137.4 U$ and for non-screening was 31.4 U$. Screening and non-screening strategy would add an average of 21.7396 and 21.7385 QALY, respectively. An increase of 0.001 QALY equates to Incremental Cost Effectiveness Ratio (ICER) of 86,4 U$ per QALY, Currently, the cost which is considered cost effective for 1 QALY point is approximately 27,548.21 U$. The two variables most influential on the model output were prevalence of RCC and US cost. Conclusions: To our knowledge, this is the sole cost benefit screening study performed for RCC in the targeted therapy era. Screening for renal tumors using abdominal ultrasonography at a cost of 35.81 U$ per exam is cost effective. Our findings are highly suggestive that early screening for RCC may be cost effective for preventing RCC metastatic disease and nevertheless will save lives.

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Effects of diverse materials-based methods on DNA extraction for Clostridium difficle from stool samples

Materials Express ◽

10.1166/mex.2019.1520 ◽

2019 ◽

Vol 9 (5) ◽

pp. 509-516 ◽

Cited By ~ 3

Author(s):

Ziqi Xiao ◽

Gaojian Yang ◽

Deng Yan ◽

Song Li ◽

Zhu Chen ◽

...

Keyword(s):

Nucleic Acid ◽

Dna Extraction ◽

Magnetic Beads ◽

Diagnostic Technique ◽

Proteinase K ◽

Molecular Diagnostic ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Spin Column ◽

Stool Samples

Nosocomial infections, including Clostridium difficile infection (CDI), and their fatality rates have increased in the past few decades. Despite emerging molecular diagnostic technologies with rapid, accurate outcomes, nucleic acid extraction from stool samples remains the first limiting step before downstream applications. Commercial nucleic acid extraction kits greatly decrease labor and time requirements, and also provide nucleic acid preparations with higher quality and purity for enzyme digestion analysis or genotyping. The magnetic bead based technique is a novel method compared with the conventional spin-column method, and currently has widespread use in nucleic acid extraction. We evaluated five DNA extraction kits with magnetic beads using materials with various properties (particle size, concentration of magnetic beads, grinding beads) and reagents (proteinase K, lysozyme, isopropanol, and absolute ethanol) to determine the cost, hands-on time, number of essential operations, and quality and purity of the DNA preparations, compared with those obtained using the QIAamp Fast DNA Stool Mini Kit. The six DNA extraction kits yielded A260/280 ratios ranging from 0.85 to 1.9 (average 1.57), and concentrations from 3.70 to 108.09 ng/μL (average 34.64 ng/μL). All the DNA samples had acceptable downstream application effects, except for those obtained using the TIANGEN Magnetic Soil and Stool DNA Kit. However, gel electrophoresis analysis of the DNA samples resulted in a light strip on the gel, indicating that the proteinaceous contaminant may not have been removed completely. A rapid and accurate molecular diagnostic technique could allow for more suitable treatment and prognosis outcomes for inpatients, depending, in large part, on the quality and purity of DNA preparations, which are frequently neglected. Our study focused on the quality of commercial kits with a primary focus on the treatment of stool samples and molecular diagnostic applications.

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Use of predictive tools in the management of COVID-19 patients: a key role of clinical laboratories

Advances in Laboratory Medicine / Avances en Medicina de Laboratorio ◽

10.1515/almed-2020-0059 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Carla Martín Grau ◽

Clara Benavent Bofill ◽

Ester Picó-Plana ◽

Gemma Recio Comí ◽

Margarida Terrón-Puig ◽

...

Keyword(s):

Leukocyte Count ◽

Area Under The Curve ◽

Molecular Diagnostic ◽

Gold Standard Method ◽

Inflammatory Parameters ◽

Emergency Units ◽

Polymerase Chain ◽

Clinical Illness ◽

Molecular Diagnostic Test

AbstractObjectivesCoronavirus disease 2019 (COVID-19) is widely spreading and represents a critical threat to global health. In the fight against this pandemic, provincial hospitals urgently need rapid diagnostic of COVID-19 infected patients to avoid collapsing of emergency units. However, the high demand of patients with severe acute respiratory symptoms limits the fast delivery of results by the gold standard method reverse transcription-polymerase chain reaction real time (rRT-PCR) for the identification of COVID-19 positive pneumonia. The principal aim is to find other useful laboratory indicators to assist rRT-PCR tests and to help controlling of this outbreak.MethodsBlood, coagulation and inflammatory parameters were collected from a total of 309 patients classified as negative (128) and positive (181) rRT-PCR test groups. Patients were classified as positive by molecular diagnostic test.ResultsLeukocyte count (WBC), neutrophils count, lymphocytes count and lactate dehydrogenase (LDH) were statistically different between both groups of patients. The use of LDH/WBC ratio increases the diagnostic performance with the best area under the curve (0.783), sensibility (82%) and the best percentage (80.5%) of correctly identified COVID-19 positive patients.ConclusionsThe combination of predictive LDH/WBC ratio with clinical illness features could help in medical management of patients and improve the technical resources of hospitals, especially in a critical scenario with a large shortage of medical equipment and lack of reagents for performing rRT-PCR.

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