2019-nCoV Assay Research Articles

A-272 Evaluation of a Real-Time Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Respiratory Pathogens in Nasopharyngeal Specimens

Abstract Background Respiratory infections present a significant challenge due to the diverse range of pathogens involved and the possibility of concurrent infections, emphasizing the importance of rapid and accurate detection for effective treatment. Advancements in molecular methods have provided cost-effective and reliable solutions for the identification of respiratory pathogens. In this study, we aimed to assess performance characteristics of the Seegene Allplex™ 2019-nCoV Assay and Seegene Novaplex™ Respiratory Panel Assays for simultaneous amplification and detection of 22 respiratory pathogens using real-time multiplex polymerase chain reaction (PCR). Methods Nucleic acid extraction was performed using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, MA) on the CyBio® FeliX liquid handler (Analytik Jena, Germany). PCR amplification and detection were performed using the Seegene Novaplex™ Respiratory Panels 1A, 2, 3, PneumoBacter Assay, and Allplex™ 2019-nCoV Assay (Seegene Inc, South Korea) on the CFX96™ System (Bio-Rad Laboratories, CA). The assays utilize Seegene MuDT™ technology that allows for the detection of multiple targets in a single channel without the need for melt curve analysis. For method validation, precision studies were performed within-run and over 4 days. The limit of detection was verified by using plasmids from Seegene containing the target sequence for each pathogen at known concentrations. Method comparison studies were carried out by extracting and analyzing a range of 41 to 79 split samples for each pathogen against the BioFire® Respiratory 2.1 Panel (bioMérieux, France), cobas® Influenza A/B & RSV Assay on the cobas® liat system (Roche Diagnostics, IN), and Lyra® SARS-CoV-2 Assay (Quidel, CA) on the CFX96™ system. Additional accuracy studies were performed by spiking negative patient samples with standards from ZeptoMetrix® (Antylia Scientific, IL) containing known concentrations of the targeted respiratory pathogens. Results Precision studies demonstrated consistent and reproducible results for all pathogens. The limit of detection was verified at 1000 copies/reaction for human parainfluenza virus 4 and human metapneumovirus, and at 100 copies/reaction for all other pathogens. Results of the spike-and-recovery studies were consistent with expected results for all targets. Patient correlation studies showed 100% agreement with expected pathogen identification for 19 pathogens, and greater than 97.9% agreement for influenza B virus, respiratory syncytial virus, human parainfluenza virus 4, and human coronavirus 229E. Conclusions The Seegene Allplex™ 2019-nCoV Assay and Novaplex™ Respiratory Panel Assays offer a rapid and reliable method for detecting respiratory pathogens in nasopharyngeal specimens. The capability of the assays to detect multiple targets in a single well enhances efficiency, cost-effectiveness, and turnaround time.

RAPID DETECTION OF SARS COV-2 INFECTION IN COMPARISION WITH RT-PCR IN TERTIARY CARE HOSPITAL

Objectives: The goal of the present study was to assess the SARS-CoV-2 antigen detection test’s performance features and compare them to the real-time reverse transcription polymerase chain reaction (RT-PCR) test, the gold standard test for the diagnosis of COVID-19 cases. Methods: From October 2020 to May 2021, patients attending the OPD, including those undergoing surgery, at a Tertiary Care Teaching Hospital in Telangana provided 1000 respiratory samples, primarily nasopharyngeal swabs. A skilled technician had collected two nasopharyngeal swabs from each person in a COVID sample collection room while wearing personal protective equipment and following strict infection control procedures. One swab was used for the rapid antigen test given by the standard Q COVID-19 Ag test kit and placed into the extraction buffer tube. Second swab was kept in the viral transport medium and used for Allplex™ 2019-nCoV Assay (Seegene, Korea), which targets envelope gene (E), and RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS CoV-2, was used for SARS-CoV-2 RNA detection according to the manufacturer’s instructions. Results: Out of 1000 samples tested for COVID-19, 623 (63.7%) were males and 377 (36.3%) were females. Out of 1000 samples, 347 samples were RT-PCR positive and 653 were RT-PCR negative. Out of 347 RT-PCR samples positive, 341 were Rapid antigen test positive samples and six were negative. Overall sensitivity and specificity are 98.27% and 99.85%, respectively. Conclusion: The real-time RT-PCR assay’s sensitivity and specificity were comparable to those of the rapid assay for SARS-CoV-2 antigen detection. It can be utilized for contact tracing measures to control the COVID-19 pandemic in places such as border crossings, airports, interregional bus and train stations, and mass testing campaigns needing quick findings. This is especially true in areas with a high prevalence of the disease.

Comparison of Two Molecular Diagnostic Tests for COVID-19: Abbott RealTime SARS-CoV-2 and Allplex™ 2019-nCoV, in the Epidemic Context in Senegal

Background: The persistence of the rapid spread of the COVID-19 pandemic is linked to the appearance of several variants of SARS-CoV2 with an impact on biological diagnosis, treatment and vaccination. The United States Food and Drug Administration (FDA) has granted several SARS-CoV-2 detection tests Emergency Use Authorization (EUA) for diagnosis and better epidemiological surveillance. Thus, multiple RT-PCR tests have been developed and brought to market in order to meet the urgent need for the diagnosis of COVID-19. However, comparative data between these tests in clinical laboratories are scarcely available to assess their performance. Objective: To compare two molecular methods for detecting SARS-CoV-2: the RT-PCR, Allplex™ 2019-nCoV tests on CFX96 Bio-Rad and the Abbott m2000sp/rt RealTime SARS-CoV-2. Materials and Methods: Nasopharyngeal and oropharyngeal swabs were taken from patients to diagnose SARS-CoV-2 infection. For each sample, we searched for the virus with two different RT-PCR tests: 1) first on Abbott m2000 SARS-CoV-2 targeting the N and RdRp genes, 2) then on Allplex™ 2019-nCoV Assay looking for the E, N and RdRp genes. Results: Percentages of the agreement were calculated. A total of 100 samples that tested negative and 90 positives on Abbott m2000 SARS-CoV-2 were retested on Allplex™ 2019-nCoV. Overall agreement was 74.74% on all samples. The specific agreement was 84% and 64.4% respectively for negative and positive samples with the RealTime SARS-CoV-2 test. A positive correlation (r2 = 0.63; p ™ 2019-nCoV and Abbott RealTime SARS-CoV-2 tests in the diagnosis of COVID-19. As the concordance is low for small viremias, the RT-PCR Allplex™ 2019-nCoV Assay would be better indicated during the acute and symptomatic phase of the disease.

SARS-CoV-2 laboratory surveillance during the first year of the COVID-19 pandemic in southern Brazil.

Brazil has one of the highest numbers of COVID-19 cases and deaths. Rio Grande do Sul (RS) in southern Brazil is one of the leading states in terms of case numbers. As part of the national public health network, the State Central Laboratory (LACEN-RS) changed its routine in 2020 to focus on the diagnosis of COVID-19. This study evaluated the laboratory surveillance of COVID-19 suspected cases analyzed at the LACEN-RS in 2020. Viral detection was performed using RT-qPCR in samples from patients with respiratory infection who met the study criteria. Viral RNA was isolated using commercial manual kits or automated extractors, and SARS-CoV-2 RT-qPCR was performed using the Bio-Manguinhos/Rio de Janeiro, IBMP/Paraná, or Allplex 2019-nCoV assay. In total, 360 representative SARS-CoV-2 samples were sequenced using the Illumina platform. In total, 31,197 of 107,578 (positivity rate = 29%) tested positive for SARS-CoV-2. The number of RT-qPCR tests performed per month followed the COVID-19 epidemic curve observed for the state, with peaks in July-August and December. Females accounted for 63% of the samples, whereas the positivity rate was higher among males (33.1% males vs. 26.5% females). The positivity rate was higher in adults aged 50-79 years compared to the overall positivity rate. The majority of cases were observed in the capital, Porto Alegre, and the metropolitan region. Ten distinct lineages were identified, with B.1.1.28, B.1.1.33, and P.2 being the most frequent. Here, we describe laboratory surveillance of COVID-19 to identify priorities for epidemiological surveillance actions in RS.

Evaluation and comparison of the sensitivity of three commercial RT-qPCR kits used for the detection of SARS-CoV-2 in Santiago, Chile.

The COVID-19 pandemic is still in force, causing global public health challenges and threats. Although vaccination and herd immunity have proven to be the most efficient way to control the pandemic, massive and early testing of patients using the RT-qPCR technique is crucial for constant genomic surveillance. The appearance of variants of SARS-CoV-2 with new mutations can reduce the efficiency of diagnostic detection. In this sense, several commercial RT-qPCR kits have been the target of extensive analysis because low assay performance could lead to false-negative diagnoses. In this study, we evaluated the performance of three commercial RT-qPCR kits; Thermo Fisher (TaqMan 2019-nCoV Assay Kit v1), BGI and Roche (LightCycler® Multiplex RNA Virus Master) used for the diagnosis of COVID-19 throughout the pandemic in Santiago de Chile. Under our best assay conditions, we found significant differences in Cq amplification values for control and viral probes, against the same nasopharyngeal swab samples (NPSs). In addition, in some cases, the sensitivity of the RT-qPCR kits decreased against viral variants. Our study suggests evaluating the RT-qPCR kits used to detect SARS-CoV-2 because variants such as Omicron, which has several mutations, can compromise their detection and underestimate viral circulation.

Clinical Evaluation of Three Commercial RT-PCR Kits for Routine COVID-19 Diagnosis

Amongst the multiple ways to diagnose coronavirus disease-2019 (COVID-19), reverse transcription polymerase chain reaction (RT-PCR) remains the reference gold standard, providing fast and accurate results. This study evaluated and compared the performance of three commercially available COVID-19 RT-PCR kits-Aridia® COVID-19 Real-Time PCR Test (CTK Biotech, Inc., Poway, CA, USA), Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit (Sansure Biotech Inc., Changsha, China) and AllplexTM 2019-nCoV assay (Seegene Inc., Seoul, Republic of Korea) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A total of 326 clinically suspected patients were enrolled for the study, and among them, 209 were diagnosed as positive and 117 as negative when tested with the reference method, US CDC 2019-Novel Coronavirus (2019-nCoV) Real Time RT-PCR Diagnostic Panel. The Aridia® kit showed total agreement with the reference test, with a sensitivity of 100% (95% CI: 98.25% to 100.0%) and a specificity of 100% (96.90% to 100.00%). The AllplexTM kit also showed 100% specificity (95% CI: 96.90% to 100.00%), but a lower sensitivity (98.09%, 95% CI: 95.17% to 99.48%). Among the three kits, the Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit showed the worst performance, with a sensitivity of 98.6% (95% CI: 95.9% to 99.7%) and a specificity of 95.73, 95% (CI: 90.31% to 98.60%). While all these kits conform to the requirement for routine molecular diagnosis with high performances, the Aridia® COVID-19 Real-Time PCR Test showed the best performance among the three kits.

SARS-CoV-2 RNA Testing Using Different Assays-Impact on Testing Strategies in a Clinical Setting.

In order to assess SARS-CoV-2 real time quantitative polymerase chain reaction (RT-qPCR) results in a real-life setting, three independent laboratories in Graz (Austria) set up a continuous cross comparison schedule. The following test systems were used: The QIAGEN NeuMoDx SARS-CoV-2 Assay, the Allplex™ 2019-nCoV Assay (Seegene) on a MicroLab Nimbus (Hamilton) platform combined with RealStar SARS-CoV-2 RT-PCR Assay (Altona Diagnostics GmbH), and the cobas SARS-CoV-2 test on a fully automated cobas 6800 system (Roche). A total of 200 samples were analysed, 184 (92%) were found to be concordant with all testing platforms, 14 (7%) discordant. Two (1%) samples tested invalid on a single platform and were excluded from further analysis. Discordant results were distributed randomly across the assays. The Ct values from all assays correlated closely with each other. All discordant samples showed Ct values ≥ 26. SARS-CoV-2 RT-qPCR assays may show considerable variability, especially in samples with low viral RNA concentrations. Decision makers should thus balance the advantages and disadvantages of RT-qPCR for mass screening and adopt suitable strategies that ensure a rational management of positive samples with high Ct values.

Clinical performance of SARS-CoV-2 detection on the cobas Liat using water gargle samples

Spring water gargle (SWG) is a suitable, non-invasive, alternative specimen for SARS-CoV-2 detection by RT-PCR. This study sought to evaluate the performance of the cobas Liat point-of-care system for the detection of SARS-CoV-2 in SWG samples. SWG samples and standard oral and nasopharyngeal swab (ONPS) were collected simultaneously from participants in a COVID-19 screening clinic, in November and December 2020. Both sample types were analyzed in parallel on the cobas Liat platform and with the Seegene Allplex 2019-nCoV assay. Among the 110 participants, 53% had compatible symptoms and 71% had a contact with a confirmed COVID-19 case. Only two (1.8%) individuals had neither symptoms nor contact. Amongst 110 paired samples, 25 (23%) were positive for SARS-CoV-2 on the cobas Liat for a least one sample type, with a kappa coefficient of 0.92. Agreement between the cobas Liat platform and the Seegene assay was also excellent (kappa coefficient values of 0.94 and 0.95). Two SWG samples failed to provide a positive result when their ONPS pair was positive, but their cycle threshold (Ct) values were >35 on the Seegene assay, reflecting a low viral load. Overall, the performance of the cobas Liat platform is excellent for the detection of SARS-CoV-2 in SWG samples in a high pre-test probability population.

Simultaneous monitoring of SARS-CoV-2 and bacterial profiles from the air of hospital environments with COVID-19-affected patients

The SARS-CoV-2 presence and the bacterial community profile in air samples collected at the Intensive Care Unit (ICU) of the Operational Unit of Infectious Diseases of Santa Caterina Novella Hospital in Galatina (Lecce, Italy) have been evaluated in this study. Air samplings were performed in different rooms of the ICU ward with and without COVID-19 patients. No sample was found positive to SARS-CoV-2, according to Allplex 2019-nCoV Assay. The airborne bacterial community profiles determined by the 16S rRNA gene metabarcoding approach up to the species level were characterized by richness and biodiversity indices, Spearman correlation coefficients, and Principal Coordinate Analysis. Pathogenic and non-pathogenic bacterial species, also detected in outdoor air samples, were found in all collected indoor samples. Staphylococcus pettenkoferi, Corynebacterium tuberculostearicum, and others coagulase-negative staphylococci, detected at high relative abundances in all the patients’ rooms, were the most abundant pathogenic species. The highest mean relative abundance of S. pettenkoferi and C. tuberculostearicum suggested that they were likely the main pathogens of COVID-19 patients at the ICU ward of this study. The identification of nosocomial pathogens representing potential patients’ risks in ICU COVID-19 rooms and the still controversial airborne transmission of the SARS-CoV-2 are the main contributions of this study.

Comparison of Allplex™ 2019-nCoV and TaqPath™ COVID-19 Assays

The clinical presentation of COVID-19 is non-specific, and to improve and limit the spread of the SARS-CoV-2 virus, an accurate diagnosis with a robust method is needed. A total of 500 nasopharyngeal swab specimens were tested for SARS-CoV-2. Of these, 184 samples were found to be positive with Allplex™ 2019-nCoV Assay, which is fully automated. All the positive samples were retested with TaqPath™ COVID-19 CE-IVD RT-PCR Kit (after this, referred to as TaqPath™ COVID-19), semi-automated. The comparison of RT-qPCR for SARS-CoV-2 genes target points shows only one target point in common, the N gene. Therefore, the N gene was used to compare both assays. We noticed different Ct values between the tests. Therefore, samples were divided into four groups depending to the Ct value results: (1) Ct < 25, (2) Ct 25–30, (3) Ct 30–35, (4) Ct > 35. TaqPath™ COVID-19 Kit reconfirmed the results obtained from Allplex™ 2019-nCoV Assay. In conclusion, both the Allplex™ 2019-nCoV assay and TaqPath™ COVID-19 tests accurately confirm the diagnosis of SARS-CoV-2 infection. Even if TaqPath™ COVID-19 has a semi-automated workflow, it does not introduce bias in the diagnostic screening of SARS-CoV-2, and it supports the indirect identification of variants of concern to undergo sequencing.

Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador)": High quality-low cost local SARS-CoV-2 tests for South America.

Dozens of commercial RT-qPCR kits for SARS-CoV-2 detection are available with or without Emergency Use Authorization (EUA) by FDA or other regulatory agencies. We evaluated the clinical performance of two SARS-CoV-2 RT-PCR kits designed and produced in South America, "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador), for RT-qPCR SARS-CoV2 detection using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique. We report a great clinical performance and analytical sensitivity for the two South American kits with sensitivity values of 96.4 and 100%, specificity of 100% and limit of detection in the range of 10 copies/uL of RNA extraction. "COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits are reliable SARS-CoV-2 tests made in South America that have been extensively used in Uruguay, Argentina, Brazil, Bolivia and Ecuador. These locally produced SARS-CoV-2 tests have contributed to overcome supply shortages and reduce diagnosis cost, while maintaining the high quality standards of FDA EUA commercially available kits. This approach could be extended for other diagnostic products to improve infectious diseases surveillance at middle and low income countries beyond COVID-19 pandemic.

Long persistence of severe acute respiratory syndrome coronavirus 2 swab positivity in a drowned corpse: a case report

BackgroundSince the beginning of the worldwide spread of severe acute respiratory syndrome coronavirus 2 to date, important knowledge has been obtained about the virus behavior in living subjects and on inanimate surfaces; however, there is still a lack of data on virus persistency on dead bodies and the risk of contagion from cadavers.Case presentationThe present case shows the persistency of the severe acute respiratory syndrome coronavirus 2 viral genome in nasopharyngeal swabs performed on a drowned Caucasian man, aged 41 years old, who was completely asymptomatic when he was alive, up to 41 days after death. Specific real-time reverse transcriptase-polymerase chain reaction (TaqMan 2019-nCoV Assay Kit v2; Thermo Fisher Scientific, Italy and Realquality RQ-SARS-CoV-2, AB Analytical) was used to evaluate the swabs.ConclusionsThis data reflect the importance of postmortem swabs in all autopsy cases, and not only in potential severe acute respiratory syndrome coronavirus 2-related death, and also highlight the necessity to evaluate virus positivity a long time after the moment of death, even if a low initial viral load was assessed.

Evaluation of Factors that Affect the Performance of COVID-19 Molecular Assays Including Presence of Symptoms, Number of Detected Genes and RNA Extraction Type.

Background and AimsRapid and accurate detection of COVID-19 is crucial for mitigation of the pandemic. We evaluated the performance of six molecular kits and the effect of several factors on the performance of the kits.Materials and MethodsTwo hundred and four nasopharyngeal samples were collected from participants aged ≥18 years at the Baruch Padeh Medical Center Poriya, Israel, between June and August 2020. Samples were tested by: Allplex 2019‐nCOV Assay (Seegene), Real‐Time Fluorescent RT‐PCR Kit for Detecting SARS-2019-nCoV (BGI Genomics), Xpert® Xpress SARS-CoV-2 test (Cepheid), Simplexa® COVID-19 Direct Kit (Focus Diagnostics), BD SARS-CoV-2 Reagents for BD MAX™ System (BD), and Logix Smart™ Coronavirus Disease 2019 (COVID-19) Test kit (CO-DIAGNOSTICS).ResultsXpert® Xpress SARS-CoV-2 test and Logix Smart™ COVID-19 Kit had the highest (91.2%) and the lowest (74.5%) sensitivity, respectively. Symptoms were a predictor of a positive result. Traditional assays had a higher minimum cycle threshold (min Ct), i.e. detected lower viral load, compared to rapid assays (p = 0.012). Samples of symptomatic participants had lower min Ct, than samples of asymptomatic participants (p < 0.001). Additionally, the more genes were detected, the lower the min Ct (p < 0.001), indicating that a greater percentage of the viral genome was amplified.ConclusionsTaken together, most assays had overall good performance. Since several factors affect the performance of kits, each laboratory must be familiar with its kit’s limitations in order to produce the most reliable results.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00574-y.

RT-PCR detection of SARS-CoV-2 in nasopharyngeal and salivary specimens: contribution of alternative collection systems and extraction processes to cope with mass screening. Interpretation of low viral loads

Abstract Objectives Due to massive screening of the persistent coronavirus SARS-CoV-2, supply difficulties emerged for swabs and extraction reagents leading to test alternative choices. Quality sampling may have an impact on the result and a low RNA detection may be difficult to interpret because it does not necessarily mean that infectious particles are present in biological samples. There is a need to understand whether the Ct value information is relevant and informative. Methods We compared the pre-analytical stability of RNA in saline solution, UTM®, Amies and Cary-Blair transport media. Expression profile of E, N and RdRp genes was assessed at various concentration levels with the Allplex™ 2019-nCoV Assay. Factors that may influence the determination of Ct were studied with several extraction reagents coupled to the GSD NovaPrime® SARS-CoV-2 RT-PCR testing kit. Results Seventy two-hour RNA stability has been demonstrated for all the transport media assessed. A matrix effect was shown, leading to a decrease in the detection of E and RdRp genes, so that only N gene was often found for Ct greater than 35.0. A follow-up over more than 67,000 patients suggests that N gene may be a sensitive indicator to detect a new active viral circulation, but establishing a correlation between a positive threshold and a low risk of infection for a given method remains difficult. Conclusions Several transport media and extraction processes are suitable for PCR-based SARS-CoV-2 detection. During periods of active virus circulation, any weakly positive results should be considered.

Clinical Evaluation of the Rapid STANDARD Q COVID-19 Ag Test for the Screening of Severe Acute Respiratory Syndrome Coronavirus 2.

Standard tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detect the presence of viral RNA using real-time reverse transcription (rRT)-PCR. Recently, convenient, rapid, and relatively inexpensive SARS-CoV-2 antigen (Ag) detection methods have been developed. The STANDARD Q COVID-19 Ag test (SD Biosensor, Inc., Suwon, Korea) is a rapid immunochromatography test that qualitatively detects the nucleocapsid protein of SARS-CoV-2 using gold conjugated antibodies. We evaluated its performance in comparison with that of Allplex 2019-nCoV Assay (Seegene, Seoul, Korea) in a retrospective case-control study using residual samples. The sensitivity and specificity of the STANDARD Q COVID-19 Ag test were 89.2% (58/65) and 96.0% (96/100), respectively. Cycle threshold (Ct) values for the three target SARS-CoV-2 genes (envelope, RNA-dependent RNA polymerase, and nucleocapsid genes) included in Allplex 2019-nCoV Assay were significantly lower in Ag test-positive patients than in Ag test-negative patients (P<0.001). The Ag test sensitivity was higher in samples with Ct≤30 and those collected one to five days post symptom onset. In conclusion, the STANDARD Q COVID-19 Ag test can serve as an alternative in high-prevalence settings, when the low sensitivity is compensated or when rRT-PCR tests are limited.

Positive, Mildly Positive, and Uncertain Nasopharyngeal Swab and Outcome in COVID-19 Patients

Introduction and Aim Diagnosis of SARS-CoV-2 infection is mainly based on gene detection through polymerase chain reaction analysis on nasopharyngeal swab. The Allplex TM 2019-nCoV assay targets 3 different viral genes: RNA-dependent RNA polymerase, envelope, and nucleocapside. A coding system was developed based on different number of genes expressed: a nasopharyngeal swab was considered “positive” if all 3 genes tested underwent amplification, “mildly positive” if only 2 out of 3 genes were detected, “uncertain” if only 1 gene and “negative” if none resulted amplified from the test. Our aim was to assess whether this classification correlates with clinical outcome in a cohort of COVID-19 patients. Methods This is a retrospective study including patients admitted with diagnosis of SARS-CoV-2 infection to a medical ward at the Montichiari Hospital, Brescia, Italy, from February 28 to April 30, 2020. All patients underwent the nasopharyngeal swab upon admission. Results A total of 204 patients were included in this study. Patients with full positive nasopharyngeal swab showed higher values of C-reactive protein and neutrophiles/lymphocytes ratio compared with patients with mildly positive or uncertain nasopharyngeal swab. Mortality did not differ between the 2 groups. A Cox multivariate analysis showed that age, male sex, and CRP values are independent predictors of in-hospital mortality. Conclusions Our study demonstrated that patients with a complete SARS-CoV-2 gene detection nasopharyngeal swab show a higher inflammatory profile, and this can be an indirect measurement of viral load in COVID-19 patients.

Characteristics of patients with suspected COVID-19 pneumonia and repeatedly negative RT-PCR.

ObjectivesChallenges remain and there are still a sufficient number of cases with epidemiological, clinical features and radiological data suggestive of COVID-19 pneumonia that persist negative in their RT-PCR results. The aim of the study was to define the distinguishing characteristics between patients developing a serological response to SARS-CoV-2 and those who did not.MethodsRT-PCR tests used were TaqPath 2019-nCoV Assay Kit v1 (ORF-1ab, N and S genes) from Thermo Fisher Diagnostics and SARS-COV-2 Kit (N and E genes) from Vircell. Serological response was tested using the rapid SARS-CoV2 IgG/IgM Test Cassette from T and D Diagnostics Canada and CMC Medical Devices and Drugs, S.L, CE.ResultsIn this cross-sectional study, we included a cohort of 52 patients recruited from 31 March 2020 to 23 April 2020. Patients with positive serology had an older average age (73.29) compared to those who were negative (54.82) (P<0.05). Sat02 in 27 of 34 patients with positive serology were below 94% (P<0.05). There was a frequency of 1.5% negative SARS-CoV-2 RT-PCRs during the study period concurring with 36.7% of positivity.ConclusionClinical features and other biomarkers in a context of a positive serology can be considered crucial for diagnosis.

Detection of SARS-CoV-2 RNA in Upper Respiratory Swap Samples by Pooling Method

Background:Widespread and effective use of molecular diagnostic tests is indispensable for protecting public health and containing the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. More than 1 year into the pandemic, as resources have reached a point of depletion, grouping samples in pools of certain sizes appears to be a reasonable method to reduce both the costs and the processing time without necessitating additional training, equipment, or materials.Aims:To assess whether the pooling strategy that was used in past outbreaks and is used in blood tests prior to transfusion for screening large populations can also be used in SARS CoV-2 tests.Study Design:Diagnostic accuracy study.Methods:This prospective study was conducted with 2815 samples, sent to the coronavirus disease 2019 (COVID-19) Laboratory of our hospital between February 12 and 21, 2021, to be tested for the presence of SARS-CoV-2. The samples were examined individually and in pools of five 100 μl taken from each sequential sample, using 3 different SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) kits, the Allplex™ 2019-nCoV Assay kit (Seegene, Republic of Korea), the GeneMAP™ 2019-nCoV detection V.3 kit (GenMark, Türkiye), and the Bio-Speedy™ SARS-CoV-2 Double Gene™ RT-qPCR kit (Bioeksen, Türkiye) on the BioRAD CFX96™ Touch (Bio-Rad Laboratories Inc., Hercules, CA, USA) platform available in our laboratory.Results:Following the extraction of serial dilutions prepared from the SARS-CoV-2 RNA positive (cycle of threshold: 20) sample, the standard curves of RT-PCR were analyzed. By evaluating the efficiency (E) values, all 3 kits showed high sensitivity and similar results; while the highest level was detected with the Allplex™ 2019-nCoV Assay kit in the nucleocapsid (N) gene (E: 124%), the lowest was detected with the Double Gene™ RT-qPCR kit in the N and ORF 1ab genes (E: 90%). Of the samples included in the study, only 1 positive sample with low viral load was found to be negative when studied by pooling. The total number of kits to be used in pooled tests and then to individually retest the 5 samples in positive pools was calculated as 827 and the savings rate as 69.91% (1968/2815).Conclusion:The pooling strategy is an effective approach to extend the impact of limited testing resources and reagents available in certain periods of the COVID-19 pandemic. Testing by pooling samples requires improvement of RNA extraction methods and careful monitoring of RT-PCR test sensitivity to avoid missing low-positive entities. Therefore, based on the prevalence of COVID-19 in their regions, laboratories should conduct their own validation of pooling studies for RNA extraction and amplification methods they use.

Establishing diagnostic algorithms for SARS-CoV-2 nucleic acid testing in clinical practice.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. Our laboratory initially used a two-step molecular assay, first reported by Corman et al, for SARS-CoV-2 identification (the Taiwan Center for Disease Control [T-CDC] method). As rapid and accurate diagnosis of COVID-19 is required to control the spread of this infectious disease, the current study evaluated three commercially available assays, including the TaqPath COVID-19 Combo kit, the cobas SARS-CoV-2 test, and the Rendu 2019-nCoV Assay kit, to establish diagnostic algorithms for clinical laboratories. A total of 790 clinical specimens, including nasopharyngeal swabs, throat swabs, sputum, saliva, stool, endotracheal aspirate, and serum were obtained from patients who were suspected or already confirmed to have COVID-19 at the Taipei Veterans General Hospital from February to May 2020. These specimens were tested for SARS-CoV-2 using the different assays and the performance variance between the assays was analyzed. Of the assays we evaluated, the T-CDC method and the TaqPath COVID-19 Combo kit require lots of hands-on practical laboratory work, while the cobas SARS-CoV-2 test and the Rendu 2019-nCoV Assay kit are fully automated detection systems. The T-CDC method and the TaqPath COVID-19 Combo kit showed similar detection sensitivity; however, the T-CDC method frequently delivered false-positive signals for envelope (E) and/or RNA-dependent RNA polymerase (RdRP) gene detection, thus increasing the risk of reporting false-positive results. A manual test-based testing strategy combining the T-CDC method and the TaqPath COVID-19 Combo kit was developed, which demonstrated excellent concordance rates (>99%) with the cobas and Rendu automatic systems. There were a few cases showing discrepant results, which may be due to the varied detection sensitivities as well as targets among the different platforms. Moreover, the concordance rate between the cobas and Rendu assays was 100%. Based on our evaluation, two SARS-CoV-2 diagnostic algorithms, one focusing on the manual assays and the other on the automatic platforms, were proposed. Our results provide valuable information that allows clinical laboratories to implement optimal diagnostic strategies for SARS-CoV-2 testing based on their clinical needs, such as test volume, turn-around time, and staff/resource limitations.

Associated screening for HCV and SARS-Cov2 infection in an urban area of Southern Italy: A cohort study.

The SARS-Cov2 pandemic led to reorganize the clinical activities in Italy, reducing the available resources to manage other diseases. In particular,important milestones achieved towards HCV elimination are now at risk of being nullified. Moreover, the burden of SARS-Cov2 asymptomatic carriers remains still an unresolved issue. To associate those two screenings, in order to respond to these clinical needs seems of actual interest. A prospective cohort study was set-up in an urban area of the Naples province, in which a contemporary screening for HCVAb and SARS-Cov-2-Ab rapid blood tests was performed. Of the 3556 eligible subjects,2740(77.05%) participated.Of them,1.4% resulted SARS-Cov2-positive, none resulting positive for SARS-Cov2-RNA, whereas 1.5% were HCVAb-positive and 0.18% HCVRNA-positive. Of those HCVAb positive,87.8% were already aware and treated in 88.8% of cases .Of the other 4 patients aware of their positivity,2(50%) were HCVRNA-positive. Of the 5 HCVAb-positive unaware of their positivity, 2 were HCVRNA-positive. HCVAb-positivity was only detected in patients of >41 years. The screening in an urban area of Southern Italy showed a seroprevalence of SARS-Cov2-Ab and HCV-Ab of 1.4% and 1.5%, respectively,whereas 0.18% had an active HCV infection. This demonstrates how the pandemic can be an opportunity to promote prevention activities for HCV.