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1.
Virol J ; 19(1): 67, 2022 Apr 11.
Article in English | MEDLINE | ID: covidwho-1785162

ABSTRACT

BACKGROUND: The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoVs) (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) still circulate worldwide. The early clinical symptoms of SARS-CoV-2 and seasonal HCoV infections are similar, so rapid and accurate identification of the subtypes of HCoVs is crucial for early diagnosis, early treatment, prevention and control of these infections. However, current multiplex molecular diagnostic techniques for HCoV subtypes including SARS-CoV-2 are limited. METHODS: We designed primers and probes specific for the S and N genes of SARS-CoV-2, the N gene of severe acute respiratory syndrome coronavirus (SARS-CoV), and the ORF1ab gene of four seasonal HCoVs, as well as the human B2M gene product. We developed and optimized a quadruple quantitative real-time PCR assay (qq-PCR) for simultaneous detection of SARS-CoV-2, SARS-CoV and four seasonal HCoVs. This assay was further tested for specificity and sensitivity, and validated using 184 clinical samples. RESULTS: The limit of detection of the qq-PCR assay was in the range 2.5 × 101 to 6.5 × 101 copies/µL for each gene and no cross-reactivity with other common respiratory viruses was observed. The intra-assay and inter-assay coefficients of variation were 0.5-2%. The qq-PCR assay had a 91.9% sensitivity and 100.0% specificity for SARS-CoV-2 and a 95.7% sensitivity and 100% specificity for seasonal HCoVs, using the approved commercial kits as the reference. Compared to the commercial kits, total detection consistency was 98.4% (181/184) for SARS-CoV-2 and 98.6% (142/144) for seasonal HCoVs. CONCLUSION: With the advantages of sensitivity, specificity, rapid detection, cost-effectiveness, and convenience, this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-CoV-2, SARS-CoV and seasonal HCoVs.


Subject(s)
COVID-19 , Coronavirus NL63, Human , Coronavirus OC43, Human , COVID-19/diagnosis , Coronavirus NL63, Human/genetics , Coronavirus OC43, Human/genetics , Humans , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics
2.
Bull Exp Biol Med ; 172(4): 495-498, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1756827

ABSTRACT

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.


Subject(s)
COVID-19/metabolism , Cell-Free Nucleic Acids/genetics , DNA, Mitochondrial/genetics , NADH Dehydrogenase/genetics , Real-Time Polymerase Chain Reaction/standards , Adult , Aged , COVID-19/virology , Case-Control Studies , Cell-Free Nucleic Acids/blood , DNA Primers/chemical synthesis , DNA, Mitochondrial/blood , Female , Humans , Male , Middle Aged , Mitochondria/genetics , Mitochondria/virology , NADH Dehydrogenase/blood , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity
3.
Crit Care ; 26(1): 63, 2022 03 21.
Article in English | MEDLINE | ID: covidwho-1753120

ABSTRACT

Infection (either community acquired or nosocomial) is a major cause of morbidity and mortality in critical care medicine. Sepsis is present in up to 30% of all ICU patients. A large fraction of sepsis cases is driven by severe community acquired pneumonia (sCAP), which incidence has dramatically increased during COVID-19 pandemics. A frequent complication of ICU patients is ventilator associated pneumonia (VAP), which affects 10-25% of all ventilated patients, and bloodstream infections (BSIs), affecting about 10% of patients. Management of these severe infections poses several challenges, including early diagnosis, severity stratification, prognosis assessment or treatment guidance. Digital PCR (dPCR) is a next-generation PCR method that offers a number of technical advantages to face these challenges: it is less affected than real time PCR by the presence of PCR inhibitors leading to higher sensitivity. In addition, dPCR offers high reproducibility, and provides absolute quantification without the need for a standard curve. In this article we reviewed the existing evidence on the applications of dPCR to the management of infection in critical care medicine. We included thirty-two articles involving critically ill patients. Twenty-three articles focused on the amplification of microbial genes: (1) four articles approached bacterial identification in blood or plasma; (2) one article used dPCR for fungal identification in blood; (3) another article focused on bacterial and fungal identification in other clinical samples; (4) three articles used dPCR for viral identification; (5) twelve articles quantified microbial burden by dPCR to assess severity, prognosis and treatment guidance; (6) two articles used dPCR to determine microbial ecology in ICU patients. The remaining nine articles used dPCR to profile host responses to infection, two of them for severity stratification in sepsis, four focused to improve diagnosis of this disease, one for detecting sCAP, one for detecting VAP, and finally one aimed to predict progression of COVID-19. This review evidences the potential of dPCR as a useful tool that could contribute to improve the detection and clinical management of infection in critical care medicine.


Subject(s)
COVID-19 , COVID-19/diagnosis , Critical Care , Humans , Real-Time Polymerase Chain Reaction/methods , Reproducibility of Results
4.
Sensors (Basel) ; 22(6)2022 Mar 17.
Article in English | MEDLINE | ID: covidwho-1753669

ABSTRACT

Global health crises due to the prevailing Coronavirus Disease 2019 (COVID-19) pandemic have placed significant strain on health care facilities such as hospitals and clinics around the world. Further, foodborne and waterborne diseases are not only spreading faster, but also appear to be emerging more rapidly than ever before and are able to circumvent conventional control measures. The Polymerase Chain Reaction (PCR) system is a well-known diagnostic tool for many applications in medical diagnostics, environmental monitoring, and food and water quality assessment. Here, we describe the design, development, and testing of a portable, low-cost, and real-time PCR system that can be used in emergency health crises and resource-poor situations. The described PCR system incorporates real-time reaction monitoring using fluorescence as an alternative to gel electrophoresis for reaction analysis, further decreasing the need of multiple reagents, reducing sample testing cost, and reducing sample analysis time. The bill of materials cost of the described system is approximately $340. The described PCR system utilizes a novel progressive selective proportional-integral-derivative controller that helps in reducing sample analysis time. In addition, the system employs a novel primer-based approach to quantify the initial target amplicon concentration, making it well-suited for food and water quality assessment. The developed PCR system performed DNA amplification at a level and speed comparable to larger and more expensive commercial table-top systems. The fluorescence detection sensitivity was also tested to be at the same level as commercially available multi-mode optical readers, thus making the PCR system an attractive solution for medical point-of-care and food and water quality assessment.


Subject(s)
COVID-19 , Point-of-Care Systems , COVID-19/diagnosis , Humans , Real-Time Polymerase Chain Reaction/methods
5.
Viruses ; 12(6)2020 06 25.
Article in English | MEDLINE | ID: covidwho-1726024

ABSTRACT

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA-Dependent RNA Polymerase/genetics , Real-Time Polymerase Chain Reaction/methods , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/virology , Coronavirus RNA-Dependent RNA Polymerase , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/analysis , SARS-CoV-2 , Sensitivity and Specificity , World Health Organization
6.
Appl Microbiol Biotechnol ; 106(5-6): 2207-2218, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1712228

ABSTRACT

The pandemic of coronavirus disease 2019 (COVID-19) continues to threaten public health. For developing countries where vaccines are still in shortage, cheaper alternative molecular methods for SARS-CoV-2 identification can be crucial to prevent the next wave. Therefore, 14 primer sets recommended by the World Health Organization (WHO) was evaluated on testing both clinical patient and environmental samples with the gold standard diagnosis method, TaqMan-based RT-qPCR, and a cheaper alternative method, SYBR Green-based RT-qPCR. Using suitable primer sets, such as ORF1ab, 2019_nCoV_N1 and 2019_nCoV_N3, the performance of the SYBR Green approach was comparable or better than the TaqMan approach, even when considering the newly dominating or emerging variants, including Delta, Eta, Kappa, Lambda, Mu, and Omicron. ORF1ab and 2019_nCoV_N3 were the best combination for sensitive and reliable SARS-CoV-2 molecular diagnostics due to their high sensitivity, specificity, and broad accessibility. KEY POINTS: • With suitable primer sets, the SYBR Green method performs better than the TaqMan one. • With suitable primer sets, both methods should still detect the new variants well. • ORF1ab and 2019_nCoV_N3 were the best combination for SARS-CoV-2 detection.


Subject(s)
COVID-19 , SARS-CoV-2 , Benzothiazoles , COVID-19/diagnosis , Diamines , Humans , Quinolines , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity
7.
Sci Rep ; 12(1): 2883, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1707349

ABSTRACT

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Containment of Biohazards/methods , Hot Temperature , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Specimen Handling/methods , Virus Inactivation , Animals , COVID-19/virology , Cell Line , Humans , Mice , Murine hepatitis virus/genetics , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity
8.
PLoS One ; 16(11): e0258263, 2021.
Article in English | MEDLINE | ID: covidwho-1700786

ABSTRACT

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.


Subject(s)
COVID-19/virology , Ribonuclease P/genetics , SARS-CoV-2/genetics , Waste Water/virology , DNA Primers/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods , Wastewater-Based Epidemiological Monitoring
9.
Sci Rep ; 12(1): 2853, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1700332

ABSTRACT

Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Humans
10.
BMC Infect Dis ; 22(1): 163, 2022 Feb 21.
Article in English | MEDLINE | ID: covidwho-1699638

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been a major public health importance and its specimen needs to be handled safely due to concerns of potential transmissibility to health care workers. Heat inactivation of the sample before nucleic acid isolation might permit safe testing processes. Hence, it is important to assess the effect of heat inactivation on SARS-CoV-2 RT-PCR detection in resource limited settings. METHODS: An experimental study was conducted at Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing. One batch of the sample was inactivated at 56 °C heat for 30 min, and the other batch was stored at 4 °C for a similar period of time. RNA extraction and detection were done by DAAN Gene kit protocols. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. p-value < 0.05 was considered as statistically significant. RESULTS: Out of 188 total samples, 119 (63.3%) were positive and 69 (36.7%) were negative in the non-inactivated group. While, 115 (61.2%) of samples were positive and 73 (38.8) were negative in heat inactivated sample batch. Rate of positivity between groups did not have statistically significant difference (p > 0.05). The mean Cycle threshold (Ct) value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95% CI - 0.247-0.331; t = 0.28; p = 0.774) and 0.38 (95% CI 0.097-0.682; t = 2.638; p = 0.010) respectively. CONCLUSION: Heat inactivation at 56 °C for 30 min did not affect the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding showed that there was statistically significant Ct value increment after heat inactivation compared to untreated samples. Therefore, false-negative results for high Ct value (Ct > 35) samples were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.


Subject(s)
COVID-19 , SARS-CoV-2 , Hot Temperature , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription
11.
Microbiol Spectr ; 10(1): e0068121, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1691411

ABSTRACT

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.


Subject(s)
COVID-19/diagnosis , Mutation, Missense , Point Mutation , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Alleles , Amino Acid Substitution , COVID-19/epidemiology , COVID-19/virology , Genes, Viral , Humans , Mass Screening , Ontario/epidemiology , Polymorphism, Single Nucleotide , Population Surveillance , Prevalence , Reproducibility of Results , Sensitivity and Specificity
12.
Front Immunol ; 12: 785599, 2021.
Article in English | MEDLINE | ID: covidwho-1643498

ABSTRACT

Zinc ion as an enzyme cofactor exhibits antiviral and anti-inflammatory activity during infection, but circulating zinc ion level during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is unclear. This study aimed to evaluate serum zinc ion level in Coronavirus Disease 2019 (COVID-19) patients and healthy subjects, as well as its correlation with antibodies against SARS-CoV-2. 114 COVID-19 patients and 48 healthy subjects (38 healthy volunteers and 10 close contacts of patients with COVID-19) were included. Zinc ion concentration and levels of antibodies against SARS-CoV-2 Spike 1 + Spike 2 proteins, nucleocapsid protein, and receptor-binding domain in serum were measured. Results showed that the concentration of zinc ion in serum from COVID-19 patients [median: 6.4 nmol/mL (IQR 1.5 - 12.0 nmol/mL)] were significantly lower than that from the healthy subjects [median: 15.0 nmol/mL (IQR 11.9 - 18.8 nmol/mL)] (p < 0.001) and the difference remained significant after age stratification (p < 0.001) or when the patients were at the recovery stage (p < 0.001). Furthermore, COVID-19 patients with more severe hypozincemia showed higher levels of IgG against the receptor-binding domain of SARS-CoV-2 spike protein. Further studies to confirm the effect of zinc supplementation on improving the outcomes of COVID-19, including antibody response against SARS-CoV-2, are warranted.


Subject(s)
Antibodies, Viral/blood , COVID-19/blood , COVID-19/immunology , Immunity , SARS-CoV-2/immunology , Zinc/blood , Adult , Antibodies, Viral/immunology , COVID-19/virology , Case-Control Studies , Cations, Divalent/blood , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Phosphoproteins/immunology , Protein Domains/immunology , Real-Time Polymerase Chain Reaction/methods , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology
13.
J Mol Diagn ; 24(2): 112-119, 2022 02.
Article in English | MEDLINE | ID: covidwho-1641441

ABSTRACT

Quantifying the detection rate of the widely used quantitative RT-PCR (RT-qPCR) test for severe acute respiratory syndrome coronavirus 2 and its dependence on patient demographic characteristics and disease progression is key in designing epidemiologic strategies. Analyzing 843,917 test results of 521,696 patients, a "positive period" was defined for each patient between diagnosis of coronavirus disease 2019 and the last positive test result. The fraction of positive test results within this period was then used to estimate detection rate. Regression analyses were used to determine associations of detection with time of sampling after diagnosis, patient demographic characteristics, and viral RNA copy number based on RT-qPCR cycle threshold values of the next positive test result. The overall detection rate in tests performed within 14 days after diagnosis was 83.1%. This rate was higher at days 0 to 5 after diagnosis (89.3%). Furthermore, detection rate was strongly associated with age and sex. Finally, the detection rate with the Allplex 2019-nCoV RT-qPCR kit was associated, at the single-patient level, with viral RNA copy number (P < 10-9). These results show that the reliability of the test result is reduced in later days as well as for women and younger patients, in whom the viral loads are typically lower.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Adult , Age Factors , COVID-19 Testing/methods , Female , Humans , Male , Middle Aged , Odds Ratio , RNA, Viral , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Sex Factors , Time Factors , Viral Load , Young Adult
14.
PLoS One ; 16(12): e0260884, 2021.
Article in English | MEDLINE | ID: covidwho-1632593

ABSTRACT

OBJECTIVES: To exploit the features of digital PCR for implementing SARS-CoV-2 observational studies by reliably including the viral load factor expressed as copies/µL. METHODS: A small cohort of 51 Covid-19 positive samples was assessed by both RT-qPCR and digital PCR assays. A linear regression model was built using a training subset, and its accuracy was assessed in the remaining evaluation subset. The model was then used to convert the stored cycle threshold values of a large dataset of 6208 diagnostic samples into copies/µL of SARS-CoV-2. The calculated viral load was used for a single cohort retrospective study. Finally, the cohort was randomly divided into a training set (n = 3095) and an evaluation set (n = 3113) to establish a logistic regression model for predicting case-fatality and to assess its accuracy. RESULTS: The model for converting the Ct values into copies/µL was suitably accurate. The calculated viral load over time in the cohort of Covid-19 positive samples showed very low viral loads during the summer inter-epidemic waves in Italy. The calculated viral load along with gender and age allowed building a predictive model of case-fatality probability which showed high specificity (99.0%) and low sensitivity (21.7%) at the optimal threshold which varied by modifying the threshold (i.e. 75% sensitivity and 83.7% specificity). Alternative models including categorised cVL or raw cycle thresholds obtained by the same diagnostic method also gave the same performance. CONCLUSION: The modelling of the cycle threshold values using digital PCR had the potential of fostering studies addressing issues regarding Sars-CoV-2; furthermore, it may allow setting up predictive tools capable of early identifying those patients at high risk of case-fatality already at diagnosis, irrespective of the diagnostic RT-qPCR platform in use. Depending upon the epidemiological situation, public health authority policies/aims, the resources available and the thresholds used, adequate sensitivity could be achieved with acceptable low specificity.


Subject(s)
COVID-19/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Load/methods , Adolescent , Adult , Aged , COVID-19/mortality , COVID-19 Nucleic Acid Testing/methods , Child , Child, Preschool , Female , Genome, Viral , Humans , Logistic Models , Male , Middle Aged , Retrospective Studies , Sensitivity and Specificity , Young Adult
15.
PLoS One ; 17(1): e0262820, 2022.
Article in English | MEDLINE | ID: covidwho-1628968

ABSTRACT

BACKGROUND: Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to prevent spread of the infection. Understanding of the antibody response to SARS-CoV-2 in patients with coronavirus disease 2019 (COVID-19) is insufficient, particularly in relation to those whose responses persist for more than 1 month after the onset of symptoms. We conducted a SARS-CoV-2 antibody test to identify factors affecting the serological response and to evaluate its diagnostic utility in patients with COVID-19. METHODS AND FINDING: We collected 1,048 residual serum samples from 396 patients with COVID-19 confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2. The samples had been used for routine admission tests in six healthcare institutions in Daegu. Antibody to SARS-CoV-2 was analyzed and the cutoff index (COI) was calculated for quantitative analysis. The patients' information was reviewed to evaluate the relationship between antibody positivity and clinical characteristics. The anti-SARS-CoV-2 antibody positivity rate was 85% and the average COI was 24·3. The positivity rate and COI increased with time elapsed since symptom onset. Anti-SARS-CoV-2 antibody persisted for at least 13 weeks after symptom onset at a high COI. There was a significant difference in anti-SARS-CoV-2 antibody positivity rate between patients with and without symptoms, but not according to sex or disease course. The descending COI pattern at weeks 1 to 5 after symptom onset was significantly more frequent in patients who died than in those who recovered. CONCLUSIONS: Anti-SARS-CoV-2 antibody persisted for at least 13 weeks at a high COI in patients with COVID-19. A decreasing COI pattern up to fifth week may be associated with a poor prognosis of COVID-19. As new treatments and vaccines are introduced, it is important to monitor continuously the usefulness of anti-SARS-CoV-2 antibody assays.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/blood , COVID-19/epidemiology , SARS-CoV-2/immunology , Aged , Antibodies, Viral/immunology , COVID-19/diagnosis , COVID-19/immunology , COVID-19 Nucleic Acid Testing/methods , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Male , Middle Aged , Real-Time Polymerase Chain Reaction/methods , Republic of Korea/epidemiology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity
16.
PLoS One ; 17(1): e0262179, 2022.
Article in English | MEDLINE | ID: covidwho-1636037

ABSTRACT

Comparisons of histopathological features and microbiological findings between decedents with respiratory symptoms due to SARS-CoV-2 infection or other causes, in settings with high prevalence of HIV and Mycobacterium tuberculosis (MTB) infections have not been reported. Deaths associated with a positive ante-mortem SARS-CoV-2 PCR test and/or respiratory disease symptoms at Chris Hani Baragwanath Academic Hospital in Soweto, South Africa from 15th April to 2nd November 2020, during the first wave of the South African COVID-19 epidemic, were investigated. Deceased adult patients had post-mortem minimally-invasive tissue sampling (MITS) performed to investigate for SARS-CoV-2 infection and molecular detection of putative pathogens on blood and lung samples, and histopathology examination of lung, liver and heart tissue. During the study period MITS were done in patients displaying symptoms of respiratory disease including 75 COVID-19-related deaths (COVID+) and 42 non-COVID-19-related deaths (COVID-). The prevalence of HIV-infection was lower in COVID+ (27%) than in the COVID- (64%), MTB detection was also less common among COVID+ (3% vs 13%). Lung histopathology findings showed differences between COVID+ and COVID- in the severity of the morphological appearance of Type-II pneumocytes, alveolar injury and repair initiated by SARS-CoV-2 infection. In the liver necrotising granulomatous inflammation was more common among COVID+. No differences were found in heart analyses. The prevalence of bacterial co-infections was higher in COVID+. Most indicators of respiratory distress syndrome were undifferentiated between COVID+ and COVID- except for Type-II pneumocytes. HIV or MTB infection does not appear in these data to have a meaningful correspondence with COVID-related deaths.


Subject(s)
Alveolar Epithelial Cells/pathology , COVID-19/epidemiology , COVID-19/mortality , Diabetes Mellitus/epidemiology , Hypertension/epidemiology , Pandemics , SARS-CoV-2/genetics , Adult , Aged , Autopsy , Biopsy, Large-Core Needle/methods , COVID-19/pathology , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , Comorbidity , Female , Humans , Length of Stay , Male , Middle Aged , Real-Time Polymerase Chain Reaction/methods , South Africa/epidemiology
17.
PLoS One ; 17(1): e0261853, 2022.
Article in English | MEDLINE | ID: covidwho-1622346

ABSTRACT

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/genetics , SARS-CoV-2/genetics , COVID-19/virology , Feasibility Studies , Humans , Nasopharynx/virology , Pandemics/prevention & control , Sensitivity and Specificity , Serologic Tests/methods , Specimen Handling/methods
18.
J Clin Lab Anal ; 36(2): e24242, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1616016

ABSTRACT

BACKGROUND: Currently, SARS-CoV-2 RNA detection using real-time reverse-transcription PCR (rRT-PCR) is the standard diagnostic test for COVID-19 infection. Various rRT-PCR assays are currently used worldwide, targeting different genes of the SARS-CoV-2. Here, we compared the analytical sensitivity and clinical performance (sensitivity and specificity) of Allplex SARS-CoV-2/FluA/FluB/RSV assay (Seegene), Standard M nCoV real-time detection kit (SD Biosensor), and U-TOP COVID-19 detection kit (Seasun Biomaterials) for SARS-CoV-2 detection. METHODS: Two hundred and forty-nine nasopharyngeal swab samples were evaluated to compare the clinical performance of the rRT-PCR assays. For the analytical performance evaluation, two RNA controls with known viral loads-SARS-CoV-2 RNA control and SARS-COV-2 B.1.351 RNA control-were used to investigate the potential impact of SARS-CoV-2 variants, particularly the B.1.351 lineage. RESULTS: Limits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (Ct ) values of the two RNA controls were within 1.0 for each target in the rRT-PCR assays (0.05-0.73), without any prominent Ct value shift or dropouts in the SARS-COV-2 B.1.351 RNA control. Using the consensus criterion as the reference standard, 89 samples were positive, whereas 160 were negative. The overall clinical performance of rRT-PCR assays was comparable (sensitivity 98.88%-100%; specificity 99.38%-100%), whereas the sensitivities of each target gene were more variable. CONCLUSIONS: The three rRT-PCR assays showed comparable analytical sensitivity and clinical performance. The analytical and clinical sensitivities of each target gene were influenced more by the primer and probe design than the target gene itself.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques , Reagent Kits, Diagnostic/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Nasopharynx/virology , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Load , Young Adult
19.
J Clin Lab Anal ; 36(2): e24226, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1611241

ABSTRACT

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.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Aged , Aged, 80 and over , False Negative Reactions , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Molecular Diagnostic Techniques/statistics & numerical data , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Real-Time Polymerase Chain Reaction/statistics & numerical data , SARS-CoV-2/isolation & purification
20.
PLoS One ; 17(1): e0262258, 2022.
Article in English | MEDLINE | ID: covidwho-1606499

ABSTRACT

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Humans , Influenza, Human/virology , Molecular Diagnostic Techniques , Nasopharynx/virology , Respiratory Syncytial Virus Infections/virology , Retrospective Studies , Sensitivity and Specificity
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