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1.
Medicine (Baltimore) ; 100(19): e25917, 2021 May 14.
Article in English | MEDLINE | ID: covidwho-2191007

ABSTRACT

ABSTRACT: The coronavirus disease (COVID-19) has become a global pandemic. Invasive mechanical ventilation is recommended for the management of patients with COVID-19 who have severe respiratory symptoms. However, various complications can develop after its use. The efficient and appropriate management of patients requires the identification of factors associated with an aggravation of COVID-19 respiratory symptoms to a degree where invasive mechanical ventilation becomes necessary, thereby enabling clinicians to prevent such ventilation. This retrospective study included 138 inpatients with COVID-19 at a tertiary hospital. We evaluated the differences in the demographic and clinical data between 27 patients who required invasive mechanical ventilation and 111 patients who did not. Multivariate logistic regression analysis indicated that the duration of fever, national early warning score (NEWS), and lactate dehydrogenase (LDH) levels on admission were significantly associated with invasive mechanical ventilation in this cohort. The optimal cut-off values were: fever duration ≥1 day (sensitivity 100.0%, specificity 54.95%), NEWS ≥7 (sensitivity 72.73%, specificity 92.52%), and LDH >810 mg/dL (sensitivity 56.0%, specificity 90.29%). These findings can assist in the early identification of patients who will require invasive mechanical ventilation. Further studies in larger patient populations are recommended to validate our findings.


Subject(s)
COVID-19/physiopathology , Early Warning Score , Respiration, Artificial/statistics & numerical data , Adult , Age Factors , Aged , Aged, 80 and over , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Female , Fever/physiopathology , Humans , Hydroxychloroquine/therapeutic use , L-Lactate Dehydrogenase/blood , Logistic Models , Male , Middle Aged , Pandemics , Real-Time Polymerase Chain Reaction , Republic of Korea , Retrospective Studies , Risk Assessment , Risk Factors , SARS-CoV-2 , Sex Factors , Socioeconomic Factors , Tertiary Care Centers , Young Adult
2.
Int J Mol Sci ; 23(16)2022 Aug 20.
Article in English | MEDLINE | ID: covidwho-2200284

ABSTRACT

Rapid identification of SARS-CoV-2 variants is essential for epidemiological surveillance. RT-qPCR-based variant differentiation tests can be used to quickly screen large sets of samples for relevant variants of concern/interest; this study was conducted on specimens collected at 11 centers located in Poland during routine SARS-CoV-2 diagnostics between August 2020 and December 2021. A total of 1096 samples (with CT < 30) were screened for Alpha, Beta, Delta, Kappa and Omicron variants using commercial assays targeting repeat mutation sites. Variants were assigned to 434 (39.6%) specimens; the remaining 662 (60.4%) samples were not classified (no tested mutations detected). Alpha (n = 289; 66.59%), Delta (n = 115; 26.5%), Kappa (n = 30; 6.91%) and Omicron (n = 2; 0.46%) variants were identified and their distribution changed over time. The first Alpha variant appeared in October 2020, and it began to gradually increase its proportion of the virus population by June 2021. In July 2021, it was replaced by the Delta variant, which already dominated by the end of the year. The first Kappa was detected in October 2021, while Omicron was found in December 2021. The screening of samples allowed the determination of epidemiological trends over a time interval reflecting the national COVID-19 waves.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19 Testing , Humans , Mutation , Poland/epidemiology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics
3.
Infect Dis Now ; 52(8): 453-455, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2179305

ABSTRACT

Real-time PCR plays a key role in the diagnosis of viral infections. Multiple kits can detect or quantify genomes of various viruses with the same thermocycling program. Detection of RNA viruses includes an additional step of reverse transcription and challenge their detection in a single run with DNA viruses. We investigated the analytical performance of HSV-1, HSV-2 and VZV DNA quantification with Altona RealStar® PCR kits using the RT-PCR program for RNA viruses instead of the PCR program for DNA viruses. For each three viruses, Bland-Altman distribution did not show differences between both programs, and quantification curves generated with both thermocycling programs confirmed high correlation (R2 ≥ 0.9983). Detection of low viral load samples was evaluated, on 10-times repeat-test. All replicate samples were detected with both thermocycling programs and were quantified at similar viral loads (bias in log10 copies/mL: +0.05 (HSV-1), -0.01 (HSV-2) and +0.25 (VZV)). This confirms the feasibility of using the RT-PCR thermocycling program to detect and quantify the genome of RNA and DNA viruses in a single run.


Subject(s)
RNA Viruses , Humans , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Real-Time Polymerase Chain Reaction , DNA Viruses
4.
Front Cell Infect Microbiol ; 12: 887754, 2022.
Article in English | MEDLINE | ID: covidwho-2154665

ABSTRACT

Candida auris continues to be a global threat for infection and transmission in hospitals and long-term care facilities. The emergence of SARS-CoV-2 has rerouted attention and resources away from this silent pandemic to the frontlines of the ongoing COVID-19 disease. Cases of C. auris continue to rise, and clinical laboratories need a contingency plan to prevent a possible outbreak amid the COVID-19 pandemic. Here, we introduce a two-tier Candida auris surveillance program that includes, first, a rapid qualitative rt-PCR for the identification of high-risk patients and, second, a method to analyze the isolated C. auris for strain typing using the Fourier-Transform Infrared spectroscopy. We have performed this two-tier surveillance for over 700 at-risk patients being admitted into our hospital and have identified 28 positive specimens (4%) over a 1-year period. Strain typing analysis by the IR spectrum acquisition typing method, supplemented by whole genome sequencing, has shown grouping of two significant clusters. The majority of our isolates belong to circulating African lineage associated with C. auris Clade III and an isolated strain grouping differently belonging to South Asian lineage C. auris Clade I. Low numbers of genomic variation point to local and ongoing transmission within the Los Angeles area not specifically within the hospital setting. Collectively, clinical laboratories having the ability to rapidly screen high-risk patients for C. auris and to participate in outbreak investigations by offering strain typing will greatly assist in the control of C. auris transmission within the hospital setting.


Subject(s)
COVID-19 , Candidiasis , Algorithms , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19 Testing , Candida , Humans , Pandemics , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics
5.
JMIR Public Health Surveill ; 7(1): e24220, 2021 01 14.
Article in English | MEDLINE | ID: covidwho-2141289

ABSTRACT

BACKGROUND: Real-time polymerase chain reaction using nasopharyngeal swabs is currently the most widely used diagnostic test for SARS-CoV-2 detection. However, false negatives and the sensitivity of this mode of testing have posed challenges in the accurate estimation of the prevalence of SARS-CoV-2 infection rates. OBJECTIVE: The purpose of this study was to evaluate whether technical and, therefore, correctable errors were being made with regard to nasopharyngeal swab procedures. METHODS: We searched a web-based video database (YouTube) for videos demonstrating SARS-CoV-2 nasopharyngeal swab tests, posted from January 1 to May 15, 2020. Videos were rated by 3 blinded rhinologists for accuracy of swab angle and depth. The overall score for swab angle and swab depth for each nasopharyngeal swab demonstration video was determined based on the majority score with agreement between at least 2 of the 3 reviewers. We then comparatively evaluated video data collected from YouTube videos demonstrating the correct nasopharyngeal swab technique with data from videos demonstrating an incorrect nasopharyngeal swab technique. Multiple linear regression analysis with statistical significance set at P=.05 was performed to determine video data variables associated with the correct nasopharyngeal swab technique. RESULTS: In all, 126 videos met the study inclusion and exclusion criteria. Of these, 52.3% (66/126) of all videos demonstrated the correct swab angle, and 46% (58/126) of the videos demonstrated an appropriate swab depth. Moreover, 45.2% (57/126) of the videos demonstrated both correct nasopharyngeal swab angle and appropriate depth, whereas 46.8% (59/126) of the videos demonstrated both incorrect nasopharyngeal swab angle and inappropriate depth. Videos with correct nasopharyngeal swab technique were associated with the swab operators identifying themselves as a medical professional or as an Ear, Nose, Throat-related medical professional. We also found an association between correct nasopharyngeal swab techniques and recency of video publication date (relative to May 15, 2020). CONCLUSIONS: Our findings show that over half of the videos documenting the nasopharyngeal swab test showed an incorrect technique, which could elevate false-negative test rates. Therefore, greater attention needs to be provided toward educating frontline health care workers who routinely perform nasopharyngeal swab procedures.


Subject(s)
COVID-19 Testing/methods , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Social Media , Specimen Handling/methods , Video Recording , Diagnostic Errors/prevention & control , Humans , Real-Time Polymerase Chain Reaction
6.
Klin Lab Diagn ; 67(11): 672-677, 2022 Nov 14.
Article in English | MEDLINE | ID: covidwho-2146783

ABSTRACT

COVID-19 is a disease caused by the new coronavirus SARS-CoV-2. Outbreaks were first reported in China on December 31, 2019. Exactly one month later, the WHO declared the outbreak a public health emergency of international concern, and on March 11, it was declared a pandemic. In February, the infection began to spread rapidly to various countries, with Europe declared the center. By April 17, 2020, cases had been confirmed in all subjects of the Russian Federation. At the beginning of September 2020, the number of cases exceeded one million; at November 19, two million; at December 26, three million. At February 10, 2021, four million; at May 23, five million; at July 20, six million; at September 5, seven million; at October 18, eight million; at November 13, nine million; and at December 12, 2021, ten million. The rapid spread of the virus, accompanied by a significant increase in the number of infections and deaths. A total of about 18.6 million cases were recorded at the end of the first half of 2022. The total number of deaths from coronavirus in Russia at that time was 382,313 (2.06% of all cases). The number of tests performed by various analytical methods amounted to over 274, 5 million, i.e. 1.9 million per 1 million population. The rapid spread and the increase in new infections caused by SARS-CoV-2 made it necessary to use new epidemiological and diagnostic approaches based on fast, accurate and reliable technology for detecting the infectious agent. One such virus detection method is polymerase chain reaction with reverse transcription and real-time detection of the results. The review presents the domestic market offerings of PCR diagnostic kits and provides their comparative consumer characteristics.


Subject(s)
COVID-19 , Reverse Transcription , Humans , COVID-19/diagnosis , COVID-19/epidemiology , SARS-CoV-2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction
7.
Klin Lab Diagn ; 67(11): 663-667, 2022 Nov 14.
Article in English | MEDLINE | ID: covidwho-2146780

ABSTRACT

The coronavirus infection continues to spread around the world. In this regard, the purpose of this work was: to develop a set of reagents for the qualitative detection of SARS-CoV-2 virus RNA. The set was developed by CJSC «Ecolab¼, 20 positive samples were used to develop the kit. The research method consisted of several stages: isolation of SARS-CoV-2 RNA, RNA reverse transcription reaction and PCR amplification of cDNA with simultaneous detection of the result in real time. The main characteristics of the kit: analytical sensitivity - 100%, specificity - 100%, accuracy - 100%. Thus, our method for diagnosing a new coronavirus infection based on real-time RT-PCR makes it possible to qualitatively and quickly detect betacoronavirus RNA in clinical material from patients and healthy individuals with suspected coronavirus infection and other symptoms of SARS.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , RNA, Viral/analysis , Indicators and Reagents , Sensitivity and Specificity , COVID-19/diagnosis , Real-Time Polymerase Chain Reaction
8.
Virol J ; 19(1): 191, 2022 11 18.
Article in English | MEDLINE | ID: covidwho-2123315

ABSTRACT

BACKGROUND: The global pandemic of coronavirus disease 2019 (COVID-19) has led to the development of multiple detection kits by national manufacturers for severe acute respiratory syndrome coronavirus 2 viral nucleic acid testing. The purpose of this study is to evaluate the performance of different kits (i.e., Maccura kit and Sansure kit) in real clinical work using clinical samples, which will help with the optimization of the test kits. METHOD: During the past three months (March-May 2022), 1399 pharyngeal swabs from suspected COVID-19 patients have been initially screened using the Maccura kit in Jilin, China, and the test results were verified using the Sansure kit. The cycle threshold (Ct) values generated by the two kits were compared at different viral load levels. Correlation and consistency of the Ct values were investigated using Spearman correlation, Deming regression, and Bland-Altman plots. The cut-off Ct values of the Maccura kit were recalculated by referencing the result of the Sansure kit as a standard. Furthermore, another 163 pharyngeal swabs from suspected COVID-19 patients were collected to verify the new cut-off values. RESULTS: As a result of the Maccura kit testing, 1192 positive cases and 207 suspected COVID-19 cases were verified. After re-examination by the Sansure kit, 1118 positive cases were confirmed. The difference between the Ct values provided by the two kits was statistically significant, except for the N gene at high viral load. The Ct values obtained from the two kits presented a linear positive correlation. The Maccura kit used new cut-off Ct values of 35.00 (ORF1ab gene) and 35.07 (N gene). Based on that, the validation pass rate for the new cut-off Ct values was 91.41%. CONCLUSION: Since the Maccura kit is found to have false positives in actual clinical work, recalculation of the cut-off values can reduce this occurrence. In order to improve the accuracy of the testing, laboratories should use two kits for COVID-19 testing, and the adjusting and optimizing of the kits for their situation are needed.


Subject(s)
COVID-19 , Nucleic Acids , Humans , SARS-CoV-2/genetics , Reagent Kits, Diagnostic , COVID-19/diagnosis , COVID-19 Testing , Real-Time Polymerase Chain Reaction/methods
9.
Int J Infect Dis ; 111: 5-9, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-2113670

ABSTRACT

OBJECTIVE: The aim of this study was to investigate the infectiousness of re-positive coronavirus disease 2019 (COVID-19) patients. METHODS: All nucleic acid testing (NAT) was performed using throat swabs, nasopharyngeal swabs, and anal swabs, which were tested by Fluorescent quantitative realtime PCR. Re-positive cases were defined as a discharged patient who re-tested positive by NAT. Micro-neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was performed based on the methods for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) viruses. IgM and IgG against the N protein of SARS-CoV-2 were determined by ELISA. RESULTS: A total 255 (16.04%) of 1590 COVID-19 patients were re-positive. The re-positive cases were more likely to occur in patients in the 20-39 years age group and in patients with disease of moderate severity. Quantitative PCR showed that cycle threshold (Ct) values and viral loads were both far lower than in the hospitalized COVID-19 patients. The viral load in re-positive cases was very low. Viral culture of the samples from re-positive patients showed no cytopathic effect, and NAT of the culture medium of viral cultures all exhibited negative results. CONCLUSION: The viral load in re-positive cases was very low; patients were not infectious and the risk of human-to-human transmission was extremely low. Discharged COVID-19 patients should undergo home health management for 3 weeks.


Subject(s)
COVID-19 , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Serologic Tests , Viral Load
10.
BMJ Case Rep ; 15(11)2022 Nov 15.
Article in English | MEDLINE | ID: covidwho-2119221

ABSTRACT

SARS-CoV-2 is now a major global health issue and manifests mainly as a respiratory disorder. Several other complications involving hypercoagulability, cardiovascular system and central nervous system have been described in the literature. Among these atypical presentations, encephalopathy associated with SARS-CoV-2 is a rare entity with heterogenous clinical and radiological findings. The direct presence of SARS-CoV-2 in cerebrospinal fluid (CSF) was rarely found in encephalopathy patients with acute SARS-Cov-2 infection.Here, we report a case of myeloencephalitis with positive real-time PCR for SARS-CoV-2 in CSF in a young woman presenting exclusively with neurological symptoms. Other differential diagnosis were extensively pursued by a comprehensive aetiological workup. To our knowledge, this is the first case report in the Omicron era. In the context of recent global explosion of SARS-Cov-2 infections, clinicians should consider this pathogen among other possible neurotropic agents and be familiar with its radiological and clinical presentations.


Subject(s)
COVID-19 , Encephalomyelitis , Female , Humans , Brain Diseases/virology , COVID-19/complications , Encephalomyelitis/diagnosis , Encephalomyelitis/virology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Real-Time Polymerase Chain Reaction
11.
Int J Mol Sci ; 23(22)2022 Nov 18.
Article in English | MEDLINE | ID: covidwho-2116018

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak posed a challenge for diagnostic laboratories worldwide, with low-middle income countries (LMICs) being the most affected. The polymerase chain reaction (PCR) is the gold standard method for detecting SARS-CoV-2 infection. However, the challenge with this method is that it is expensive, which has resulted in under-testing for SARS-CoV-2 infection in many LMICs. Hence, this study aimed to compare and evaluate alternative methods for the mass testing of SARS-CoV-2 infection in laboratories with limited resources to identify cost-effective, faster, and accurate alternatives to the internationally approved kits. A total of 50 residual nasopharyngeal swab samples were used for evaluation and comparison between internationally approved kits (Thermo Fisher PureLink™ RNA Isolation Kit and Thermo Fisher TaqPath™ COVID-19 Assay Kit) and alternative methods (three RNA extraction and four commercial SARS-CoV-2 RT-PCR assay kits) in terms of the cost analysis, diagnostic accuracy, and turnaround time. In terms of performance, all of the alternative RNA extraction methods evaluated were comparable to the internationally approved kits but were more cost-effective (Lucigen QuickExtract™ RNA Extraction Kit, Bosphore EX-Tract Dry Swab RNA Solution and Sonicator method) and four commercial SARS-CoV-2 RT-PCR assay kits (Nucleic Acid COVID-19 Test Kit (SARS-CoV-2), abTESTM COVID-19 qPCR I Kit, PCL COVID19 Speedy RT-PCR Kit, and PCLMD nCoV One-Step RT-PCR Kit) with a sensitivity range of 76-100% and specificity of 96-100%. The cost per sample was reduced by more than 50% when compared to internationally approved kits. When compared to the Thermo Fisher PureLink™ Kit and Thermo Fisher TaqPath™ COVID-19 Assay Kit, the alternative methods had a faster turnaround time, indicating that laboratories with limited resources may be able to process more samples in a day. The above-mentioned cost-effective, fast, and accurate evaluated alternative methods can be used in routine diagnostic laboratories with limited resources for mass testing for SARS-CoV-2 because these were comparable to the internationally approved kits, Thermo Fisher PureLink™ Kit and Thermo Fisher TaqPath™ COVID-19 Assay Kit. The implementation of alternative methods will be the most cost-effective option for testing SARS-CoV-2 infection in LMICs.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19 Testing , Laboratories , Real-Time Polymerase Chain Reaction
12.
Microbiol Spectr ; 10(2): e0235121, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-2114947

ABSTRACT

Microbiological diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a challenge. Although real-time reverse transcription PCR (RT-PCR) represents the gold standard method, strategies that allow rapid and simple diagnosis are necessary for the early identification of cases. In this study, we evaluated the diagnostic performance of six different commercial rapid antigen tests (Coronavirus antigen [Ag] rapid test cassette [Healgen Scientific, Houston, TX, USA], COVID-19 Ag FIA [Vircell, SD Biosensor Inc., Gyeonggi-do, Republic of Korea], Clinitest rapid COVID-19 antigen test [Siemens, Healthineers, Erlangen, Germany], SARS-CoV-2 rapid antigen test [SD Biosensor; Roche Diagnostics, Basel, Switzerland], Panbio COVID-19 Ag rapid test device [Abbott, Chicago, IL, USA], and SARS-CoV-2 test [MonLab, Barcelona, Spain]) in 130 nasopharyngeal swab samples tested previously by RT-PCR. The overall sensitivity of the rapid tests ranged from 65% to 79%, and the specificity was 100% for all of them. The sensitivity was higher for those samples with RT-PCR cycle threshold (CT) values below 25 and those from patients presenting within the first week of symptoms. The Siemens test showed the highest sensitivity for patients with high viral loads while the Vircell test performed better than the rest for CT values of ≥25. IMPORTANCE The rapid detection of people infected with SARS-CoV-2 is essential for a correct and effective control of the disease it causes. This process must be sensitive, fast, and simple, and it must be possible to carry out in any type of health center. Rapid antigen tests are the answer to this need. Knowing its ability to detect the virus in different stages of the disease is essential for a correct diagnosis, which is why this study has been carried out to evaluate the sensitivity and specificity of 6 different antigens tests in nasopharyngeal smear samples.


Subject(s)
COVID-19 , SARS-CoV-2 , Antigens, Viral/analysis , COVID-19/diagnosis , COVID-19 Testing , Humans , Real-Time Polymerase Chain Reaction
13.
PLoS One ; 17(11): e0271860, 2022.
Article in English | MEDLINE | ID: covidwho-2109301

ABSTRACT

Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Pandemics , COVID-19/diagnosis , COVID-19 Testing , Specimen Handling/methods , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity
15.
Int J Mol Sci ; 23(21)2022 Oct 25.
Article in English | MEDLINE | ID: covidwho-2081940

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , RNA, Viral/genetics , COVID-19 Testing , COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity
16.
Clin Lab ; 68(10)2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-2080870

ABSTRACT

BACKGROUND: We retrospectively examined all initial positive SARS-CoV-2 test results using three real-time PCR tests from patients without a history of COVID-19 collected from September to October 2021 at a university-affiliated hospital. METHODS: We defined a possible false-positive (PFP) case as a positive case that showed negative results upon per-forming a confirmatory test on the same specimen. Positivity% and PFP% were defined as the number of first positive and the number of PFP cases divided by the total test numbers, respectively. RESULTS: The positivity%/PFP% values were 0.76%/0.10%, 0.29%/0.02%, and 0.21%/0.03% for the Xpert, Allplex, and cobas tests, respectively. Six (75%) cobas PFP cases were RdRp-only positive. All PFP cases analyzed by Xpert except one had cycle threshold values ≥ 40. Contamination during extraction was suspected in five of the 10 PFP cases analyzed by Allplex, which requires a separate extraction step. CONCLUSIONS: Care must be taken when analyzing first-positive cases as these may be false-positive signals.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/diagnosis , COVID-19 Testing , Real-Time Polymerase Chain Reaction , Retrospective Studies , SARS-CoV-2/genetics , Sensitivity and Specificity
17.
PLoS One ; 17(10): e0276464, 2022.
Article in English | MEDLINE | ID: covidwho-2079770

ABSTRACT

The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19.


Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , Ribonuclease P , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Multiplex Polymerase Chain Reaction/methods , RNA, Messenger
18.
PLoS One ; 17(9): e0273697, 2022.
Article in English | MEDLINE | ID: covidwho-2065114

ABSTRACT

BACKGROUND: Severe acute respiratory infections (SARI) pose a great global burden. The contribution of respiratory viruses to adult SARI is relatively understudied in Asia. We aimed to determine viral aetiology of adult SARI patients in Kuala Lumpur, Malaysia. METHODS: The prevalence of 20 common (mainly viral) respiratory pathogens, and MERS-CoV, SARS-CoV and 5 bacterial select agents was investigated from May 2017 to October 2019 in 489 SARI adult patients in Kuala Lumpur, Malaysia, using molecular assays (Luminex NxTAG-RPP kit and qPCR assays). Viral metagenomics analysis was performed on 105 negative samples. RESULTS: Viral respiratory pathogens were detected by PCR in 279 cases (57.1%), including 10 (2.0%) additional detections by metagenomics analysis. The most detected viruses were rhinovirus/enterovirus (RV/EV) (49.1%) and influenza virus (7.4%). Three melioidosis cases were detected but no SARS-CoV, MERS-CoV or other bacterial select agents. Bacterial/viral co-detections and viral co-detections were found in 44 (9.0%) and 27 (5.5%) cases respectively, mostly involving RV/EV. Independent predictors of critical disease were male gender, chronic lung disease, lack of runny nose and positive blood culture with a significant bacterial pathogen. Asthma and sore throat were associated with increased risk of RV/EV detection, while among RV/EV cases, males and those with neurological disease were at increased risk of critical disease. CONCLUSIONS: Prior to the COVID-19 pandemic, the high prevalence of respiratory viruses in adults with SARI was mainly attributed to RV/EV. Continued surveillance of respiratory virus trends contributes to effective diagnostic, prevention, and treatment strategies.


Subject(s)
COVID-19 , Enterovirus , Respiratory Tract Infections , Viruses , Adult , COVID-19/epidemiology , Enterovirus/genetics , Female , Humans , Malaysia/epidemiology , Male , Pandemics , Real-Time Polymerase Chain Reaction , Rhinovirus/genetics , Viruses/genetics
19.
BMC Vet Res ; 18(1): 369, 2022 Oct 11.
Article in English | MEDLINE | ID: covidwho-2064800

ABSTRACT

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.


Subject(s)
Alphacoronavirus , Coronavirus Infections , Nucleic Acids , Swine Diseases , Alphacoronavirus/genetics , Animals , Coronavirus Infections/diagnosis , Coronavirus Infections/veterinary , Diarrhea/diagnosis , Diarrhea/veterinary , Real-Time Polymerase Chain Reaction/veterinary , Recombinases , Sensitivity and Specificity , Swine , Swine Diseases/diagnosis
20.
Arch Argent Pediatr ; 120(5): 336-339, 2022 10.
Article in English, Spanish | MEDLINE | ID: covidwho-2056105

ABSTRACT

Stopping the spread of coronavirus disease 2019 (COVID-19) is critical and can be achieved through rapid and effective detection techniques. Our objective was to compare the diagnostic accuracy of rapid antigen tests (RAgT) and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) and to describe amplification cycle thresholds (Cts). Participants were children aged 1 month to 11 years with symptoms for less than 7 days, who did not have a detectable result in the past 90 days, and were immunocompetent. A total of 1855 patients were included; the prevalence of COVID-19 was 4.7%. For the RAgT, overall sensitivity was 60.2% and specificity, 99.8%; in children older than 5 years, values were 69.8% and 99.8%, respectively. Ct values for discordant samples were higher. To conclude, the diagnostic accuracy indicated that the specificity of RAgT is similar to that of RT-qPCR, but its sensitivity is notably lower, especially in children younger than 5 years.


Frenar la propagación de la enfermedad por el coronavirus 2019 (COVID-19, por su sigla en inglés) es fundamental, y se puede realizar mediante técnicas de detección rápidas y efectivas. El objetivo fue comparar la precisión diagnóstica de un test rápido de antígeno (TRAg,) con la reacción en cadena de polimerasa con retrotranscripción (RT-qPCR, por su sigla en inglés) y describir los umbrales de amplificación (Ct, por su sigla en inglés). Participaron niños de 1 mes a 11 años que tuvieran menos de 7 días de síntomas, sin resultado detectable en los últimos 90 días, e inmunocompetentes. Se incluyeron 1855 pacientes con una prevalencia de COVID-19 del 4,7 %. La sensibilidad global del TRAg fue del 60,2 % y su especificidad, del 99,8 %; en niños mayores de 5 años los valores fueron de 69,8 % y 99,8 %, respectivamente. Los valores de Ct de las muestras discordantes fueron más altos. En conclusión, la precisión diagnóstica muestra que TRAg tiene una especificidad similar a la RT-qPCR, pero una sensibilidad considerablemente menor, sobre todo en niños de menos de 5 años.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Child , Child, Preschool , Clinical Laboratory Techniques/methods , Humans , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
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