Reverse Transcription-polymerase Chain Reaction Kit Research Articles

Evaluation of a commercial Immunochromatographic Strip Assay (ICT) for rapid detection of bovine, porcine and human Rotavirus A

The most frequent diarrhoeal pathogen “Rotavirus” is commonly detected by Ribo Nucleic Acid- Polyacrylamide Gel Electrophoresis (RNA-PAGE), Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Immunoassay (Enzyme Linked Immunosorbent Assay) in humans. However, use of these tests is limited in animals therefore, penside test like Immunochromatographic test/Lateral Flow Assay (ICT/LFA) is necessary. The present study was carried out to compare the diagnostic efficacy of rapid commercial ICT strip assay, RNA-PAGE and RT-PCR for specific detection of Rotavirus A (RVA) from stool samples of calves, piglets and children. A total of 313 faecal samples were collected between November 2022 to April 2023 from children below 5years of age (n=100), calves (n=100) and piglets (n=113) ≤ 3 months of age and were screened by RNA-PAGE, RT-PCR and commercial ICT kit for rotavirus A.The overall positivity of RVA from human, calves and piglets using RNA-PAGE, RT-PCR and LFA was found to be 35% (35/100), 8% (8/100) and 14.16% (16/113), respectively. Higher positivity of rotavirus was recorded in male children (44.26%, 27/61) than in female children (20.51%, 8/39). The kappa agreement between LFA and RT-PCR was 0.79 (substantial agreement); between LFA and RNA-PAGE was 0.61 (substantially low agreement) and between RNA-PAGE and RT-PCR was found to be 0.75 (substantial agreement). Relative diagnostic sensitivity and specificity of LFA in comparison with RT-PCR was 74.54% and 98.44%, respectively and in comparison with RNA-PAGE was 72.97% and 93.47%, respectively. RNA-PAGE in comparison with RT-PCR is having diagnostic sensitivity and specificity of 65.45% and 99.6%, respectively. In conclusion, the ICT strip assay is a rapid, convenient and effective method with satisfactory efficacy for detection of RVA from children, calves and piglets. ICT fulfilled the WHO ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Delivered) criteria for point-of-care testing. It can be useful in determining rotavirus outbreaks in resource-limited settings.

Cost-effective in-house COVID-19 reverse transcription-polymerase chain reaction testing with yeast-derived Taq polymerase.

Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

Sensitivity and specificity of a rapid antigen assay for the detection of SARS-CoV-2 during the surge of B.1.1.529 variant

Background: As COVID-19 spread across the globe, the new variants continued to emerge, leading to a rapid surge in the cases and thereby overwhelming the testing laboratories. As Rapid antigen test kits became available, they complimented the reverse transcription polymerase chain reaction (RT-PCR) based testing and thereby moderately disburdening the testing laboratories. However, to increase the testing capacities, the regulatory authorities approved these rapid kits with little scrutiny. Therefore, validation of rapid antigen testing kits becomes essential before they are used as viable alternatives to RT-PCR based testing. Aims and Objectives: The aim of this study was to compare the sensitivity and specificity of OSKIT SARS-CoV-2 corona antigen test (Oscar Medicare Pvt. Ltd., India) to that of COVID-19 ONE-STEP RT-PCR KIT (Meril Diagnostics Pvt. Ltd., India). Materials and Methods: In this prospective study, spanning the peak of third wave in India (Between December 1 and January 31, 2022), 242 specimens were collected and analyzed by comparing their results both by OSKIT SARS-CoV-2 Corona Antigen Test and COVID-19 ONE-STEP RT-PCR KIT. The various clinico-epidemiological attributes of the patients were taken into consideration and used to analyze the results. Results: Rapid antigen tests (RAT) was positive in 44 individuals, giving an overall sensitivity of 67.31%. Sensitivity increased when Ct value of below 30 was taken a positive or when only symptomatic individuals were taken for analysis. Sensitivity was highest in case the duration of symptoms was <5 days (92.86%). When comparing RAT results with Ct value of the screening gene (N gene), a higher proportion of positive cases was observed for lower Ct values. Further analysis revealed that majority of the RAT positive cases were from that subset of symptomatic patients who had a history of symptoms of <5 days. Conclusion: The COVID-19 rapid antigen test evaluated in this study was able to detect SARS-CoV-2 infection with high viral loads in both asymptomatic and symptomatic individuals. Thus, this test can serve as a rapid tool for reducing community spread of the virus. The study concludes that the duration since symptom onset greatly affects sensitivity of antigen testing.

Investigation of SARS-CoV-2 RNA contamination in water supply resources of Tabriz metropolitan during a peak of COVID-19 pandemic.

It is crucial to have access to clean water resources during the COVID-19 pandemic for hygiene, since virus infection through wastewater leaks in metropolitan areas can be a threat. Accurate monitoring of urban water resources during the pandemic seems to be the only way to confirm safe and infected resources. Here, in this study, the amount of Severe Acute Respiratory Syndrome Coronavirus 2's Ribonucleic Acid (SARS-CoV-2 RNA) in the Tabriz urban water network located in the northwest of Iran was investigated by an extensive sampling of the city's water sources at a severe peak of the COVID-19 pandemic. The sampling process comprised a range of water sources, including wells, qanats, water treatment facilities, dams, and reservoirs. For each sample, a combination of polyethylene glycol (PEG) and sodium chloride (NaCl) was used for concentration and a laboratory RNA-based method was conducted for quantification. Before applying the extraction and quantification procedure to real samples, the proposed concentration method was verified with synthetic serum samples for the first time. After the concentration, RNA extraction was done by the BehPrep extraction column method, and Reverse Transcription Polymerase Chain Reaction(RT-PCR) detection of the virus was done by Covitech COVID-19 RT-PCR kit. In none of the water supply resources, SARS-COV-2 RNA has been detected except in a sample grabbed from a well adjacent to an urban wastewater discharge point downstream. The results of molecular analysis for the positive sample showed that the CT value and concentration of the virus genome were equal to 32.57 and 5720copies/L, respectively. Quantitative analysis of real samples shows that the city's water network was safe at the time of the study. However, given that the positive sample was exposed to wastewater leakage, periodic sampling from wells and qanats is suggested during the pandemic until it can be proven that the leakage to these water sources is impossible.

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.

Discordant results of SARS-CoV-2 PCR-based tests in the early phase of pandemic in Indonesia: Infection control consequences

IntroductionGrowing evidence suggest that cycle threshold (CT)-value of reverse transcription polymerase chain reaction (RT-PCR) is correlated with transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and some kits set different CT-value cut-off. This report presents the discordant results of two widely used RT-PCR kits in Indonesia due to different CT-value cut-offs and highlights its potential consequence in SARS-CoV-2 containment. MethodsNasopharyngeal swab samples with SARS-CoV-2 negative with a RT-PCR kit (manufacture pre-set CT-value cut-off was 35 amplification cycles) were retested with another RT-PCR kit with a higher pre-set CT-value of 40 amplification cycles. All procedures were performed according to the manufacturer protocols. ResultsIn total, 30 samples with SARS-CoV-2 negative for the first kit were retested. We found that 25 out of 33 samples (75.5%) were positive using the second RT-PCR kit that had a higher manufacture pre-set CT-value cut-off. In addition, among 500 RT-PCR tests using the first RT-PCR kit, 103 of them (20.6%) were categorized as inconclusive results based on the second manufacturer’ guideline. Discussion and conclusionOur data suggest the possibility of discordant results of SARS-CoV-2 detection due to different pre-set cut-offs by the companies. As consequence, this could leave a fraction of individuals who were misclassified that could act as source of virus transmission within community.

Evaluation of the Rapid Antigen Detection Kit with the Polymerase Chain Reaction for Detection of SARS-CoV-2 in Respiratory Samples

The disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was named as coronavirus disease-2019 (COVID-19) by the World Health Organization (WHO) in February 11, 2020. The rapid diagnosis of COVID-19 patients is essential to reduce the disease spread. The reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard test to diagnose SARS-CoV-2 acute infection. The rapid antigen test which can detect the presence of viral protein antigens in respiratory tract samples is being investigated as an alternative option, especially in cases where RT-PCR is not available or the test capacity is exceeded, due to its faster results, ease of application, low cost and lack of special equipment and personnel. In this study, it was aimed to evaluate the performance of a commercial rapid antigen test using nasopharyngeal samples of COVID-19 patients confirmed with RT-PCR. From the first day of the research, the first 80 consecutively SARS-CoV-2 RT-PCR positive and 40 RT-PCR negative respiratory samples sent to the Medical Microbiology Laboratory for routine SARS-CoV-2 RT-PCR testing were included in the study. RT-PCR tests of the samples were performed in routine studies with the BioSpeedy SARS-CoV-2 RT-PCR kit (Bioeksen, Turkey). Rapid antigen tests were performed with the Wesail COVID-19 antigen test kit (Guangdong, China) simultaneously with RT-PCR tests. Amongst the 80 positive RT-PCR samples, 56 were detected by the rapid antigen test. All the samples detected as positive with the rapid antigen tests were also positive with RT-PCR. There was a moderate agreement between the qualitative results of both tests (Kappa= 0.609, p<0.001). According to the PCR test, the sensitivity, specificity, positive predictive value (PPV), negative predictive value, and accuracy of the rapid antigen test were; 70%, 100%, 100%, 62.5%, and 80% (96/120), respectively. The sensitivities of the rapid antigen test were calculated as 92.6% in 54 samples with a cycle threshold (Ct) value of <17, 88.7% in 62 samples with a Ct value of <20, 77.8% in 72 samples with a Ct value of <22, and 74.7% in 75 samples with a Ct value of <25. According to our study data; the rapid antigen test was found less sensitive than the RT-PCR test. Negative results obtained with rapid antigen testing cannot exclude SARS-CoV-2 infection and must be confirmed by RT-PCR. In addition, according to the ROC analysis of rapid antigen test positivity obtained according to RT-PCR Ct values, the clinical performance of the rapid antigen test is good in samples with Ct values <20. The rapid antigen test should be evaluated as a reliable screening test in patients with high viral load. To the best of our knowledge, there is no other study in the literature performed with the Wesail COVID-19 rapid antigen test kit (Guangdong, China) used in our study. The fact that PPV was found to be 100% even at a low prevalence period of the pandemic will enable positive patients to be screened quickly and effectively with rapid antigen tests in the first step during the high prevalence period of the pandemic. In the light of these data and our results, it can be predicted that using the rapid antigen test as a screening test in the first step and confirming only negative patients with RT-PCR will contribute to the effective management of the pandemic process in terms of both time and cost. As a result of the study, the rapid antigen test with low sensitivity but high PPD can be included as a facilitating test in the first step of the diagnostic algorithm in terms of rapid identification of the patients with high viral load, initiation of treatment and providing filiation.

Clinical Comparative Evaluation of the LabTurboTM AIO® Reverse Transcription-Polymerase Chain Reaction and World Health Organization-Recommended Assays for the Detection of Emerging SARS-CoV-2 Variants of Concern.

PurposeSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent behind coronavirus disease-2019 (COVID-19). Single-plex reverse transcription-polymerase chain reaction (RT-PCR)-based assays are widely used for COVID-19 detection but exhibit decreased sensitivity and specificity in detecting the rapidly spreading SARS-CoV-2 variants; in contrast, multiplex RT-PCR reportedly yields better results. Here, we aimed at comparatively analyzing the clinical performance of the LabTurboTM AIO COVID-19 RNA testing kit, a multiplex quantitative RT-PCR kit, including a three-target (E, N1, and RNase P), single-reaction, triplex assay used for SARS-CoV-2 detection, with that of the WHO-recommended RT-PCR assay.Materials and MethodsResidual, natural, nasopharyngeal swabs obtained from universal transport medium specimens at SARS-CoV-2 testing centers (n = 414) were collected from May to October 2021. For SARS-CoV-2 qRT-PCR, total viral nucleic acid was extracted. The limit of detection (LOD) and the comparative clinical performances of the LabTurboTM AIO COVID-19 RNA kit and the WHO-recommended RT-PCR assay were assessed. Statistical analysis of the correlation was performed and results with R2 values >0.9 were considered to be highly correlated.ResultsThe LOD of the LabTurboTM AIO COVID-19 RNA kit was 9.4 copies/reaction for the target genes N1 and E. The results obtained from 102 SARS-CoV-2-positive and 312 SARS-CoV-2-negative samples showed 100% correlation with previous WHO-recommended RT-PCR assay results.ConclusionMultiplex qRT-PCR is a critical tool for detecting unknown pathogens and employs multiple target genes. The LabTurboTM AIO COVID-19 RNA testing kit provides an effective and efficient assay for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants.

Comparative Evaluation of Truenat Reverse Transcription-Polymerase Chain Reaction with Commercially Available Reverse Transcription-Polymerase Chain Reaction Kits for COVID-19 Diagnosis

Introduction: Early and correct identification of the betacoronavirus is important for effective isolation treatment and case management. Real-time polymerase chain reaction (PCR) are consider as a gold standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, for that, there are a requirement of skilled workforce and elaborate infrastructure. A rapid point of care test known as Truenat Beta CoV and Truenat SARS COV assay were recommended by the Government of India. The aim of the study was to find out the performance of Truenat assay in comparison to four RT-PCR assay kits. Materials and Methods: The cross sectional study was conducted in a COVID-19 testing laboratory in Central India. Forty known Truenat positive sample with different viral load were analyze in selected rtPCR kits from 4 different manufacturers. Results: Of the total of ten very low viral load samples, BGI kit was able to detect six samples, followed by TruePCR six samples, TaqPath five samples and NIV kit were able to detect three samples. Similarly, in the case of low viral load sample, BGI and TaqPath kit were able to detect all the 10 samples followed by NIV kit five samples and TruePCR nine samples respectably. In the case of medium and high viral load samples, all four reverse transcription-PCR (RT-PCR) kits were shown a 100% detection rate. Conclusions: Based on our findings, we believe truenat RT-PCR is a more reliable technique for the detection of SARC-CoV-2. Hence, it should be installed in the healthcare setup for better control of the pandemic.

Investigation of Tomato ringspot virus (ToRSV) by Real-Time TaqMan RT-PCR in Hakkari province, Turkey

Tomato ringspot virus (ToRSV) belongs to the Nepovirus genus in the family Secoviridae. It has a wide host range and is listed as a quarantine virus in Turkey. In this study, 80 leaf samples were collected from tomato, pepper, cucumber and grapevine cultivation sites located in three different parts of Hakkari province: Şemdinli, Çukurca and Center districts. Real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) method was used for the detection of the virus. Amplification was carried out in reaction mix including QuantiNova Probe RT-PCR kit (Qiagen, Germany) using primers and TaqMan probe based on 3'-UTR (untranslated region) of virus, which amplified a 182 bp product of the genome. ToRSV was detected in 13 of the 80 samples and threshold cycle (CT) values ranged from 23.9 to 37.4. It was found that 16.25% of the samples collected from the districts of Hakkari province were found to be infected with ToRSV whereas no ToRSV was detected in the samples collected from the center of the city. The virus was detected on pepper and cucumber samples in Çukurca district, and it was also detected in tomato, pepper, cucumber and grapevine samples in Şemdinli district. To our knowledge, this study is the first report of molecular detection of ToRSV by real-time TaqMan RT-PCR in Turkey.

Review of Current Diagnostics Developed for COVID-19

COVID-19 emerged in 2019 as a pandemic that affected the world in many aspects and it have been the biggest challenge for many countries in the past year. Due to the lack of approved treatment methods for the disease, and more importantly, to inhibit its spread, the critical task is to detect it fast and reliable. Many research groups and companies have been developing different methods and products for the diagnosis of COVID-19. Each method has advantages and disadvantages, while rapid, inexpensive, and high-throughput detection methods are needed. In this regard, significant progress has been achieved so far. In this article, we reviewed in-vitro Diagnostics (IVD) and Commercial Kits in three main categories including real-time reverse transcription-polymerase chain reaction kits, serology-based tests, and point-of-care diagnostic tests. In addition, familiarizing with coronavirus and its detection methods, genome study, Cell entrance and antigenicity, and Specimen, Lab Biosafety, and Authorization of Medical Devices for COVID-19 were discussed.

Local binary pattern and deep learning feature extraction fusion for COVID‐19 detection on computed tomography images

The deadly coronavirus virus (COVID‐19) was confirmed as a pandemic by the World Health Organization (WHO) in December 2019. It is important to identify suspected patients as early as possible in order to control the spread of the virus, improve the efficacy of medical treatment, and, as a result, lower the mortality rate. The adopted method of detecting COVID‐19 is the reverse‐transcription polymerase chain reaction (RT‐PCR), the process is affected by a scarcity of RT‐PCR kits as well as its complexities. Medical imaging using machine learning and deep learning has proved to be one of the most efficient methods of detecting respiratory diseases, but to train machine learning features needs to be extracted manually, and in deep learning, efficiency is affected by deep learning architecture and low data. In this study, handcrafted local binary pattern (LBP) and automatic seven deep learning models extracted features were used to train support vector machines (SVM) and K‐nearest neighbour (KNN) classifiers, to improve the performance of the classifier, a concatenated LBP and deep learning feature was proposed to train the KNN and SVM, based on the performance criteria, the models VGG‐19 + LBP achieved the highest accuracy of 99.4%. The SVM and KNN classifiers trained on the hybrid feature outperform the state of the art model. This shows that the proposed feature can improve the performance of the classifiers in detecting COVID‐19.

Comparative Evaluation of Nasopharyngeal and Oropharyngeal Swab Based Rapid SARS-CoV-2 Antigen Detection and Real-Time RT-PCR for Diagnosis of COVID-19 in Tertiary Care Hospital.

Purpose: To assess and compare the diagnostic accuracy of the GenBody COVID-19 Antigen kit (GenBody Inc., Cheonan, South Korea) available in the market with the gold standard reverse transcription-polymerase chain reaction (RT-PCR) assay to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).Methods: Nasopharyngeal and oropharyngeal swabs were collected from suspected coronavirus disease 2019 (COVID-19) patients and tested by RT-PCR and GenBody Rapid antigen kit. Performance characteristic of the antigen kit was calculated.Results: We tested nasopharyngeal swabs and oropharyngeal swabs (n=240). Amongst the 102 positive RT-PCR samples, the rapid antigen test detected 36 as positive, showing an overall sensitivity of 35.3%. All the samples detected positive with the antigen rapid test were also detected positive by RT-PCR.Conclusion: The performance of the rapid antigen kit was good with respect to high viral load samples, whereas those with lower levels were missed. Unfortunately, the overall low sensitivity of the antigen kit does not allow using it alone as the frontline testing kit for COVID-19 diagnosis.

Automatic Detection of Covid-19 with Bidirectional LSTM Network Using Deep Features Extracted from Chest X-ray Images.

Coronavirus disease, which comes up in China at the end of 2019 and showed different symptoms in people infected, affected millions of people. Computer-aided expert systems are needed due to the inadequacy of the reverse transcription-polymerase chain reaction kit, which is widely used in the diagnosis of this disease. Undoubtedly, expert systems that provide effective solutions to many problems will be very useful in the detection of Covid-19 disease, especially when unskilled personnel and financial deficiencies in underdeveloped countries are taken into consideration. In the literature, there are numerous machine learning approaches built with different classifiers in the detection of this disease. This paper proposes an approach based on deep learning which detects Covid-19 and no-finding cases using chest X-ray images. Here, the classification performance of the Bi-LSTM network on the deep features was compared with the Deep Neural Network within the frame of the fivefold cross-validation technique. Accuracy, sensitivity, specificity and precision metrics were used to evaluate the classification performance of the trained models. Bi-LSTM network presented better performance compare to DNN with 97.6% value of high accuracy despite the few numbers of Covid-19 images in the dataset. In addition, it is understood that concatenated deep features more meaningful than deep features obtained with pre-trained networks by one by, as well. Consequently, it is thought that the proposed study based on the Bi-LSTM network and concatenated deep features will be noteworthy in the design of highly sensitive automated Covid-19 monitoring systems.

Discordant results of SARS-CoV-2 PCR-based tests in the early phase of pandemic in Indonesia: Infection control consequences

IntroductionGrowing evidence suggest that cycle threshold (CT)-value of reverse transcription polymerase chain reaction (RT-PCR) is correlated with transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and some kits set different CT-value cut-off. This report presents the discordant results of two widely used RT-PCR kits in Indonesia due to different CT-value cut-offs and highlights its potential consequence in SARS-CoV-2 containment. MethodsNasopharyngeal swab samples with SARS-CoV-2 negative with a RT-PCR kit (manufacture pre-set CT-value cut-off was 35 amplification cycles) were retested with another RT-PCR kit with a higher pre-set CT-value of 40 amplification cycles. All procedures were performed according to the manufacturer protocols. ResultsIn total, 30 samples with SARS-CoV-2 negative for the first kit were retested. We found that 25 out of 33 samples (75.5%) were positive using the second RT-PCR kit that had a higher manufacture pre-set CT-value cut-off. In addition, among 500 RT-PCR tests using the first RT-PCR kit, 103 of them (20.6%) were categorized as inconclusive results based on the second manufacturer’ guideline. Discussion and conclusionOur data suggest the possibility of discordant results of SARS-CoV-2 detection due to different pre-set cut-offs by the companies. As consequence, this could leave a fraction of individuals who were misclassified that could act as source of virus transmission within community.

Characterization and Recombinant Genotypes of HIV-1 in Gansu Province, China

It is important to monitor the diversity and evolution of HIV-1 genotypes, especially in some remote and undeveloped regions in China where the diversity and distribution of HIV-1 genotypes are not fully clear. To investigate the genotypes and distribution of HIV-1 in far Northwestern Gansu Province of China, we selected 220 HIV-1-positive plasma samples from the Center for Disease Control and Prevention (CDC) in Gansu from January 2016 to December 2018. The viral load of inclusion samples were over 1,000 copies per milliliter. The gag, pol, and env gene of HIV-1 were amplified by nested reverse transcription-polymerase chain reaction kit, sequenced, and then identified genotypes using HIV-BLAST tool and the neighbor-joining method. One hundred fifty of 220 inclusion samples were successfully determined HIV-1 genotypes. Our results show that circulating recombinant forms (CRF) 07_BC and CRF01_AE are predominant and accounted for 46.7% and 28.0%, respectively. Other HIV-1 subtypes and genotypes included B/B' (6.0%), CRF08_BC (4.0%), and C (1.3%). In addition, we reported CRF65_cpx and CRF55_01B subtypes in Gansu for the first time. Phylogenetic tree analysis showed that the sequences of different samples are scattered in different genotype groups, and no obvious aggregation occurs. Our results indicate the genetic variety and complexity of HIV-1 and provide critical information for HIV/AIDS control and prevention in Gansu Province.

Evaluation of the diagnostic performance of nine commercial RT-PCR kits for the detection of SARS-CoV-2 in Colombia.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic has led to the design and development of multiple reverse‐transcription polymerase chain reaction kits aimed to facilitate the rapid scale‐up of molecular testing for massive screening. We evaluated the diagnostic performance of nine commercial kits, which showed optimal performance and high discriminatory power. However, we observed differences in terms of sensitivity, specificity, and E gene Ct Values and discuss these results in light of the influence of SARS‐CoV‐2 genetic variability and its potential impact in current molecular diagnostic assays.

Evaluation of Deep Architectures for Automatic Lung Infection Segmentation of COVID-19 HRCT Images

Deep learning has been one of the widely used techniques in biomedical image segmentation. COVID-19 is spreading rapidly all over the world. Early diagnosis of disease is necessary to control the pandemic. The common method of diagnosis of COVID-19 infection is RT-PCR (Reverse Transcription - Polymerase Chain Reaction) method. However, RT-PCR is prone to produce a number of false results and availability of RT-PCR kits is limited. Computed Tomography (CT) and X-Ray of lung can be used as an alternative tool to make the diagnosis, as the disease primarily targets the epithelial cells of the lung. The CT scanners considered to be diagnostic tools for COVID-19. The number of images to be analyzed by the radiologist is large therefore it is necessary to automate the task of CT Image processing. In this paper, we have developed an Automated biomedical segmentation system using five different deep learning architectures which performs the COVID-19 lung infection segmentation. The models are trained using open source datasets. Five deep learning models selected for study are U-Net, LinkNet, ResU-Net, ResU-Net++ and U-Net++. The U-Net++ model shown better results with a dice coefficient of 84.69%, sensitivity of 78.92%, specificity of 99.51% and precision of 92.12%. Experimental results are generated using PSG IMSR (Institute of Medical Sciences & Research) dataset, which is a collection of 75,000 images of nearly 125 patients. The radiologists have verified the results of infection segmentation. Automated infection segmentation of HRCT scans using deep learning models can be used for faster diagnosis of COVID-19. Impact Statement — RT-PCR method used for COVID-19 infection diagnosis is prone to produce a number of false results and the availability of a number of RT-PCR testing kits is limited. Medical imaging like chest CT scan can be used as alternative tool as the disease primarily targets lung. Due to wide availability of CT scanners, they are considered to be diagnosis of COVID-19. The number of patients who undergo scans and images to be analyzed by the radiologist is very large therefore it is necessary to automate the task. Deep learning approach can be used effectively to aid the Radiologists and Physicians. We have proposed deep learning based automated system which can greatly reduce the time in analyzing HRCT lung images of patients to ensure proper treatment to the needy patients. The system can assists in monitor the progress in patients recovery by comparing the infection present in the lung. This paper has been submitted on 14-July-2021. This project work is carried out as part master program in Computer science and Engineering.

Prevalence of Rotavirus Genotypes after Introduction of Monovalent Rotavirus Vaccine in Uzbekistan during 2014-2016

Introduction: Rotaviruses are one of the most leading causes of severe gastroenteritis in children less than five years of age worldwide. This study describes prevalence of rotavirus A (RVA) genotypes in Uzbekistan during for the period October-December in 2014 and2015-2016 after introduction of rotavirus vaccination. Methods: In total, 17546 stool specimens testing for the presence of rotavirus antigen by EIA was performed by using the Prospect Rotavirus Kit (Oxoid Ltd.UK). In total 318 EIA positive samples were randomly selected and genotyped by using one-step conventional reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was performed using a Qiagen One-Step RT-PCR kit (Qiagen, Inc., Valencia, CA) and Rotavirus Genotyping Oligonucleotide Primers (CDC, Atlanta). Results: The results showed a change in the circulating genotypes towards the prevalence of the genotype G2P[4] and a decrease in the prevalence of the genotype G1P[8]. Conclusion: The prevalence of the genotype G2P[4] is not necessarily due to vaccine escape, but can also occur in the course of the natural fluctuation of RVA genotypes, both geographically and temporally and this tendency requires further monitoring.

Laboratory verification of an RT-PCR assay for SARS-CoV-2.

BackgroundReverse transcription‐polymerase chain reaction (RT‐PCR) is an extremely common clinical method for detecting pathogens, particularly for emerging infectious diseases such as the new coronavirus disease (COVID‐19). Currently, detection of the RNA from the novel coronavirus SARS‐CoV‐2 is the gold standard for establishing a COVID‐19 diagnosis. This study evaluates the characteristic performance of the analytical system in a clinical laboratory.MethodsA commercial SARS‐CoV‐2 RNA RT‐PCR Kit used in a clinical laboratory is assessed based on ISO 15189 verification requirements. A multiple real‐time RT‐PCR assay for the RdRP, N, and E genes in SARS‐CoV‐2 is verified.ResultsThe analytical system exhibits good analytical sensitivity (1000 copies/mL) and specificity (100%); however, the values of 86.7% and 100% for analytical accuracy deserved attention, compared with two other types of methods. Overall, the kit is potentially useful for SARS‐CoV‐2 diagnostic testing and meets the verification requirements.ConclusionCompliance with international standards, such as ISO 15189, is valuable for clinical laboratories and for improving laboratory medicine quality and safety. Normalization is essential for obtaining reliable results from the SARS‐CoV‐2 RNA RT‐PCR assay. This study aims to develop an improved SARS‐CoV‐2 verification framework compared with traditional molecular diagnostic methods, given the urgency of implementing new assays in clinical laboratories.