TUMOR MARKERS EXPRESSION LEVELS IN GASTRIC CANCER PATIENT'S PERIPHERAL BLOOD BY RT-PCR ASSESSMENT.

BACKGROUND: Hematological recurrence is the second most frequent cause of failure in the treatment of gastric cancer. The detection of circulating tumor markers in peripheral blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method may be a useful tool to predict recurrence and determine the patient's prognosis. However, no consensus has been reached regarding the association between the tumor markers level in peripheral blood and its impact on patient survival. AIMS: To evaluate the expression of the circulating tumor markers CK20 and MUC1 in peripheral blood samples from patients with gastric cancer by qRT-PCR, and to verify the association of their expression levels with clinicopathological characteristics and survival. METHODS: A total of 31 patients with gastric adenocarcinoma were prospectively included in this study. CK20 and MUC1 expression levels were analyzed from peripheral blood by the qRT-PCR technique. RESULTS: There was no statistically significant (p>0.05) association between CK20 expression levels and clinical, pathological, and surgical features. Higher MUC1 expression levels were associated with female patients (p=0.01). There was a correlation between both gene levels (R=0.81, p<0.001), and CK20 level and tumor size (R=0.39, p=0.034). CONCLUSIONS: CK20 and MUC1 expression levels could be assessed by qRT-PCR from total peripheral blood samples of patients with gastric cancer. CK20 levels were correlated to MUC1 levels as well as to tumor size. There was no difference in disease-free survival and overall survival regarding both genetic markers expression in this series.

Assessing the comparability of cycle threshold values derived from five external quality assessment rounds for omicron nucleic acid testing.

BACKGROUND: A variety of open-system real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays for several acute respiratory syndrome coronavirus 2 are currently in use. This study aimed to ensure the quality of omicron nucleic acid testing and to assess the comparability of cycle threshold (Ct) values derived from RT-PCR. METHODS: Five external quality assessment (EQA) rounds using the omicron virus-like particles were organized between February 2022 and June 2022. RESULTS: A total of 1401 qualitative EQA reports have been collected. The overall positive percentage agreement was 99.72%, the negative percentage agreement was 99.75%, and the percent agreement was 99.73%. This study observed a significant variance in Ct values derived from different test systems. There was a wide heterogeneity in PCR efficiency among different RT-PCR kits and inter-laboratories. CONCLUSION: There was strong concordance among laboratories performing qualitative omicron nucleic acid testing. Ct values from qualitative RT-PCR tests should not be used for clinical or epidemiological decision-making to avoid the potential for misinterpretation of the results.

Ultrasensitive NGS MRD assessment in Ph+ ALL: Prognostic impact and correlation with RT-PCR for BCR::ABL1.

Reverse transcription polymerase chain reaction (RT-PCR) for BCR::ABL1 is the most common and widely accepted method of measurable residual disease (MRD) assessment in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL); however, RT-PCR may not be an optimal measure of MRD in many cases of Ph+ ALL. We evaluated the clinical impact of a highly sensitive next-generation sequencing (NGS) MRD assay (sensitivity of 10-6 ) and its correlation with RT-PCR for BCR::ABL1 in patients with Ph+ ALL. Overall, 32% of patients had a discordance between MRD assessment by RT-PCR and NGS, and 31% of patients who achieved NGS MRD negativity were PCR+ at the same timepoint. Among eight patients with long-term detectable BCR::ABL1 by PCR, six were PCR+/NGS-. These patients generally had stable PCR levels that persisted despite therapeutic interventions, and none subsequently relapsed; in contrast, patients who were PCR+/NGS+ had more variable PCR values that responded to therapeutic intervention. In a separate cohort of prospectively collected clinical samples, 11 of 65 patients (17%) with Ph+ ALL who achieved NGS MRD negativity had detectable BCR::ABL1 by PCR, and none of these patients relapsed. Relapse-free survival and overall survival were similar in patients who were PCR+/NGS- and PCR-/NGS-, suggesting that PCR for BCR::ABL1 did not provide additional prognostic information in patients who achieved NGS MRD negativity. NGS-based assessment of MRD is prognostic in Ph+ ALL and identifies patients with low-level detectable BCR::ABL1 who are unlikely to relapse nor to benefit from therapeutic interventions.

RT-PCR-based assessment of the SD Bioline Rota/Adeno Antigen-based test in infants with and without diarrhea.

BACKGROUND: Rotavirus A (RVA) infections remain a major cause of severe acute diarrhea affecting children worldwide. To date, rapid diagnostic tests (RDT) are widely used to detect RVA. However, paediatricians question whether the RDT can still detect the virus accurately. Therefore, this study aimed to evaluate the performance of the rapid rotavirus test in comparison to the one-step RT-qPCR method. METHODS: A cross-sectional study was conducted in Lambaréné, Gabon, from April 2018 to November 2019. Stool samples were collected from children under 5 years of age with diarrhoea or a history of diarrhoea within the last 24 h, and from asymptomatic children from the same communities. All stool samples were processed and analysed using the SD BIOLINE Rota/Adeno Ag RDT against a quantitative reverse transcription PCR (RT-qPCR), which is considered the gold standard. RESULTS: For a total of 218 collected stool samples, the overall sensitivity of the RDT was 46.46% (confidence interval (CI) 36.38-56.77), with a specificity of 96.64% (CI 91.62-99.08) compared to one-step RT-qPCR. After confirming the presence or absence of RVA gastroenteritis, the RDT showed suitable results in detecting rotavirus A-associated disease, with a 91% concordance with the RT-qPCR. Furthermore, the performance of this test varied when correlated with seasonality, symptoms, and rotavirus genotype. CONCLUSION: This RDT showed high sensitivity and was suitable for the detection of RVA in patients with RVA gastroenteritis, although some asymptomatic RVA shedding was missed by RT-qPCR. It could be a useful diagnostic tool, especially in low-income countries.

Cost-analysis of real time RT-PCR test performed for COVID-19 diagnosis at India's national reference laboratory during the early stages of pandemic mitigation.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) is one of the most accurate and extensively used laboratory procedures for diagnosing COVID-19. This molecular test has high diagnostic accuracy (sensitivity and specificity) and is considered as the gold standard for COVID-19 diagnosis. During COVID-19 surge in India, rRT-PCR service was encouraged and supported by the government of India through existing healthcare setup at various levels of healthcare facilities. The primary purpose of this research was to determine the per-unit cost of providing COVID-19 rRT-PCR services at the national reference laboratory at ICMR-National Institute of Virology in Pune during the early phase of COVID-19 pandemic mitigation, from the provider's perspective. The monthly cost for rRT-PCR testing as well as an estimated annual average unit cost for testing that takes account of peaks and troughs in pandemic were investigated. The time frame used to estimate unit cost was one year (July 2020-June 2021). For data collection on all resources spent during the early phase of pandemic, a conventional activity-based bottom-up costing technique was used. Capital costs were discounted and annualized over the estimated life of the item. Apportioning statistics were selected for cost heads like human resources, capital, and equipment based on time allocation, sharing of services, and utilization data. The data was also used to understand the breakdown of costs across inputs and over time and different levels of testing activity. During the initial phase of pandemic mitigation, the per unit cost of providing the COVID-19 rRT-PCR test was estimated to be ₹566 ($7.5) in the month of July 2020, where the total 56318 COVID-19 rRT-PCR tests was performed. The major proportion (87%) of funds was utilized for procuring laboratory consumables, followed by HR (10%), and it was least for stationary & allied items (0.02%). Unit cost was found to be the most sensitive to price variations in lab consumables (21.7%), followed by the number of samples tested (3.9%), salaries paid to HR (2.6%), price of equipment (0.23%), and building rental price (0.14%) in a univariate sensitivity analysis. The unit cost varies over the period of the pandemic in proportion with the prices of consumables and inversely proportional with number of tests performed. Our study would help the Government to understand the value for money they invested for laboratory diagnosis of COVID-19, budget allocation, integration and decentralization of laboratory services so as to help for achieving universal health coverage.

Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR.

Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

Performance and Cost-effectiveness of Pooling COVID-19 Samples Using Real time Polymerase Chain Reaction Test.

BACKGROUND: Rapid detection of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) by real-time polymerase chain reaction (RT-PCR) is the most reliable method used worldwide. Although the incidence of the disease has increased globally, the limited availability of PCR kits has become the major bottleneck for the diagnosis of COVID positive patients. METHODS: Random samples were pooled for two months in group of two-five and tested for SARS-CoV-2. If the pool was negative, all individuals in the pool were reported negative. If the pool was positive, then the individual samples were retested to identify the positive individual. RESULTS: The mean cycle threshold (Ct) value of pooled samples was not significantly different with that of individual samples for N, ORF-1ab and E genes. Also, pooling saved more than 60% of reagents, time and effort, workforce and cost. CONCLUSIONS: In this study, the positivity rate was around 5% and saving of reagent, cost, time and manpower was more than 60%.

Comparison and Harmonization of Different Semi-Automated and Automated qRT-PCR Assays in the Assessment of SARS-CoV-2.

In SARS-CoV-2 diagnostics, cycle threshold (Ct) values from qRT-PCRs semi-quantitatively estimate a patient's viral load. However, relevant analytical differences between qRT-PCR assays are often neglected. This study was designed (i) to identify such differences between five commonly used assays and (ii) to demonstrate a straightforward strategy to harmonize them. QRT-PCRs for SARS-CoV-2 were carried out in 85 oropharyngeal swab samples using three fully automated (Alinity m, cobas®6800 and GeneXpert) and two semi-automated (genesig® and RIDA®GENE) assays. Qualitative results (positive/negative) showed excellent comparability between the fully automated assays, but not between the Alinity m and semi-automated methods. Ct values significantly varied between all the methods, with the median values ranging from 22.76 (Alinity m) to 30.89 (RIDA®GENE) and 31.50 (genesig®), indicating the lowest sensitivity for semi-automated methods. Passing-Bablok analysis further revealed systemic biases. Assay-specific viral load concentration calculations-based on generated individual standard curves-resulted in much better comparability between the assays. Applying these calculations, significant differences were no longer detectable. This study highlights relevant analytical differences between SARS-CoV-2 qRT-PCR assays, leading to divergent decisions about the mandatory isolation of infected individuals. Secondly, we propose a strategy to harmonize qRT-PCR assays to achieve better comparability. Our findings are of particular interest for laboratories utilizing different assays.

Introducing a simple and cost-effective RT-PCR protocol for detection of DPYD*2A polymorphism: the first study in Kurdish population.

PURPOSE: Fluoropyrimidines, the major chemotherapeutic agents in various malignancies treatment, are metabolized by dihydropyrimidine dehydrogenase (DPD). DPD deficiency can lead to severe and sometimes fatal toxicity. In the present study, we developed a simple protocol to detect the DPYD*2A variant. Common side effects in patients treated with these drugs were also evaluated in a Kurdish population. METHOD: We established a reverse-transcriptase polymerase chain reaction (RT-PCR) technique for detection of DPYD*2A. Sanger sequencing was used to confirm the results. 121 Kurdish patients receiving fluoropyrimidine derivatives were enrolled, and clinical information regarding the dosage and toxicity was analyzed. RESULTS: Our RT-PCR method was able to detect one patient with heterozygous state for DPYD*2A (0.8%). The most observed adverse drug reactions were tingling, nausea, and hair loss. The frequency of patients with the toxicity of grade 3 or worse was 6.6%. CONCLUSION: This was the first study that detect DPYD*2A polymorphism in the Kurdish population. Our method was successfully able to detect the DPYD*2A variant and, due to its simplicity and cost-effectiveness, it may be considered as an alternative to the current methods, especially in developing countries. Our detected polymorphism rate at 0.8% is comparable with other studies. Despite the low rate of DPYD*2A polymorphism, pharmacogenetics assessment before beginning the treatment process is highly recommended due to its association with a high risk of severe toxicity.

Eastern Mediterranean Health Journal [2022; Vol.28, Issue 9]

Eastern Mediterranean Health Journal is the official health journal published by the Eastern Mediterranean Regional Office of the World Health Organization. It is a forum for the presentation and promotion of new policies and initiatives in health services; and for the exchange of ideas concepts epidemiological data research findings and other information with special reference to the Eastern Mediterranean Region. It addresses all members of the health profession medical and other health educational institutes interested NGOs WHO Collaborating Centres and individuals within and outside the Region

المجلة الصحية لشرق المتوسط هى المجلة الرسمية التى تصدرعن المكتب الاقليمى لشرق المتوسط بمنظمة الصحة العالمية. وهى منبر لتقديم السياسات والمبادرات الجديدة فى الصحة العامة والخدمات الصحية والترويج لها، و لتبادل الاراء و المفاهيم والمعطيات الوبائية ونتائج الابحاث وغير ذلك من المعلومات، و خاصة ما يتعلق منها باقليم شرق المتوسط. وهى موجهة الى كل اعضاء المهن الصحية، والكليات الطبية وسائر المعاهد التعليمية، و كذا المنظمات غير الحكومية المعنية، والمراكز المتعاونة مع منظمة الصحة العالمية والافراد المهتمين بالصحة فى الاقليم و خارجه

La Revue de Santé de la Méditerranée Orientale est une revue de santé officielle publiée par le Bureau régional de l’Organisation mondiale de la Santé pour la Méditerranée orientale. Elle offre une tribune pour la présentation et la promotion de nouvelles politiques et initiatives dans le domaine de la santé publique et des services de santé ainsi qu’à l’échange d’idées de concepts de données épidémiologiques de résultats de recherches et d’autres informations se rapportant plus particulièrement à la Région de la Méditerranée orientale. Elle s’adresse à tous les professionnels de la santé aux membres des instituts médicaux et autres instituts de formation médico-sanitaire aux ONG Centres collaborateurs de l’OMS et personnes concernés au sein et hors de la Région

Inter-laboratory testing as a strategy for external quality assessment for qualitative detection of SARS-CoV-2 by real-time RT-PCR testing in India.

To implement the strategy of test, track and treat to tackle the ongoing COVID-19 pandemic, the number of real-time RT-PCR-based testing laboratories was increased for diagnosis of SARS-CoV-2 in the country. To ensure reliability of the laboratory results, the Indian Council of Medical Research initiated external quality assessment (EQA) by deploying inter-laboratory quality control (ILQC) activity for these laboratories by nominating 34 quality control (QC) laboratories. This report presents the results of this activity for a period of September 2020 till November 2020. A total of 597 laboratories participated in this activity and 86 per cent of these scored ≥90 per cent concordance with QC laboratories. This ILQC activity showcased India's preparedness in quality diagnosis of SARS-CoV-2.

Diagnostic Performance Assessment of Saliva RT-PCR and Nasopharyngeal Antigen for the Detection of SARS-CoV-2 in Peru.

Widely available and reliable testing for SARS-CoV-2 is essential for the public health response to the COVID-19 pandemic. We estimated the diagnostic performance of reverse transcription PCR (RT-PCR) performed on saliva and the SD Biosensor STANDARD Q antigen test performed on nasopharyngeal swab compared to the reference standard, nasopharyngeal swab (NP) RT-PCR. We enrolled participants living and/or seeking care in health facilities in North Lima, Peru from November 2020 to January 2021. Consenting participants underwent same-day RT-PCR on both saliva and nasopharyngeal swab specimens, antigen testing on a nasopharyngeal swab specimen, pulse oximetry, and standardized symptom assessment. We calculated sensitivity, specificity, and predictive values for the nasopharyngeal antigen and saliva RT-PCR compared to nasopharyngeal RT-PCR. Of 896 participants analyzed, 567 (63.3%) had acute signs/symptoms of COVID-19. The overall sensitivity and specificity of saliva RT-PCR were 85.8% and 98.1%, respectively. Among participants with and without acute signs/symptoms of COVID-19, saliva sensitivity was 87.3% and 37.5%, respectively. Saliva sensitivity was 97.4% and 56.0% among participants with cycle threshold (CT) values of ≤30 and >30 on nasopharyngeal RT-PCR, respectively. The overall sensitivity and specificity of nasopharyngeal antigen were 73.2% and 99.4%, respectively. The sensitivity of the nasopharyngeal antigen test was 75.1% and 12.5% among participants with and without acute signs/symptoms of COVID-19, and 91.2% and 26.7% among participants with CT values of ≤30 and >30 on nasopharyngeal RT-PCR, respectively. Saliva RT-PCR achieved the WHO-recommended threshold of >80% for sensitivity for the detection of SARS-CoV-2, while the SD Biosensor nasopharyngeal antigen test did not. IMPORTANCE In this diagnostic validation study of 896 participants in Peru, saliva reverse transcription PCR (RT-PCR) had >80% sensitivity for the detection of SARS-CoV-2 among all-comers and symptomatic individuals, while the SD Biosensor STANDARD Q antigen test performed on nasopharyngeal swab had <80% sensitivity, except for participants whose same-day nasopharyngeal RT-PCR results showed cycle threshold values of <30, consistent with a high viral load in the nasopharynx. The specificity was high for both tests. Our results demonstrate that saliva sampling could serve as an alternative noninvasive technique for RT-PCR diagnosis of SARS-CoV-2. The role of nasopharyngeal antigen testing is more limited; when community transmission is low, it may be used for mass screenings among asymptomatic individuals with high testing frequency. Among symptomatic individuals, the nasopharyngeal antigen test may be relied upon for 4 to 8 days after symptom onset, or in those likely to have high viral load, whereupon it showed >80% sensitivity.

A rapid and cost-effective diagnostic algorithm for the detection of SARS-CoV-2 infection in the emergency area by combining highly sensitive antigenic test and RT-PCR.

A diagnostic algorithm for SARS-CoV-2 infection in patients admitted to the emergency area, based on a combination of rapid antigen and molecular testing, has been evaluated with 3070 nasopharyngeal swabs. Compared to molecular test alone, the proposed algorithm allowed to significantly reduce costs and average time to results.

RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS.

BACKGROUND: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. AIM: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene. MATERIALS AND METHODS: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. RESULTS: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. CONCLUSION: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.

Virological and Serological Assessment of US Army Trainees Isolated for Coronavirus Disease 2019.

BACKGROUND: Laboratory screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key mitigation measure to avoid the spread of infection among recruits starting basic combat training in a congregate setting. Because viral nucleic acid can be detected persistently after recovery, we evaluated other laboratory markers to distinguish recruits who could proceed with training from those who were infected. METHODS: Recruits isolated for coronavirus disease 2019 (COVID-19) were serially tested for SARS-CoV-2 subgenomic ribonucleic acid (sgRNA), and viral load (VL) by reverse-transcriptase polymerase chain reaction (RT-PCR), and for anti- SARS-CoV-2. Cluster and quadratic discriminant analyses of results were performed. RESULTS: Among 229 recruits isolated for COVID-19, those with a RT-PCR cycle threshold >30.49 (sensitivity 95%, specificity 96%) or having sgRNA log10 RNA copies/mL <3.09 (sensitivity and specificity 96%) at entry into isolation were likely SARS-CoV-2 uninfected. Viral load >4.58 log10 RNA copies/mL or anti-SARS-CoV-2 signal-to-cutoff ratio <1.38 (VL: sensitivity and specificity 93%; anti-SARS-CoV-2: sensitivity 83%, specificity 79%) had comparatively lower sensitivity and specificity when used alone for discrimination of infected from uninfected. CONCLUSIONS: Orthogonal laboratory assays used in combination with RT-PCR may have utility in determining SARS-CoV-2 infection status for decisions regarding isolation.

Cost analysis of coronavirus disease 2019 test strategies using pooled reverse transcriptase-polymerase chain reaction technique.

BACKGROUND: This study aimed to compare the testing strategies for COVID-19 (i.e., individual, simple pooling, and matrix pooling) in terms of cost. METHODS: We simulated the total expenditures of each testing strategy for running 10,000 tests. Three parameters were used: positive rate (PR), pool size, and test cost. We compared the total testing costs under two hypothetical scenarios in South Korea. We also simulated country-specific circumstances in India, South Africa, South Korea, the UK, and the USA. RESULTS: At extreme PRs of 0.01% and 10%, simple pooling was the most economic option and resulted in cost reductions of 98.0% (pool size ≥80) and 36.7% (pool size = 3), respectively. At moderate PRs of 0.1%, 1%, 2%, and 5%, the matrix pooling strategy was the most economic option and resulted in cost reductions of 97.0% (pool size ≥88), 86.1% (pool size = 22), 77.9% (pool size = 14), and 59.2% (pool size = 7), respectively. In both hypothetical scenarios of South Korea, simple pooling costs less than matrix pooling. However, the preferable options for achieving cost savings differed depending on each country's cost per test and PRs. CONCLUSIONS: Both pooling strategies resulted in notable cost reductions compared with individual testing in most scenarios pertinent to real-life situations. The appropriate type of testing strategy should be chosen by considering the PR of COVID-19 in the community and the test cost while using an appropriate pooling size such as five specimens.

Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis.

TaqMan probe-based commercial real-time (RT) PCR kits are expensive but most frequently used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections needs to meet the challenge of testing more persons at a reasonable cost. This study developed a simple and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay targeting two virus-specific genes along with a host-specific internal control. A total of 180 randomly selected samples portioning into two subsets based on crude and high-quality RNA extraction were used to compare this assay with a nationwide available commercial kit (Sansure Biotech Inc., (Hunan, China)), so that we could analyze the variation and validity of this in-house developed method. Our customized-designed primers can specifically detect the viral RNA likewise Sansure. We separately optimized SYBR Green RT-PCR reaction of N, E, S, and RdRp genes based on singleplex melting curve analysis at the initial stage. After several rounds of optimization on multiplex assays of different primer combinations, the optimized method finally targeted N and E genes of the SARS-CoV-2 virus, together with the ß-actin gene of the host as an internal control. Comparing with the Sansure commercial kit, our proposed assay provided up to 97% specificity and 93% sensitivity. The cost of each sample processing ranged between ~2 and ~6 USD depending on the purification level of extracted RNA template. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in low-income countries.

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

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

External quality assessment of COVID-19 real time reverse transcription PCR laboratories in India.

Sudden emergence and rapid spread of COVID-19 created an inevitable need for expansion of the COVID-19 laboratory testing network across the world. The strategy to test-track-treat was advocated for quick detection and containment of the disease. Being the second most populous country in the world, India was challenged to make COVID-19 testing available and accessible in all parts of the country. The molecular laboratory testing network was augmented expeditiously, and number of laboratories was increased from one in January 2020 to 2951 till mid-September, 2021. This rapid expansion warranted the need to have inbuilt systems of quality control/ quality assurance. In addition to the ongoing inter-laboratory quality control (ILQC), India implemented an External Quality Assurance Program (EQAP) with assistance from World Health Organization (WHO) and Royal College of Pathologists, Australasia. Out of the 953 open system rRTPCR laboratories in both public and private sector who participated in the first round of EQAP, 891(93.4%) laboratories obtained a passing score of > = 80%. The satisfactory performance of Indian COVID-19 testing laboratories has boosted the confidence of the public and policy makers in the quality of testing. ILQC and EQAP need to continue to ensure adherence of the testing laboratories to the desired quality standards.

Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES: In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS: We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS: We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.