Direct PCR with the CDC 2019 SARS-CoV-2 assay: optimization for limited-resource settings
Ontology highlight
ABSTRACT:
SUBMITTER: Victoriano C
PROVIDER: S-EPMC9272867 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.
| S-EPMC7598596 | biostudies-literature
Project description:The rapid emergence and spread of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants across the globe underscores the crucial need for continuous SARS-CoV-2 surveillance to ensure that potentially more pathogenic variants are detected early and contained. Whole genome sequencing (WGS) is currently the gold standard for COVID-19 surveillance; however, it remains cost-prohibitive and requires specialized technical skills. To increase surveillance capacity, especially in resource-scarce settings, supplementary methods that are cost- and time-effective are needed. Real-time multiplex PCR genotyping assays offer an economical and fast solution for screening circulating and emerging variants while simultaneously complementing existing WGS approaches. In this study we evaluated the AllplexTM SARS-CoV-2 Variants II multiplex real-time PCR genotyping assay, Seegene (South Korea), and implemented it in retrospectively characterizing circulating SARS-CoV-2 variants in a rural South African setting between April and October 2021, prior to the emergence of the Omicron variant in South Africa. The AllplexTM SARS-CoV-2 Variants II real-time PCR assay demonstrated perfect concordance with whole-genome sequencing in detecting Beta and Delta variants and exhibited high specificity, sensitivity and reproducibility. Implementation of the assay in characterization of SARS-CoV-2 variants between April and October 2021 in a rural South African setting revealed a rapid shift from the Beta to the Delta variant between April and June. All specimens successfully genotyped in April were Beta variants and the Delta variant was not detected until May. By June, 78% of samples genotyped were Delta variants and in July >95% of all genotyped samples were Delta variants. The Delta variant continued to predominate through to the end of our analysis in October 2021. Taken together, a commercial SARS-CoV-2 variant genotyping assay detected the rapid rate at which the Delta variant displaced the Beta variant in Limpopo, an under-monitored province in South Africa. Such assays provide a quick and cost-effective method of monitoring circulating variants and should be used to complement genomic sequencing for COVID-19 surveillance especially in resource-scarce settings.
| S-EPMC9231807 | biostudies-literature
Project description:Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N?=?96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N?=?87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N?=?230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ? and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N?=?46) and 78.6% (N?=?14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX.The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.
| S-EPMC3223235 | biostudies-literature
Project description:BackgroundTo retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR.MethodsFour previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay.ResultsIn this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site.ConclusionSARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.
| S-EPMC9059459 | biostudies-literature
Project description:Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 disease. RT-qPCR has been the primary method of diagnosis; however, the required infrastructure is lacking in many developing countries and the virus has remained a global challenge. More inexpensive and immediate test methods are required to facilitate local, regional, and national management strategies to re-open world economies. Here we have developed a SARS-CoV-2 antigen test in an inexpensive lateral flow format to generate a chromatographic result identifying the presence of the SARS-CoV-2 antigen, and thus an active infection, within a patient anterior nares swab sample. Our 15-min test requires no equipment or laboratory infrastructure to administer with a limit of detection of 2.0 × 102 TCID50/mL and 87.5% sensitivity, 100% specificity when tested against 40 known positive and 40 known negative patient samples established by a validated RT-qPCR test.
| S-EPMC8626481 | biostudies-literature
Project description:Despite increasing availability of anti-retroviral therapy, invasive cryptococcal disease continues to be a leading cause of death among HIV-infected individuals in resource-limited settings. Screening asymptomatic HIV-infected individuals with advanced immunosuppression for serum cryptococcal antigen clearly identifies a population at high risk of cryptococcal meningitis and death. However, screening with serum cryptococcal antigen alone identifies a heterogeneous clinical population, many of whom have mild clinical symptoms, sub-clinical meningeal infection, or fungemia. Currently, there is wide variation in practice and little evidence to guide the use of anti-fungal and anti-retroviral treatment for asymptomatic cryptococcal antigenemia (ACA). Furthermore, implementing a targeted screening and treatment intervention for ACA presents numerous operational challenges for already overburdened health care systems in resource-limited settings. While such an intervention shows promise, there are critical gaps in our understanding of ACA and its implications in the outpatient setting and an urgent need for additional research in this area.
| S-EPMC3809900 | biostudies-literature
Project description:BackgroundAlthough laparoscopic surgery has made remarkable progress and become the standard approach for various surgical procedures worldwide over the past 30 years, its establishment in low-resource settings, particularly in public hospitals, has been challenging. The lack of equipment and trained expertise has hindered its widespread adoption in these settings. Cholecystectomy is one of the most commonly performed procedures using laparoscopy world wide AIM: The aim of the study is to determine whether laparoscopic cholecystectomy is feasible in a resource challenged setting METHODS: The research focused on individuals who underwent laparoscopic or open cholecystectomies at Yekatit 12 Hospital in Addis Ababa, Ethiopia, over a one-year period. Comprehensive data collection was conducted prospectively, encompassing both intraoperative and postoperative parameters. Follow-up was carried out via phone calls. The surgical procedures employed innovative techniques, including the reuse of sterilized single-use equipment and the utilization of local resources. The evaluation involved a comparison of demographic information, intraoperative details (such as critical view determination and operative duration), and postoperative complications, including assessments of pain and wound infections RESULTS: From August 2021 to September 2022, 119 patients were assessed. Among these patients, 65 (54.6%) underwent open cholecystectomies, while the remaining 54 (45.4%) underwent laparoscopic cholecystectomies. The average duration of the laparoscopic cholecystectomies was 90.7 min, which is 17.7 min behind the open. Patients in the laparoscopy group had significantly shorter hospital stays than the open group, and 94% were discharged by post operative day 2. The conversion rate from laparoscopic to open surgery was determined to be 3.3% CONCLUSION: To sum up, the safe execution of laparoscopic cholecystectomies is feasible in public hospitals and settings with limited resources, given adequate training and resource distribution. The study findings showcased superior outcomes, including reduced hospitalization duration and fewer complications, while maintaining comparable levels of operative duration and patient satisfaction in both groups.
| S-EPMC10964563 | biostudies-literature
Project description:BackgroundCOVID-19 is an ongoing public health pandemic regardless of the countless efforts made by various actors. Quality diagnostic tests are important for early detection and control. Notably, several commercially available one step RT-PCR based assays have been recommended by the WHO. Yet, their analytic and diagnostic performances have not been well documented in resource-limited settings. Hence, this study aimed to evaluate the diagnostic sensitivities and specificities of three commercially available one step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in Ethiopia in clinical setting.MethodsA cross-sectional study was conducted from April to June, 2021 on 279 respiratory swabs originating from community surveillance, contact cases and suspect cases. RNA was extracted using manual extraction method. Master-mix preparation, amplification and result interpretation was done as per the respective manufacturer. Agreements between RT-PCRs were analyzed using kappa values. Bayesian latent class models (BLCM) were fitted to obtain reliable estimates of diagnostic sensitivities, specificities of the three assays and prevalence in the absence of a true gold standard.ResultsAmong the 279 respiratory samples, 50(18%), 59(21.2%), and 69(24.7%) were tested positive by TIB, Da An, and BGI assays, respectively. Moderate to substantial level of agreement was reported among the three assays with kappa value between 0 .55 and 0.72. Based on the BLCM relatively high specificities (95% CI) of 0.991(0.973-1.000), 0.961(0.930-0.991) and 0.916(0.875-0.952) and considerably lower sensitivities with 0.813(0.658-0.938), 0.836(0.712-0.940) and 0.810(0.687-0.920) for TIB MOLBIOL, Da An and BGI respectively were found.ConclusionsWhile all the three RT-PCR assays displayed comparable sensitivities, the specificities of TIB MOLBIOL and Da An were considerably higher than BGI. These results help adjust the apparent prevalence determined by the three RT-PCRs and thus support public health decisions in resource limited settings and consider alternatives as per their prioritization matrix.
| S-EPMC8775315 | biostudies-literature
Project description:Alternatives to nasopharyngeal sampling are needed to increase capacity for SARS-CoV-2 testing. Among 275 participants, we piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including polyester flocked swabs as well as 3D-printed plastic lattice swabs, placed into viral transport media or an RNA stabilization agent. Flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), missing those with low viral load (<106 viral copies/mL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into RNA preservative, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. We also evaluated pooling strategies that involved pooling specimens in the lab versus pooling swabs at the point of collection, finding both successfully detected samples with >105 viral copies/mL.
| S-EPMC8230941 | biostudies-literature
Project description:Standard nasopharyngeal swab testing for SARS-CoV-2 detection by PCR is not always feasible due to limitations in trained personnel, personal protective equipment, swabs, PCR reagents, and access to cold chain and biosafety hoods. We piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including both standard polyester flocked swabs as well as 3D printed plastic lattice swabs, placed into either viral transport media or an RNA stabilization agent. Quantitative SARS-CoV-2 viral detection by RT-qPCR was compared to that obtained by nasopharyngeal sampling as the reference standard. Pooling specimens in the lab versus pooling swabs at the point of collection was also evaluated. Among 275 participants, flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), mostly missing those with low viral load (<10^3 viral copies/uL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into an RNA stabilizer, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. Pooling sample specimens or swabs both successfully detected samples >102 viral copies/uL. Nasal swabs are likely adequate for clinical diagnosis of acute infections to help expand testing capacity in resource-constrained settings. When collected into an RNA preservative that also inactivates infectious virus, nasal swabs yielded quantitative viral loads approximating those obtained by nasopharyngeal sampling.
| S-EPMC7924294 | biostudies-literature