Viral Load Samples Research Articles

Use of RT-PCR thermocycling program for the quantification of DNA viruses in a single run with RNA viruses: Example of Altona RealStar® HSV or VZV PCR kits

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.

Application of a ddRT-PCR to quantify seasonal influenza virus for viral isolation

Viral isolation in cell cultures has been regarded for decades as the “gold standard” for the laboratory diagnosis of influenza viral infections. Not all viral strains could be isolated from clinical samples. This study aimed to quantify the viral load in the samples before isolation to save working time and improve working efficiency. Four hundred samples from patients with influenza-like cases were confirmed pdmH1N1 positive (200 cases) and B Victoria (BV) positive (200 cases) by whole-genome sequencing and analyzed by ddPCR for viral load in samples before isolation, and isolation results were verified by hemagglutination (HA) assay and hemagglutination-inhibition (HI) tests. Probit regression analysis was used to calculate the isolation viral load limit with a 95% probability level by SPSS 19.0 software. The results showed that the isolation limit of viral load was 4.9 × 104 (95% CI: 2.5 × 104–9.0 × 104) copies/mL for pdmH1N1 and 1.9 × 104 (95% CI: 7.8 × 103–3.6 × 104) copies/mL for BV. The isolation rate of clinical samples is positively correlated with the viral load in clinical samples, which can be used for viral culture, providing important guidance for daily work.

Detección y cuantificación de VHB y VHC en muestras de plasma mediante la utilización de diferentes ensayos moleculares: estudio comparativo

IntroductionViral load is a very useful marker for monitoring patients infected with HBV and HCV. This work compares assays based on transcription-mediated amplification and on real-time PCR to verify whether they can be interchangeable. Material and methodsa bicentric study, in which 147 plasma samples from patients infected with HBV and 229 with HCV were analyzed, was carried out. Transcription-mediated amplification-based assays (Aptima® HBV Quant and Aptima® HCV Quant Dx, employing Panther system (Hologic®)) and on real-time PCR (COBAS® AmpliPrep / COBAS® TaqMan® and COBAS® 6800) were used and the degree of concordance between them was calculated. ResultsViral load was detected in both systems in 60 (40.82%) HBV samples (median log viral load: COBAS®: 2.51IU/mL (IQR 2.20-3.17), Panther: 2.71IU/mL (IQR 2.21-3.22)) and in 39 (16.96%) HCV samples (median log viral load: COBAS®: 3.93IU/mL (IQR 2.24-6.01), Panther: 3.80IU/mL (IQR 1.99-6.14)). The agreement between both systems was κ=0.943 for HBV and κ=0.925 for HCV. Comparison of viral load samples detected by both assays showed a hight correlation for HBV (R2=0.86) and for HCV (R2=0.97). ConclusionsBoth transcription-mediated amplification and on real-time PCR based assays may be interchangeable for the management of patients infected with HBV and HCV.

Time-of-Day Variation in SARS-CoV-2 RNA Levels during the Second Wave of COVID-19.

Circadian rhythms influence and coordinate an organism’s response to its environment and to invading pathogens. We studied the diurnal variation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasal/throat swabs collected in late 2020 to spring 2021 in a population immunologically naïve to SARS-CoV-2 and prior to widespread vaccination. SARS-CoV-2 diagnostic PCR data from 1698 participants showed a significantly higher viral load in samples obtained in the afternoon, in males, and in hospitalised patients when linear mixed modelling was applied. This study illustrates the importance of recording sample collection times when measuring viral replication parameters in clinical and research studies.

HIV suppression was maintained during the COVID-19 pandemic in Malawi: a program-level cohort study

Background and ObjectivesMeasures introduced to reduce the spread of SARS-CoV-2 by the Malawi government and the national HIV care program might have compromised treatment outcomes of patients living with HIV on antiretroviral therapy (ART). We studied viral load (VL) outcomes before and during the COVID-19 epidemic in Malawi. MethodsIn this population-based cohort study, we included all routine VL measurements collected from July 2019 to December 2020 in about 650 ART clinics in Malawi. We examined differences between pandemic periods (before/during COVID-19) for i) VL monitoring, and ii) VL suppression (VLS: <1,000 copies/ml). For i) we studied the number of VL measurements over time and assessed predictors of missed measurements before and during COVID-19 in logistic regression models. For ii) we estimated the odds of VLS before and during the COVID-19 epidemic stratified by treatment regimen using generalized estimation equations adjusted for age, sex, time on ART, and type of biological sample. We imputed missing treatment regimens by population-calibrated multiple imputation. ResultsWe included 607,894 routine VL samples from 556,281 patients. VL testing declined during COVID-19 (243,729; 40%) compared to before COVID-19 (365,265; 60%), but predictors of missing tests were similar in the two periods. VLS rates increased slightly from 93% before to 94% during COVID-19. Compared to before COVID-19, the odds of VLS increased during COVID-19 for patients on protease inhibitor-based (PI) regimens (adjusted odds ratio [aOR] 1.22, 95% CI: 0.99-1.49) and for patients on integrase strand transfer inhibitor-based (INSTI) regimens (aOR 1.10, 95% CI: 1.03-1.17). There was no difference in VLS between the two periods among patients on nonnucleoside reverse transcriptase inhibitor-based (NNRTI) regimens. VLS varied by age, sex, regimen, and duration on ART, ranging from 45.1% (95% CI 40.3-50.0%) to 97.2% (95% CI 96.9-97.4%). ConclusionThere was a significant decline in VL monitoring during COVID-19, but we did not find clear evidence that the pandemic reduced VL suppression rates. Routine scheduled VL monitoring, targeted adherence support, and timely regimen switches for patients with treatment failure remain critical to improving VLS.

Accuracy of rapid point-of-care antigen-based diagnostics for SARS-CoV-2: An updated systematic review and meta-analysis with meta-regression analyzing influencing factors.

Comprehensive information about the accuracy of antigen rapid diagnostic tests (Ag-RDTs) for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is essential to guide public health decision makers in choosing the best tests and testing policies. In August 2021, we published a systematic review and meta-analysis about the accuracy of Ag-RDTs. We now update this work and analyze the factors influencing test sensitivity in further detail. We registered the review on PROSPERO (registration number: CRD42020225140). We systematically searched preprint and peer-reviewed databases for publications evaluating the accuracy of Ag-RDTs for SARS-CoV-2 until August 31, 2021. Descriptive analyses of all studies were performed, and when more than 4 studies were available, a random-effects meta-analysis was used to estimate pooled sensitivity and specificity with reverse transcription polymerase chain reaction (RT-PCR) testing as a reference. To evaluate factors influencing test sensitivity, we performed 3 different analyses using multivariable mixed-effects meta-regression models. We included 194 studies with 221,878 Ag-RDTs performed. Overall, the pooled estimates of Ag-RDT sensitivity and specificity were 72.0% (95% confidence interval [CI] 69.8 to 74.2) and 98.9% (95% CI 98.6 to 99.1). When manufacturer instructions were followed, sensitivity increased to 76.3% (95% CI 73.7 to 78.7). Sensitivity was markedly better on samples with lower RT-PCR cycle threshold (Ct) values (97.9% [95% CI 96.9 to 98.9] and 90.6% [95% CI 88.3 to 93.0] for Ct-values <20 and <25, compared to 54.4% [95% CI 47.3 to 61.5] and 18.7% [95% CI 13.9 to 23.4] for Ct-values ≥25 and ≥30) and was estimated to increase by 2.9 percentage points (95% CI 1.7 to 4.0) for every unit decrease in mean Ct-value when adjusting for testing procedure and patients' symptom status. Concordantly, we found the mean Ct-value to be lower for true positive (22.2 [95% CI 21.5 to 22.8]) compared to false negative (30.4 [95% CI 29.7 to 31.1]) results. Testing in the first week from symptom onset resulted in substantially higher sensitivity (81.9% [95% CI 77.7 to 85.5]) compared to testing after 1 week (51.8%, 95% CI 41.5 to 61.9). Similarly, sensitivity was higher in symptomatic (76.2% [95% CI 73.3 to 78.9]) compared to asymptomatic (56.8% [95% CI 50.9 to 62.4]) persons. However, both effects were mainly driven by the Ct-value of the sample. With regards to sample type, highest sensitivity was found for nasopharyngeal (NP) and combined NP/oropharyngeal samples (70.8% [95% CI 68.3 to 73.2]), as well as in anterior nasal/mid-turbinate samples (77.3% [95% CI 73.0 to 81.0]). Our analysis was limited by the included studies' heterogeneity in viral load assessment and sample origination. Ag-RDTs detect most of the individuals infected with SARS-CoV-2, and almost all (>90%) when high viral loads are present. With viral load, as estimated by Ct-value, being the most influential factor on their sensitivity, they are especially useful to detect persons with high viral load who are most likely to transmit the virus. To further quantify the effects of other factors influencing test sensitivity, standardization of clinical accuracy studies and access to patient level Ct-values and duration of symptoms are needed.

Viral load scale-up in south Sudan: Strategic implementation of tools to monitor HIV treatment success among people living with HIV.

As access to antiretroviral therapy (ART) for people with HIV (PWH) in the Republic of South Sudan (RSS) increases, viral load (VL) suppression is critical to protect global HIV response investments. We describe VL scale-up between 2017-2020 in the RSS President's Emergency Plan for AIDS Relief (PEPFAR)-supported program. President's Emergency Plan for AIDS Relief (PEPFAR) South Sudan developed a VL scale-up plan and tools spanning the VL cascade: pre-test, test and post-test and included assessment of clinical facility and laboratory readiness; clinical and laboratory forms and standard operating procedures for test ordering, specimen collection, processing, results return and utilization; procedures to map clients, monitor turn-around-times (TAT), and an electronic system to monitor VL performance. Between 2017 to 2020, VL monitoring was established in 58 facilities, with 59,600 VL samples processed, and improvements in TAT (150-28days) and rejection rates (1.9%-0.8%). VL documentation improved for dates of ART initiation, VL test request and dispatch, and HIV regimen. Total average time from high VL to repeat VL decreased from 15.9months to 6.4months in 2017 and 2019, respectively. A concerted approach to VL scale-up has been fundamental as South Sudan strives towards UNAIDS 95-95-95 targets for PWH on ART.

Systems Analysis and Improvement Approach to optimize the pediatric and adolescent HIV Cascade (SAIA-PEDS): a pilot study

IntroductionChildren and adolescents lag behind adults in achieving UNAIDS 95-95-95 targets for HIV testing, treatment, and viral suppression. The Systems Analysis and Improvement Approach (SAIA) is a multi-component implementation strategy previously shown to improve the HIV care cascade for pregnant women and infants. SAIA merits adaptation and testing to reduce gaps in the pediatric and adolescent HIV cascade.MethodsWe adapted the SAIA strategy components to be applicable to the pediatric and adolescent HIV care cascade (SAIA-PEDS) in Nairobi and western Kenya. We tested whether this SAIA-PEDS strategy improved HIV testing, linkage to care, antiretroviral treatment (ART), viral load (VL) testing, and viral load suppression for children and adolescents ages 0–24 years at 5 facilities. We conducted a pre-post analysis with 6 months pre- and 6 months post-implementation strategy (coupled with an interrupted time series sensitivity analysis) using abstracted routine program data to determine changes attributable to SAIA-PEDS.ResultsBaseline levels of HIV testing and care cascade indicators were heterogeneous between facilities. Per facility, the monthly average number of children/adolescents attending outpatient and inpatient services eligible for HIV testing was 842; on average, 253 received HIV testing services, 6 tested positive, 6 were linked to care, and 5 initiated ART. Among those on treatment at the facility, an average of 15 had a VL sample taken and 13 had suppressed VL results returned.Following the SAIA-PEDS training and mentorship, there was no substantial or significant change in the ratio of HIV testing (RR: 0.803 [95% CI: 0.420, 1.532]) and linkage to care (RR: 0.831 [95% CI: 0.546, 1.266]). The ratio of ART initiation increased substantially and trended towards significance (RR: 1.412 [95% CI: 0.999, 1.996]). There were significant and substantial improvements in the ratio of VL tests ordered (RR: 1.939 [95% CI: 1.230, 3.055]) but no substantial or significant change in the ratio of VL results suppressed (RR: 0.851 [95% CI: 0.554, 1.306]).ConclusionsThe piloted SAIA-PEDS implementation strategy was associated with increases in health system performance for indicators later in the HIV care cascade, but not for HIV testing and treatment indicators. This strategy merits further rigorous testing for effectiveness and sustainment.

The Role of Subgenomic RNA in Discordant Results From Reverse Transcription-Polymerase Chain Reaction Tests for COVID-19.

Reverse transcription-polymerase chain reaction (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when 1 RT-PCR target is positive for SARS-CoV-2 and 1 RT-PCR target is negative for SARS-CoV-2 within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. To understand the role of subgenomic RNA transcripts in discordant results from RT-PCR tests for COVID-19. A panel of 6 droplet digital PCR assays was designed to quantify the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity. Eleven clinical nasopharyngeal swabs were also tested with this panel. In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. The same trends in the relative abundance of copies across different targets observed in infected cells were observed in clinical samples, although trends were more pronounced in infected cells. This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by these data.

Performance assessment of the new Xpert® HIV-1 viral load XC assay for quantification of HIV-1 viral loads

BackgroundHIV-1 RNA quantification is a key component of treatment monitoring. ObjectivesTo assess the performance of a redesigned HIV-1 RNA quantitative assay that uses a dual-target approach: Xpert® HIV-1 Viral Load (VL) XC. Study designFresh and frozen samples (N = 533) from HIV-1 positive patients tested with Abbott HIV-1 assays (Alinity m and RealTime [m2000]) were retested using the new Xpert XC assay. Three samples with known underquantification using the previous single-target Xpert assay were retested. ResultsThe Xpert XC assay yielded valid results in 98.5% (N = 528/536) of cases and showed high sensitivity in 80 fresh samples that had undetectable VLs or ≤1.7 log copies/mL with Alinity m. Linear regression and Bland-Altman analyses showed high concordance with the Abbott tests for quantified samples over a wide VL range (1.6–6.9 copies/mL), including non-B subtypes (mean difference=-0.1±0.23 log copies/mL). Mutations associated with integrase resistance did not impact the results. Very good linearity and reproducibility was shown for the tested subtypes B, CRF06_cpx, and CRF02_AG. Xpert XC VLs in samples that were previously underquantified using the original single-target Xpert assay were similar to those detected by the Abbott assays (±0.11 log copies/mL). ConclusionsThe Xpert XC assay showed excellent correlation with the Abbott assays for all tested HIV-1 subtypes. Sensitivity, linearity and accuracy were high in the therapeutically relevant VL range. With a time to result of only 90 min, this on-demand decentralized assay is a safe, reliable and fast option for VL monitoring in HIV-1-infected patients.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks.

SUMMARYThe ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

Assessment of the feasibility of pool testing for SARS-CoV-2 infection screening

Background SARS-CoV-2 pandemic represented a huge challenge for national health systems worldwide. Pooling nasopharyngeal (NP) swabs seems to be a promising strategy, saving time and resources, but it could reduce the sensitivity of the RT-PCR and exacerbate samples management in terms of automation and tracing. In this study, taking advantage of the routine implementation of a screening plan on health workers, we evaluated the feasibility of pool testing for SARS-CoV-2 infection diagnosis in the presence of low viral load samples. Method Pools were prepared with an automated instrument, mixing 4, 6 or 20 NP specimens, including one, two or none positive samples. Ct values of positive samples were on average about 35 for the four genes analyzed. Results The overall sensitivity of 4-samples and 6-samples pools was 93.1 and 90.0%, respectively. Focussing on pools including one sample with Ct value ≥35 for all analyzed genes, sensitivity decreased to 77.8 and 75.0% for 4- and 6-samples, respectively; pools including two positive samples, resulted positive in any size as well as pools including positive samples with Ct values <35. Conclusion Pool testing strategy should account the balance between cost-effectiveness, dilution effect and prevalence of the infection. Our study demonstrated the good performances in terms of sensitivity and saving resources of pool testing mixing 4 or 6 samples, even including low viral load specimens, in a real screening context possibly affected by prevalence fluctuation. In conclusion, pool testing strategy represents an efficient and resources saving surveillance and tracing tool, especially in specific context like schools, even for monitoring changes in prevalence associated to vaccination campaign.

Tenofovir diphosphate in dried blood spots predicts future viremia in persons with HIV taking antiretroviral therapy in South Africa.

Tenofovir diphosphate (TFV-DP) in dried blood spots (DBS) is used as a biomarker of antiretroviral therapy (ART) adherence. Recent treatment studies have shown that TFV-DP predicts future viremia in persons with HIV (PWH) but there are few data from high-burden settings. We investigated whether TFV-DP in DBS predicts future viral breakthrough in South African PWH. Prospective observational cohort. We enrolled 250 adults receiving tenofovir-containing regimens, currently virally suppressed (<50 copies/ml) but at risk of future viral breakthrough, from four primary health clinics in Cape Town. Paired viral load and DBS for TFV-DP were collected monthly for 12 months. Viral breakthrough was the first confirmed viral load greater than 400 copies/ml. Logistic regression estimated the odds ratio (OR) and 95% confidence intervals for future viral breakthrough at the next visit. Participants provided 2944 paired DBS and viral load samples. Median (IQR) age was 34 (27-42) years; median duration on ART at study entry was 11 (4-12) months;78% were women. Twenty-one (8%) participants developed viral breakthrough. Participants with TFV-DP 400 fmol/punch or less had an adjusted OR of 16.1 (95% CI: 3.9-67.4; P < 0.001) for developing viral breakthrough 1 month later compared with participants with TFV-DP greater 800 fmol/punch. TFV-DP in DBS strongly predicted future viral breakthrough in a clinical cohort of South African PWH. A biomarker able to identify PWH at risk for future viral breakthrough has the potential to improve health outcomes through timely intervention. Future studies exploring the clinical use of TFV-DP in DBS in conjunction with viral load in ART monitoring are warranted.

Importance of sample input volume for accurate SARS-CoV-2 qPCR testing

Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/μL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 μL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 μL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 samples with low viral loads can be improved by increasing the sample input volume.

Comparison of the pathogenicity of porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 in pregnant sows.

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.

Use of Tenofovir Diphosphate Levels to Predict Viremia During the Postpartum Period in Women Living With Human Immunodeficiency Virus (HIV): A Nested Case-Control Study.

There are few data on the utility of tenofovir diphosphate (TFV-DP) in dried blood spots (DBSs) to predict future viral load (VL) in postpartum women with HIV on antiretroviral therapy (ART). We conducted a nested case-control study within a trial of postpartum ART delivery strategies. Participants started ART containing tenofovir disoproxil fumarate (TDF) in pregnancy, were <10 weeks postpartum, and had a VL <400 copies/mL. VL and TFV-DP samples were taken every 3-6 months over 24 months. Cases had ≥1 VL ≥20 copies/mL; controls were randomly sampled from women with persistent viral suppression (VS; VL <20 copies/mL). Generalized estimating equations were used to calculate likelihood odds ratios (LORs) for future VL ≥20 copies/mL by TFV-DP concentration at the preceding visit. 61 cases and 20 controls contributed 365 DBS-VL pairs (median ART duration, 16 months). Sensitivity and specificity of TFV-DP <700fmol/punch to detect future viremia were 62.9% (95% CI, 54.7-70.6%) and 89.7% (84.9-93.4%), respectively. Adjusting for age, ART duration, previous VL, and duration between the TFV-DP and VL measures, LORs of viremia for TFV-DP concentrations 350-699 and <350fmol/punch versus TFV-DP ≥1850fmol/punch were 3.5 (95% CI, 1.1-10.8; P = .033) and 12.9 (3.6-46.6; P < .0001), respectively. Including only samples taken during VS, the LOR of future viremia for TFV-DP concentration <350fmol/punch versus TFV-DP ≥1850fmol/punch was 9.5 (1.9-47.0). TFV-DP concentrations in DBSs were strongly associated with future viremia and appear useful to identify nonadherence and predict future elevated VL.

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.

Evaluation of an Antigen Detection Rapid Diagnostic Test for Detection of SARS-CoV-2 in Clinical Samples

Antigen detection rapid diagnostic tests have been developed for first-line large-scale screening given their rapidity, simplicity, and accuracy. This study evaluates the diagnostic performance of an antigen detection rapid diagnostic test (BLOK BioScience, London, UK) detecting SARS-CoV-2 nucleocapsid protein. Serially diluted SARS-CoV-2 isolate and 110 NPS from COVID-19 patients were tested to determine the test’s sensitivity, and other viral isolates and 20 NPS from non-infected individuals were, for specificity, also tested. Ten clinical samples from COVID-19 patients with SARS-CoV-2 variants, including alpha, beta, gamma, delta, and eta variants, were collected to evaluate the test’s potential application in detecting emerging variants. Overall sensitivity was 92%, and stratifying into viral loads yielded 100% for Ct &lt; 25 samples including SARS-CoV-2 variants, but 11.11% for Ct ≥ 30 samples. The analytical sensitivity of log10 TCID50/mL 2.0 was identified for SARS-CoV-2. Ninety-seven percent specificity with only SARS-CoV cross-reactivity lead to the Youden index of 0.89. The rapid diagnostic test has a high sensitivity for detecting SARS-CoV-2 in high viral load samples, possibly including emerging SARS-CoV-2 variants, but reduced sensitivity in low viral load samples suggests its optimized usage as a complementary testing method to other tests, including RT-PCR or a point-of-care test for large-scale screening, particularly for pandemic areas or airport border infection control.

Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection.

RNA amplification tests sensitively detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but their complexity and cost are prohibitive for expanding coronavirus disease 2019 (COVID-19) testing. We developed “Harmony COVID-19,” a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device. Our ready-to-use, multiplexed reverse transcription, loop-mediated isothermal amplification (RT-LAMP) can detect down to 0.38 SARS-CoV-2 RNA copies/μl and can report in 17 min for high–viral load samples (5000 copies/μl). Harmony detected 97 or 83% of contrived samples with ≥0.5 viral particles/μl in nasal matrix or saliva, respectively. Evaluation in clinical nasal specimens (n = 101) showed 100% detection of RNA extracted from specimens with ≥0.5 SARS-CoV-2 RNA copies/μl, with 100% specificity in specimens positive for other respiratory pathogens. Extraction-free analysis (n = 29) had 95% success in specimens with ≥1 RNA copies/μl. Usability testing performed first time by health care workers showed 95% accuracy.

SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

Background: Surveillance of SARS-CoV-2 across the globe has enabled detection of new variants and informed the public health response. With highly sensitive methods like qPCR widely adopted for diagnosis, the ability to sequence and characterize specimens with low titers needs to keep pace.Methods: Nucleic acids extracted from nasopharyngeal swabs collected from four sites in the United States in early 2020 were converted to NGS libraries to sequence SARS-CoV-2 genomes using metagenomic and xGen target enrichment approaches. Single nucleotide polymorphism (SNP) analysis and phylogeny were used to determine clade assignments and geographic origins of strains.Results: SARS-CoV-2-specific xGen enrichment enabled full genome coverage for 87 specimens with Ct values &amp;lt;29, corresponding to viral loads of &amp;gt;10,000 cp/ml. For samples with viral loads between 103 and 106 cp/ml, the median genome coverage for xGen was 99.1%, sequence depth was 605X, and the “on-target” rate was 57 ± 21%, compared to 13%, 2X and 0.001 ± 0.016%, respectively, for metagenomic sequencing alone. Phylogenetic analysis revealed the presence of most clades that existed at the time of the study, though clade GH dominated in the Midwest.Conclusions: Even as vaccines are being widely distributed, a high case load of SARS-CoV-2 infection persists around the world. Viral genetic surveillance has succeeded in warning the public of new variants in circulation and ensured that diagnostic tools remain resilient to a steadily increasing number of mutations. Target capture offers a means of characterizing low viral load samples which would normally pose a challenge for metagenomic sequencing.