Analytical and Clinical Performance of the NeuMoDx™ Platform for Cytomegalovirus and Epstein-Barr Virus Viral Load Testing.

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR.

Though pooling samples for SARS-CoV-2 detection has effectively met the need for rapid diagnostic and screening tests, many factors can influence the sensitivity of a pooled test. In this study, we conducted a simulation experiment to evaluate modes of pooling specimens and aimed at formulating an optimal pooling strategy. We focussed on the type of swab, their solvent adsorption ability, pool size, pooling volume, and different factors affecting the quality of preserving RNA by different virus solutions. Both quantitative PCR and digital PCR were used to evaluate the sampling performance. In addition, we determined the detection limit by sampling which is simulated from the virus of different titers and evaluated the effect of sample-storage conditions by determining the viral load after storage. We found that flocked swabs were better than fibre swabs. The RNA-preserving ability of the non-inactivating virus solution was slightly better than that of the inactivating virus solution. The optimal pooling strategy was a pool size of 10 samples in a total volume of 9 mL. Storing the collected samples at 4 °C or 25 °C for up to 48 h had little effect on the detection sensitivity. Further, we observed that our optimal pooling strategy performed equally well as the single-tube test did. In clinical applications, we recommend adopting this pooling strategy for low-risk populations to improve screening efficiency and shape future strategies for detecting and managing other respiratory pathogens, thus contributing to preparedness for future public health challenges.

A solution to achieve sequencing from SARS-CoV-2 specimens with low viral loads: concatenation of reads from independent reactions.

BACKGROUND: During the pandemic, whole genome sequencing was critical to characterize SARS-CoV-2 for surveillance, clinical and therapeutical purposes. However, low viral loads in specimens often led to suboptimal sequencing, making lineage assignment and phylogenetic analysis difficult. We propose an alternative approach to sequencing these specimens that involves sequencing in triplicate and concatenation of the reads obtained using bioinformatics. This proposal is based on the hypothesis that the uncovered regions in each replicate differ and that concatenation would compensate for these gaps and recover a larger percentage of the sequenced genome. RESULTS: Whole genome sequencing was performed in triplicate on 30 samples with Ct > 32 and the benefit of replicate read concatenation was assessed. After concatenation: i) 28% of samples reached the standard quality coverage threshold (> 90% genome covered > 30x); ii) 39% of samples did not reach the coverage quality thresholds but coverage improved by more than 40%; and iii) SARS-CoV-2 lineage assignment was possible in 68.7% of samples where it had been impaired. CONCLUSIONS: Concatenation of reads from replicate sequencing reactions provides a simple way to access hidden information in the large proportion of SARS-CoV-2-positive specimens eliminated from analysis in standard sequencing schemes. This approach will enhance our potential to rule out involvement in outbreaks, to characterize reinfections and to identify lineages of concern for surveillance or therapeutical purposes.

Analytical and clinical evaluation of the cobas Epstein-Barr virus test at a tertiary care cancer hospital.

BACKGROUND: Epstein-Barr Virus (EBV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Cobas EBV test (Roche Molecular, Pleasanton, CA) has recently been FDA-cleared for the monitoring of EBV viral loads in plasma samples of transplant patients. In this study, we compared the viral loads obtained by a laboratory-developed test (EBV LDT) using Altona Analyte specific reagents (ASR) to those obtained on the Cobas EBV test. METHODS: The analytical performance of the assay was established using the EBV verification panel from Exact Diagnostics and the EBV ATCC strain B95-8. The clinical evaluation was performed using 343 plasma samples initially tested on the EBV LDT. RESULTS: The analytical sensitivity (<18.8 IU/mL), precision (SD < 0.17 log) and linear range (35.0 IU/mL to 1E + 08 IU/mL) of the Cobas EBV assay established by the manufacturers were confirmed. The strength of the qualitative agreement was substantial between the cobas EBV and the EBV LDT (85.6 %; κ = 0.71) and almost perfect when discordant results were resolved (96.4 %; κ = 0.93). The quantitative agreement was moderate (82.9 %; κ = 0.53) with the viral load obtained on the Cobas EBV test being lower across the linear range of the two tests (mean log difference of 1.0). While the absolute values of the viral loads were markedly different, the overall trends observed in patients with multiple consecutive results were similar between the two tests. CONCLUSIONS: The Cobas EBV test provides an accurate and valid, in vitro diagnostic (IVD) option for monitoring of EBV viral loads in transplant patients and should provide an opportunity for increased standardization and commutability of tests results across laboratories.

Comparative evaluation of RT-PCR and antigen-based rapid diagnostic tests (Ag-RDTs) for SARS-CoV-2 detection: performance, variant specificity, and clinical implications.

The COVID-19 pandemic has highlighted the critical need for accurate and efficient diagnostic tools for detecting severe acute respiratory coronavirus 2 (SARS-CoV-2) infections. This study presents a comparison of two diagnostic tests: RT-PCR and antigen detection rapid diagnostic tests (Ag-RDTs). This study focused on their performance, variant specificity, and their clinical implications. A simultaneous testing of 268 samples was carried out for SARS-CoV-2 using RT-PCR and Ag-RDTs [flourescence immunoassay (FIA) and lateral flow immunoassay (LFIA)]. Viral load was quantified, and variant identification was performed using a PCR-based assay. The prevalence was found to be 30.2% using reverse transcription PCR (RT-PCR), 26.5% using FIA, and 25% using LFIA. When comparing the FIA and LFIA, the overall diagnostic performance was found to be 80.25% vs 76.54%, 96.79% vs 97.33%, 91.55% vs 90.51%, and 91.88% vs 92.56% for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. Both Ag-RDTs showed a strong agreement with RT-PCR (κ = 0.78-0.80). The overall accuracies of the FIA and LFIA were 92.41% and 92.13%, respectively. The FIA showed higher sensitivity (73.68%) and PPV (92.08%) than the LFIA (65.79% and 90.56%, respectively) in asymptomatic patients. At low Ct values (<25), both Ag-RDTs had 100% sensitivity, but the sensitivity reduced to 31.82% for FIA and 27.27% for LFIA at Ct values > 30. The diagnostic sensitivity of FIA compared to LFIA for detecting the Alpha variant was 78.85% vs. 69.23% and 72.22% vs. 83.33% for the Delta variant. Both Ag-RDTs had 100% sensitivity for detecting Omicron. Both Ag-RDTs performed well in patients with high viral loads and Omicron variant infections compared to those infected with Alpha and Delta variants. This study confirms the comparable performance of RT-PCR and Ag-RDTs, specifically FIA and LFIA, for SARS-CoV-2 detection. The FIA showed higher sensitivity and PPV in asymptomatic cases, while both Ag-RDTs exhibited strong agreement with RT-PCR results. Notably, Ag-RDTs, particularly FIA, proved effective in detecting the Omicron variant and cases with high viral loads, highlighting their potential clinical utility in managing the COVID-19 pandemic.IMPORTANCEThis study is of utmost importance in providing effective responses to manage the COVID-19 pandemic. It rigorously compares the diagnostic accuracy, variant specificity, and practical considerations of reverse transcription PCR (RT-PCR) and antigen detection rapid diagnostic tests (Ag-RDTs) for severe acute respiratory coronavirus 2 (SARS-CoV-2), answering critical questions. The results of this study will help healthcare professionals choose the appropriate testing methods, allocate resources effectively, and enhance public health strategies. Given the evolution of the virus, understanding the performance of these diagnostic tools is crucial to adapting to emerging variants. Additionally, the study provides insights into logistical challenges and accessibility issues, which will contribute to refining testing workflows, particularly in resource-limited settings. Ultimately, the study's impact extends to global healthcare, providing valuable information for policymakers, clinicians, and public health officials as they work together for mitigating the impact of the pandemic.

Real-world performance of the NeuMoDx™ HCV Quant Test for quantification of hepatitis C virus (HCV)-RNA.

Quantification of hepatitis C virus (HCV)-RNA in serum or plasma samples is an essential parameter in HCV diagnostics. Here, the NeuMoDx™Molecular System (Qiagen) was tested for the most common HCV genotypes and compared to the cobas c6800 system (Roche). HCV-RNA from 131 plasma/serum samples from chronically infected patients was determined in parallel on the NeuMoDx and c6800 systems. Linearity was analysed using the four most common HCV genotypes (1-4) in our cohort. The coefficient of variation (CV) within (intra-assay) and between (inter-assay) runs was calculated based on HCV-RNA concentration. Quantitative HCV-RNA results were highly correlated on both test systems (R2 = 0.7947; y = 0.94 x + 0.37). On average, the NeuMoDx and c6800 HCV RNA levels showed a mean difference of only 0.05 log10 IU/mL but with a broad distribution (±1.2 2 x SD). The NeuMoDx demonstrated very good linearity across all HCV genotypes tested at concentrations between 1.7 and 6.2 log10 IU/mL (R2 range: 0.9257-0.9991) with the highest mean coefficient of determination for genotype 1 (R2 = 0.9909). The mean intra- and inter-assay CV for both serum and plasma samples was <5 %. The NeuMoDx HCV-RNA Assay demonstrates high subtype-independent comparability, linearity, and reproducibility for the quantification of HCV-RNA in serum and plasma samples from chronically infected patients.

The development, evaluation, performance, and validation of micro-PCR and extractor for the quantification of HIV-1 &-2 RNA.

HIV infection has been a global public health threat and overall reported ~ 40 million deaths. Acquired immunodeficiency syndrome (AIDS) is attributed to the retroviruses (HIV-1/2), disseminated through various body fluids. The temporal progression of AIDS is in context to the rate of HIV-1 infection, which is twice as protracted in HIV-2 transmission. Q-PCR is the only available method that requires a well-developed lab infrastructure and trained personnel. Micro-PCR, a portable Q-PCR device, was developed by Bigtec Labs, Bangalore, India. It is simple, accurate, fast, and operationalised in remote places where diagnostic services are inaccessible in developing countries. This novel micro-PCR determines HIV-1 and HIV-2 viral load using a TruePrep™ extractor device for RNA isolation. Five ml blood samples were collected at the blood collection centre at AIIMS, New Delhi, India. Samples were screened for serology, and a comparison of HIV-1/2 RNA was done between qPCR and micro-PCR in the samples. The micro-PCR assay of HIV-RNA has compared well with those from real-time PCR (r = 0.99, i < 0.002). Micro-PCR has good inter and intra-assay reproducibility over a wide dynamic range (1.0 × 102-1.0 × 108 IU/ml). The linear dynamic range was 102-108 IU/ml. The clinical and analytical specificity of the assay was comparable, i.e., 100%. Intra-assay and inter-assay coefficients of variation ranged from 1.17% to 3.15% and from 0.02% to 0.46%, respectively. Moreover, due to the robust, simple, and empirical method, the Probit analysis has also been done for qPCR LODs to avoid uncertainties in target recoveries. The micro-PCR is reliable, accurate, and reproducible for early detection of HIV-1 and HIV-2 viral loads simultaneously. Thus, it can easily be used in the field and in remote places where quantification of both HIV-1/2 is not reachable.

Performance evaluation of the SMG HHV-6 Q Real-Time PCR Kit for quantitative detection and differentiation of human herpesvirus 6A and 6B.

The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log10 copies/mL for HHV-6A DNA and 2.88 log10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40-9.00 log10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.IMPORTANCEQuantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.

Assessment of the performance of the plasma separation card for HIV-1 viral load monitoring in South Africa.

Scaling up of newer innovations that address the limitations of the dried blood spot and the logistics of plasma monitoring is needed. We employed a multi-site, cross-sectional assessment of the plasma separation card (PSC) on blood specimens collected from all consenting adults, assenting young and pediatric patients living with HIV from 10 primary healthcare clinics in South Africa. Venous blood for EDTA-plasma samples was collected and analyzed according to the standard of care assay, while collected capillary blood for the PSC samples was analyzed using the Roche COBAS AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 Test at the National Reference laboratories. McNemar tests assessed the differences in concordance between the centrifuged plasma and dried plasma spots. The usability of PSC by blood spotting, PSC preparation, and pre-analytical work was assessed by collecting seven-point Likert-scale data from healthcare and laboratory workers. We enrolled 538 patients, mostly adults [n = 515, 95.7% (95% CI: 93.7%-97.1%)] and females [n = 322, 64.2% (95% CI: 60.0%-68.1%)]. Overall, 536 paired samples were collected using both PSC- and EDTA-plasma diagnostics, and 502 paired PSC- and EDTA-plasma samples assessed. Concordance between the paired samples was obtained for 446 samples. Analysis of these 446 paired samples at 1,000 copies per milliliter threshold yielded an overall sensitivity of 87.5% [95% CI: 73.2%-95.8%] and specificity of 99.3% [95% CI: 97.9%-99.8%]. Laboratory staff reported technical difficulties in most tasks. The usability of the PSC by healthcare workers was favorable. For policymakers to consider PSC scale-up for viral load monitoring, technical challenges around using PSC at the clinic and laboratory level need to be addressed. IMPORTANCE: Findings from this manuscript emphasize the reliability of the plasma separation card (PSC), a novel diagnostic method that can be implemented in healthcare facilities in resource-constrained settings. The agreement of the PSC with the standard of care EDTA plasma for viral load monitoring is high. Since the findings showed that these tests were highly specific, we recommend a scale-up of PSC in South Africa for diagnosis of treatment failure.

Performance evaluation of the high-throughput quantitative Alinity m BK virus assay.

BK virus (BKV) infection or reactivation in immunocompromised individuals can lead to adverse health consequences including BKV-associated nephropathy (BKVAN) in kidney transplant patients and BKV-associated hemorrhagic cystitis (BKV-HC) in allogeneic hematopoietic stem cell transplant recipients. Monitoring BKV viral load plays an important role in post-transplant patient care. This study evaluates the performance of the Alinity m BKV Investigational Use Only (IUO) assay. The linearity of the Alinity m BKV IUO assay had a correlation coefficient of 1.000 and precision of SD ≤ 0.25 Log IU/mL for all panel members tested (2.0-7.3 Log IU/mL). Detection rate at 50 IU/mL was 100%. Clinical plasma specimens tested comparing Alinity m BKV IUO to ELITech MGB Alert BKV lab-developed test (LDT) on the Abbott m2000 platform using specimen extraction protocols for DNA or total nucleic acid (TNA) resulted in coefficient of correlation of 0.900 and 0.963, respectively, and mean bias of 0.03 and -0.54 Log IU/mL, respectively. Alinity m BKV IUO compared with Altona RealStar BKV and Roche cobas BKV assays demonstrated coefficient of correlation of 0.941 and 0.980, respectively, and mean bias of -0.47 and -0.31 Log IU/mL, respectively. Urine specimens tested on Alintiy m BKV IUO and ELITech BKV LDT using TNA specimen extraction had a coefficient of correlation of 0.917 and mean bias of 0.29 Log IU/mL. The Alinity m BKV IUO assay was performed with high precision across the dynamic range and correlated well with other available BKV assays. IMPORTANCE: BK virus (BKV) in transplant patients can lead to adverse health consequences. Viral load monitoring is important in post-transplant patient care. This study evaluates the Alinity m BKV assay with currently available assays.

Development and validation of the first HBV qRT-PCR assay in the Mediterranean area targeting the X region.

Hepatitis B virus (HBV) infection is a global public health burden and affects approximatively 300 million people around the world. Since, HBV population is represented with genetic diversity, having different viral effects. Development of a new prognosis method play a key role on the efficiency of the different treatment. The HBx protein of HBV has a potential role in Hepatocellular Carcinoma (HCC), which makes it a valuable target for HCC prognosis. In this context, the first quantitative real-time PCR (qRT-PCR) assay in the Mediterranean area was developed and validated. Specific primers and probes of a conserved X region across all HBV genotypes were designed and the qRT-PCR was performed with the TaqPath 1-Step Multiplex Master Mix on 441 Moroccan plasma samples in Pasteur Institute of Morocco. The assay demonstrated a linear quantification range of 1010-101 IU/reaction (R2 = 0.99) and a quantification limit of 15 IU/mL. Comparative evaluations with the COBAS Ampliprep/COBAS TaqMan (CAP/CTM) HBV, v2.0 and the artus HBV QS-RGQ assays showed strong correlations (R2 = 0.92 and R2 = 0.89, respectively). Our test is fast, highly sensitive, specific, reproducible, and labor-saving. This system will be of great advantage to Mediterranean countries in their efforts to eliminate viral hepatitis B and C by 2030, enabling precise monitoring and effective treatment of HBV infections.

The challenge of standardizing CAR-T cell monitoring: A comparison of two flow-cytometry methods and correlation with qPCR technique.

Chimeric antigen receptor (CAR) T-cell therapy is a breakthrough in hematologic malignancies, such as acute B lymphoblastic leukemia (B-ALL). Monitoring this treatment is recommended, although standardized protocols have not been developed yet. This work compares two flow cytometry monitoring strategies and correlates this technique with qPCR method. CAR-T cells were detected by two different flow-cytometry protocols (A and B) in nine blood samples from one healthy donor and five B-ALL patients treated with Tisagenlecleucel (Kymriah®, USA). HIV-1 viral load allowed CAR detection by qPCR, using samples from seven healthy donors and nine B-ALL patients. CAR detection by protocol A and B did not yield statistically significant differences (1.9% vs. 11.8% CD3 + CAR+, p = 0.07). However, protocol B showed a better discrimination of the CD3 + CAR+ population. A strong correlation was observed between protocol B and qPCR (r = 0.7, p < 0.0001). CD3 + CAR+ cells were detected by flow cytometry only when HIV-1 viral load was above 104 copies/mL. In conclusion, protocol B was the most specific flow-cytometry procedure for the identification of CAR-T cells and showed a high correlation with qPCR. Further efforts are needed to achieve a standardized monitoring approach.

Evaluation of GeneXpert and advanced biological laboratories UltraGene HCV diagnostic detection and performance against Roche real time PCR in Myanmar.

BACKGROUND: Developing countries experience limited access to HCV laboratory tests for different reasons. Providing near to real-time HCV testing and results especially to at-risk populations including those in rural settings for timely initiation to treatment is key. Within a rural Myanmar setting, we compared HCV diagnostic detection and quantification of the GeneXpert, and Advanced Biological Laboratories UltraGene-HCV assays against the gold standard and reference method Roche real-time HCV in Myanmar. METHODS: Blood samples from 158 high-risk individuals were assessed using three different methods at baseline. Results were checked for normality and log transformed. Log differences and bias between methods were calculated and correlated. Pearson's correlation coefficient was used to determine the association of HCV viral loads across all methods. The level of agreement with the standard method (Roche real time HCV) was assessed using Bland-Altman analyses. RESULTS: There was a strong positive correlation coefficient between all three methods with GeneXpert and Roche having the strongest, r = 0.96, (p<0.001). Compared to Roche, ABL (mean difference, 95 % limits of agreement; -0.063 and -1.4 to 1.3 Log10IU/mL) and GeneXpert (mean difference, 95 % limits of agreement; -0.28 and -0.7 to 1.8 Log10IU/mL) showed a good level of agreement with the GeneXpert being slightly superior. CONCLUSION: We demonstrate the excellent performance and no-inferiority, in terms of levels of agreements of both GeneXpert and ABL compared to the Roche platform and supporting the use of the POC assays as alternative a cost-effective methods in HCV detection and diagnosis in developing and low resource settings countries.

Accuracy of quantitative viral secondary standards: a re-examination.

Interlaboratory agreement of viral load assays depends on the accuracy and uniformity of quantitative calibrators. Previous work demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values in both log10 copies/mL and log10 international unit (IU)/mL, with bias from manufacturer-assigned nominal values of 0.0-0.9 log10 units (either copies or IU)/mL. Standards normalized to IU and those values assigned by dPCR rather than by real-time PCR (qPCR) showed better agreement with nominal values. The latter reinforces prior conclusions regarding the utility of using such methods for quantitative value assignment in reference materials. Quantitative standards have improved over the last several years, and the remaining bias from nominal values might be further reduced by universal implementation of dPCR methods for value assignment, normalized to IU. IMPORTANCE: Interlaboratory agreement of viral load assays depends on accuracy and uniformity of quantitative calibrators. Previous work, published in JCM several years ago, demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values, indicating a substantial improvement in the production of accurate secondary viral standards, while supporting the need for further work in this area and for the broad adaption of international unit (IU) as a reporting standard for quantitative viral load results.

Context-dependent accuracy of the cobas plasma separation card for HCV RNA viral load measurement.

Collection and preservation of plasma are challenging in remote or under-resourced settings. The cobas® Plasma Separation Card (PSC) is an alternative specimen type for blood-borne pathogen nucleic acid quantitation. We assessed PSC as a specimen type for HCV RNA quantitation in Pakistan. Plasma from venous blood and PSC from finger prick blood were prepared at two sites: Site 1 (in Lahore, n = 199) consisted of laboratory-based outpatient clinics. Specimens were prepared in the same facility and stored frozen. Site 2 was a catchment area within a resource-limited, semi-urban locality of Islamabad with limited access to healthcare services (n = 151). Community public health outreach staff collected blood and prepared the PSC in the participants' homes. Specimens were transported to the central hepatitis laboratory in Lahore to be stored frozen until tested. HCV RNA testing was performed using the cobas HCV RNA test in a central laboratory. Concordance with respect to RNA detectability was high at Site 1 (97.4%), but lower at Site 2 (82.4%). At Site 1, HCV viral load in plasma and PSC were well correlated across the linear range with a 0.21 log10 IU/mL mean bias toward higher concentrations in PSC. At Site 2, HCV viral load in plasma and PSC were poorly correlated. There was a 0.11 log10 IU/mL mean bias toward higher concentrations in PSC. PSC performance can be excellent in underserved settings where refrigerated transport of traditional specimens is difficult. In very challenging field settings, extra support must be provided to ensure correct specimen collection and handling.

Evaluation of diagnostic performance of SARS-CoV-2 infection using digital droplet polymerase chain reaction in individuals with or without COVID-19 symptoms.

BACKGROUND: Reverse transcription-quantitative PCR (RT-qPCR) is commonly used to diagnose SARS-CoV-2, but it has limited sensitivity in detecting the virus in asymptomatic close contacts and convalescent patients. In this study, we propose the use of reverse transcription-digital droplet PCR (RT-ddPCR) to detect SARS-CoV-2 in clinical samples. METHODS: The clinical performance of RT-ddPCR targeting of ORF1ab and N genes was evaluated in parallel with RT-qPCR using 200 respiratory samples collected from close contacts and patients at different phases of infection. RESULTS: The limits of detection (LODs) for RT-ddPCR assays were determined using six dilutions of ACCUPLEX SARS-Cov-2 reference material. The LODs of ORF1ab and N genes were 3.7 copies/reaction and 2.2 copies/reaction, respectively. Compared to RT-qPCR, RT-ddPCR increased the positive rate by 12.0% in 142 samples from SARS-CoV-2-infected patients. Additionally, RT-ddPCR detected SARS-CoV-2 in three of 26 specimens from close contacts that tested negative by RT-qPCR, and infection was confirmed using follow-up samples. Finally, RT-ddPCR improved the equivocal results from RT-qPCR in 56.3% (9/16) of convalescent patient samples. CONCLUSIONS: Detecting SARS-CoV-2 in samples with low viral loads using RT-qPCR can be challenging. However, our study suggests that RT-ddPCR, with its higher sensitivity and accuracy, is better suited for detecting low viral copies in samples, particularly those from close contacts and convalescent patients.

Challenges and Pragmatic Solutions for Assessing the Reliability of HIV-1 Viral Load Monitoring in Resource-Constrained Settings.

INTRODUCTION: HIV-1 RNA detection is the most reliable method for monitoring treatment response among people living with HIV. Effective quality control measures that include internal quality control (IQC) are challenging in resource-constrained settings. METHODS: We ascertained the utility of the kit low positive control (LPC) as an effective IQC to monitor the reliability of the HIV-1 viral load assay. Variations in LPC values were measured for 390 different runs over 10 years (2011-2021) and compared to in-house IQC data using Levey-Jennings control chart. RESULTS: Overall, the Levey-Jennings analysis showed minimal variation (±0.5 log) for both the LPC and IQC data. The mean LPC value for first 20 runs (20 days) was 2.91. The mean LPC value for the 390 runs comprising 35 different lots was 3.01 ± 0.1 log. CONCLUSION: Our decadal data reveal that Abbott RealTime HIV-1 assay (Abbott Molecular Inc., IL, USA) LPC exhibited no significant biological variation over 390 runs distributed over 10 years. Hence, assay LPC can supplant the IQC for monitoring assay trends as a stable and commutable material in resource-constrained settings.

Evaluation of two automated real-time PCR-based quantification methods for whole blood Epstein-Barr viral load.

Quantification of EBV DNA is important in transplantation settings for the diagnosis of post-transplantation. We evaluated the performance of the AltoStar® EBV PCR Kit 1.5 on whole blood specimens: limit of detection, linearity, accuracy, and precision were determined using the WHO NIBSC 09/260 international standard. Results of 69 clinical samples were compared between the AltoStar® EBV PCR Kit 1.5 (altona Diagnostics) and the RealTime EBV assay (Abbott). The LoD of the AltoStar® Kit was 148 IU/mL and linearity was between 375 and 500000. A high concordance was found between nominal value of the NIBSC dilutions and the AltoStar EBV result. The total variation ranged from 2.2% to 9.6%. Out of 69 clinical samples tested, there was a high concordance between the 22 paired results within the overlapping linear ranges of both tests. The AltoStar® EBV assay is reliable and accurate for EBV viral load determination on whole blood samples.

Using queueing models as a decision support tool in allocating point-of-care HIV viral load testing machines in Kisumu County, Kenya.

Point-of-care (POC) technologies-including HIV viral load (VL) monitoring-are expanding globally, including in resource-limited settings. Modelling could allow decision-makers to consider the optimal strategy(ies) to maximize coverage and access, minimize turnaround time (TAT) and minimize cost with limited machines. Informed by formative qualitative focus group discussions with stakeholders focused on model inputs, outputs and format, we created an optimization model incorporating queueing theory and solved it using integer programming methods to reflect HIV VL monitoring in Kisumu County, Kenya. We modelled three scenarios for sample processing: (1) centralized laboratories only, (2) centralized labs with 7 existing POC 'hub' facilities and (3) centralized labs with 7 existing and 1-7 new 'hub' facilities. We calculated total TAT using the existing referral network for scenario 1 and solved for the optimal referral network by minimizing TAT for scenarios 2 and 3. We conducted one-way sensitivity analyses, including distributional fairness in each sub-county. Through two focus groups, stakeholders endorsed the provisionally selected model inputs, outputs and format with modifications incorporated during model-building. In all three scenarios, the largest component of TAT was time spent at a facility awaiting sample batching and transport (scenarios 1-3: 78.7%, 89.9%, 91.8%) and waiting time at the testing site (18.7%, 8.7%, 7.5%); transportation time contributed minimally to overall time (2.6%, 1.3%, 0.7%). In scenario 1, the average TAT was 39.8 h (SD: 2.9), with 1077 h that samples spent cumulatively in the VL processing system. In scenario 2, the average TAT decreased to 33.8 h (SD: 4.8), totalling 430 h. In scenario 3, the average TAT decreased nearly monotonically with each new machine to 31.1 h (SD: 8.4) and 346 total hours. Frequency of sample batching and processing rate most impacted TAT, and inclusion of distributional fairness minimally impacted TAT. In conclusion, a stakeholder-informed resource allocation model identified optimal POC VL hub allocations and referral networks. Using existing-and adding new-POC machines could markedly decrease TAT, as could operational changes.

m-PIMA™ HIV1/2 VL: A suitable tool for HIV-1 and HIV-2 viral load quantification in West Africa.

Point-of-Care for HIV viral RNA quantification seems to be a complementary strategy to the existing conventional systems. This study evaluated the performance of the m-PIMA™ HIV1/2 Viral Load for the quantification of both HIV-1 and HIV-2 RNA viral load. A total of 555 HIV-1 and 90 HIV-2 samples previously tested by Abbott RealTime HIV-1 (Abbott, Chicago, USA) and Generic HIV-2® Charge virale (Biocentric, France) were tested using the m-PIMA™ HIV1/2 Viral Load at the HIV National Reference lab in Senegal. For HIV-1, Pearson correlation and Bland-Altman plots showed a coefficient r = 0.97 and a bias of -0.11 log10 copies/ml (95% confidence interval [CI]: -0.086 to -0.133 log10 copies/ml) for the m-PIMA™ HIV1/2 Viral Load, respectively. Sensitivity and specificity at 3 log10 copies/ml (threshold of virological failure) were 93.6% (95%[CI]: 91.5% to 95.6%) and 99.1% (95%[CI]: 98.3% to 99.9%), respectively. For HIV-2, a correlation of r = 0.95 was also noted with a bias of - 0.229 log10 copies/ml (95%[CI]: -0.161 to -0.297 log10 copies/ml). Sensitivity and specificity at 3 log10 copies/ml were 97.6% (95%[CI]: 94.3% to 100%) and 93.9% (95%[CI]: 88.9% to 98.8%), respectively. These results confirmed that m-PIMA™ HIV1/2 VL could be a good alternative for HIV-1 and HIV-2 viral load testing in decentralized settings in Senegal.