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BACKGROUND Long-term impact of drug resistance in perinatally-infected children and adolescents living with HIV (CALWH) is poorly understood. We determined drug resistance and examined its long-term impact on failure and mortality in Kenyan CALWH failing 1st-line NNRTI-based ART. SETTING Academic Model Providing Access to Healthcare; western Kenya. METHODS Participants were enrolled in 2010-13 (timepoint-1) and a subsample re-enrolled after 4-7 years (timepoint-2). Viral load was performed on timepoint-1 samples, with genotyping of those with detectable viral load. Primary endpoints were treatment failure (viral load>1,000 copies/mL) at and death before timepoint-2. Multinomial regression analysis was used to characterize resistance effect on death, failure and loss-to-follow-up, adjusting for key variables. RESULTS The initial cohort (n=480) was 52% (n=251) female, median age eight years, median CD4% 31, 79% (n=379) on zidovudine/abacavir+lamivudine+efavirenz/nevirapine for median two years. Of these, 31% (n=149) failed at timepoint-1. Genotypes at timepoint-1, available on n=128, demonstrated 93% (n=119) extensive resistance, impacting 2nd-line. Of 128, 22 failed at timepoint-2, 17 died and 32 were lost-to-follow-up before timepoint-2. Having ≥5 resistance mutations at timepoint-1 was associated with higher mortality (relative risk ratio=8.7, confidence interval 2.1-36.3) and loss-to-follow-up (relative risk ratio=3.2, confidence interval 1.1-9.2). Switching to 2nd-line was associated with lower mortality (relative risk ratio

Quantification of Cytomegalovirus (CMV) DNA has become the standard of care in the diagnosis and management of CMV infection in transplant recipients. The objective of the study was to evaluate performance characteristics of the Aptima CMV Quant assay in comparison to Abbott RealTi m e CMV assay, Qiagen Artus CMV RGQ MDx assay, and Roche cobas CMV test using plasma samples.

This prospective study of 39 women living with human immunodeficiency virus (HIV) on antiretroviral therapy in Western Kenya aimed to quantify genital tract HIV-1 RNA (GT-HIV RNA) shedding before and after cryotherapy for cervical intraepithelial neoplasia. Most GT-HIV RNA shedding was detected precryotherapy, suggesting that cryotherapy was not the primary cause of shedding.

Viral infections are major causes of morbidity and mortality in solid-organ and hematopoietic stem cell transplant recipients. This study evaluated the performance of the Galileo Pathogen Solution metagenomics Next-Generation sequencing assay to detect and quantify 11 DNA viruses (cytomegalovirus, Epstein–Barr virus, BK virus, human adenovirus, JC virus, herpes simplex virus 1 and 2, varicella zoster virus, human herpesvirus 6A and 6B, and parvovirus B19) and to qualitatively detect torque teno virus. DNA extracted from 47 plasma samples of viremic transplant recipients were subjected to DNA library preparation with pathogen enrichment/human background depletion, sequencing, and automated data analysis. The viral loads were determined with the Galileo assay using a standard curve generated from a calibration panel. All of the samples tested had a 100% agreement with the real-time quantitative PCR (qPCR) assays in detecting the primary virus targets and the majority of the quantified samples had a viral load difference within 0.46 log10 IU/mL or copies/mL. The mean difference for cytomegalovirus between the Galileo and qPCR assays was 0.21 log10 IU/mL (SD, ±0.43 log10 IU/mL). The mean difference for BK virus between the Galileo and qPCR assays was 0.17 log10 cp/mL (SD, ±0.67 log10 cp/mL). Additionally, 75 co-infections were detected in 31 samples by the Galileo assay. The study findings show that the Galileo assay can simultaneously detect and quantify multiple viruses in transplant recipients with results that are comparable with standard-of-care qPCR assays.

BACKGROUND: COVID-19 and its social responses threaten the health of people living with HIV. We conducted a rapid-response interview to assess COVID-19 protective behaviors of people living with HIV and the impact of their responses on HIV-related health care. METHOD: Men and women living with HIV (N = 162) aged 20-37 years participating in a longitudinal study of HIV treatment and care completed routine study measures and an assessment of COVID-19-related experiences. RESULTS: At baseline, most participants demonstrated HIV viremia, markers indicative of renal disorders, and biologically confirmed substance use. At follow-up, in the first month of responding to COVID-19, engaging in more social distancing behaviors was related to difficulty accessing food and medications and increased cancelation of health care appointments, both by self and providers. We observed antiretroviral therapy adherence had improved during the initial month of COVID-19 response. CONCLUSIONS: Factors that may pose added risk for COVID-19 severity were prevalent among people living with HIV, and those with greater risk factors did not practice more COVID-19 protective behaviors. Social distancing and other practices intended to mitigate the spread of COVID-19 interfered with HIV care, and impeded access to food and medications, although an immediate adverse impact on medication adherence was not evident. These results suggest social responses to COVID-19 adversely impacted the health care of people living with HIV, supporting continued monitoring to determine the long-term effects of co-occurring HIV and COVID-19 pandemics.

Despite the adaptation of international standards, quantitative viral load testing of transplant-associated viruses continues to be limited by interlaboratory disagreement. Studies have suggested that this disagreement and the poor commutability of standards may, in some cases, be linked to amplicon size and the fragmentation of circulating viral DNA. We evaluated target fragmentation as a cause of noncommutability and pretest fragmentation of quantitative standards as a potential means of increasing commutability and interassay agreement. Forty-two cytomegalovirus (CMV)-positive and 41 Epstein-Barr virus (EBV)-positive plasma samples, together with two different quantitative standards for each virus, were tested as unknowns using 10 different quantitative PCR assays at 5 different laboratories. Standards were tested both intact and after intentional fragmentation by ultrasonication. Quantitative agreement between methods was assessed, together with commutability, using multiple statistical approaches. Most assays yielded results within 0.5 log10 IU/ml of the mean for CMV, while for EBV a greater variability of up to 1.5 log10 IU/ml of the mean was shown. Commutability showed marked improvement following fragmentation of both CMV standards but not after fragmentation of the EBV standards. These findings confirm the impact of amplicon size and target fragmentation on commutability for CMV and suggest that for some (but not all) viruses, interlaboratory harmonization can be improved through the use of fragmented quantitative standards.

An element essential for PCR detection of microbial agents in many sample types is the extraction step, designed to purify nucleic acids. Despite the importance of this step, yields have not been extensively compared across methods to determine whether the method used contributes to quantitative differences and the lack of commutability seen with existing clinical methods. This may in part explain why plasma and blood viral load assays have proven difficult to standardize. Also, studies have identified small DNA fragments of

Accurate and rapid diagnosis is needed for timely intervention and clinical management of acute respiratory infections. This study evaluated performance characteristics of the Panther Fusion assay for the detection of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3 (Para 1 to 3), human metapneumovirus (hMPV), rhinovirus (RV), and adenovirus (Adeno) targets in comparison to those of the eSensor and Lyra assays using 395 nasopharyngeal (NP) and 104 lower respiratory tract (LRT) specimens. Based on the consensus positive result established (positive result in 2 of the 3 assays), the NP specimens for the Fusion and eSensor assays had 100% positive percent agreement (PPA) for all the analytes and the Lyra assays had 100% PPA for Flu A and Adeno analytes. A 100% negative percent agreement (NPA) was observed for all the Lyra analytes, whereas those for the Fusion targets ranged from 98.4 to 100% and those for the eSensor ranged from 99.4 to 100% for all the analytes except RV. For the LRT specimens, Fusion had 100% PPA and 100% NPA for all the targets except hMPV. There was a 100% PPA for eSensor analytes; the NPA ranged from 98 to 100%, except for RV. For the Lyra assays, the PPA ranged between 50 and 100%, while the NPA was 100% for all the targets except Adeno. The Fusion assay performed similarly to the eSensor assay for majority of the targets tested and provides laboratories with a fully automated random-access system to test for a broad array of viral respiratory pathogens.

A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV). Both commercial quantitative viral standards and 16 patient samples ( n = 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results. Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results. The three reagent sets, when used to assay quantitative standards on the Bio-Rad system, all showed a high degree of accuracy, low variability, and close agreement with one another. When used on the RainDance system, one of the three reagent sets appeared to have a much better correlation to nominal values than did the other two. Quantitative results for patient samples showed good correlation in most pairwise comparisons, with some showing poorer correlations when testing samples with low viral loads. Digital PCR is a robust method for measuring CMV viral load. Some degree of result variation may be seen, depending on platform and reagents used; this variation appears to be greater in samples with low viral load values.

Quantification of HIV-1 RNA has become the standard of care in the clinical management of HIV-1-infected individuals. The objective of this study was to evaluate performance characteristics and relative workflow of the Aptima HIV-1 Quant Dx assay in comparison with the Abbott RealTime HIV-1 assay using plasma and cervicovaginal lavage (CVL) specimens. Assay performance was evaluated by using an AcroMetrix HIV-1 panel, AcroMetrix positive controls, Qnostics and SeraCare HIV-1 evaluation panels, 208 clinical plasma samples, and 205 matched CVL specimens on the Panther and m2000 platforms. The Aptima assay demonstrated good linearity over the quantification range tested (2 to 5 log 10 copies/ml), and there was strong linear correlation between the assays ( R 2 = 0.99), with a comparable coefficient of variance of 10 copies/ml higher than those determined by the RealTime assay. The assays differed in their sensitivity for quantifying HIV-1 RNA loads in CVL samples, with the Aptima and RealTime assays detecting 30% and 20%, respectively. Aptima had fewer invalid results, and on average, the viral loads in CVL samples quantified by the Aptima assay were 0.072 log 10 copies/ml higher than those of the RealTime assay. Our results demonstrate that the Aptima assay is sensitive and accurate in quantifying viral loads in both plasma and CVL specimens and that the fully automated Panther system has all the necessary features suitable for clinical laboratories demanding high-throughput sample processing.

The recent development of the 1st WHO International Standard for human cytomegalovirus (CMV) and the introduction of commercially produced secondary standards have raised hopes of improved agreement among laboratories performing quantitative PCR for CMV. However, data to evaluate the trueness and uniformity of secondary standards and the consistency of results achieved when these materials are run on various assays are lacking. Three concentrations of each of the three commercially prepared secondary CMV standards were tested in quadruplicate by three real-time and two digital PCR methods. The mean results were compared in a pairwise fashion with nominal values provided by each manufacturer. The agreement of results among all methods for each sample and for like concentrations of each standard was also assessed. The relationship between the nominal values of standards and the measured values varied, depending upon the assay used and the manufacturer of the standards, with the degree of bias ranging from +0.6 to −1.0 log 10 IU/ml. The mean digital PCR result differed significantly among the secondary standards, as did the results of the real-time PCRs, particularly when plotted against nominal log 10 IU values. Commercially available quantitative secondary CMV standards produce variable results when tested by different real-time and digital PCR assays, with various magnitudes of bias compared to nominal values. These findings suggest that the use of such materials may not achieve the intended uniformity among laboratories measuring CMV viral load, as envisioned by adaptation of the WHO standard.