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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.

HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments. A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil–water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples. Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%. From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.

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.

Background Human cytomegalovirus (CMV) is the leading cause of intrauterine and perinatal viral infection. The most common route of CMV transmission in newborns is through breastmilk and this can lead to infant morbidity and mortality. Breast milk that has been frozen for an extended period may need to be tested for CMV DNA to determine the source of infection. It has been a challenge for clinical laboratories to ensure the stability of CMV DNA in frozen breast milk for accurate viral load measurement. Objectives To evaluate the stability of CMV DNA in breast milk by testing quantitative viral loads over a 28-day period for breast milk stored at 4 °C and a 90-day period for breast milk stored at −20 °C. Study design Baseline viral loads were determined on day 0 and the samples stored at 4 °C underwent extraction and amplification at four time points, up to 28 days. The samples stored at −20 °C underwent extraction and amplification at five time points up to 90 days. Log10 values were calculated and t-test, Pearson’s coefficient, and concordance correlation coefficient were calculated. Results There was no statistically significant difference between the time points by t-test, and correlation coefficients showed greater than 90% concordance for days 0 and 28 as well as days 0 and 90 at both storage temperatures tested. Conclusions The concentration of CMV DNA in breast milk was stable for 28 days at 4 °C and 90 days at −20 °C as the concentrations did not differ significantly from the baseline viral loads.

Background Timely and precise laboratory diagnosis of Herpes simplex viruses (HSV) is required to guide clinical management. Objectives The study evaluated limit of detection (LOD) and performance characteristics of the Aptima HSV 1 & 2 assay in comparison to four assays. Study design The multi-center study compared qualitative detection of HSV-1 and 2 by the Aptima HSV-1 and 2 assay (Hologic) to ELVIS culture, Lyra Direct (Quidel), AmpliVue (Quidel) and a laboratory developed test (LDT). LOD was performed using VTM and STM diluted viral concentrations and clinical performance was evaluated using 505 swab specimens. Results The Aptima LOD studies performed showed a lower detection limit for STM specimens as 1450 copies/mL and 430 copies/mL for HSV1 and HSV-2 respectively; the LOD for VTM specimens was 9370 copies/mL and 8045 copies/mL for HSV-1 and HSV-2 respectively. When the assays were analyzed based on the positive consensus result established the Aptima had 95% of percent positive agreement (PPA) and 100% negative percent agreement (NPA) for the HSV-1. For the HSV-2, the PPA and NPA for Aptima were 96% and 100% respectively. AmpliVue had 1.8% invalid rate, while Lyra had no invalid results but an inhibition rate of 0.8%. Aptima and LDT did not have any invalid or inhibited results. Conclusion The results indicate that the Aptima HSV-1 & 2 assay is sensitive and the performance characteristics of the Aptima assay is comparable to the assays analyzed for the detection and differentiation of HSV-1 and 2 from cutaneous and mucocutaneous lesions.

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.

Background The single-nucleotide polymorphism (SNP) 3435C > T in exon 26 of the MDR1 gene has been shown to correlate with the functioning of P-glycoprotein. We studied the frequency of SNP in exon 26 of the MDR1 gene in breast cancer and its role in predicting response to neoadjuvant chemotherapy in breast cancer. Patients and Methods Ninety-six patients with locally advanced breast carcinoma were enrolled. Genotyping of exon 26 of the MDR1 gene was performed, and computed tomography scans were performed before and after neoadjuvant chemotherapy. Response to 3 cycles of the 5-fluorouracil/doxorubicin/cyclophosphamide (FAC) regimen was assessed. The prevalence of SNP was compared with that of historical controls. Association of the response was compared with the genotypes. Results The frequency of genotypes was different from that of healthy sex-matched historical controls. Prevalence of TT genotype was significantly increased in breast cancer patients ( P = .025). The patients with TT genotype had 2.26 times the chance of responding to neoadjuvant chemotherapy when compared with patients with the CC genotype ( P = .44). Conclusion Significantly higher prevalence of 3435TT genotype in exon 26 of the MDR1 gene in patients with breast cancer might suggest the possibility of increased breast cancer susceptibility. The genotypes did not show any significant association to response to chemotherapy in the population studied.

Background. The inter-individual differences in upper aerodigestive tract (UADT) cancer risk may be partly attributed to the polymorphic variability in the CYP1A1 gene that is involved in the metabolic activation of xenobiotics to carcinogenic reactive metabolites. Methods. The hospital-based case-control study evaluated CYP1A1*2A and CYP1A1*2C polymorphisms in 408 histopathologically confirmed cases and 220 controls using polymerasechain reaction-restriction fragment length polymorphism methods. Results. The multivariate logistic regression analyses demonstrated that CYP1A1 *1A/*2A (odds ratio [OR] 1.76; 95% Confidence interval [CI] 1.19–2.60) and *2A/*2A (OR 2.83; 95% CI 1.43–5.61) genotypes were significantly associated with increased risk for UADT cancers. The gene–environment interaction analyses revealed a significant interaction among tobacco smokers and chewers carrying CYP1A1*2A mutant genotypes on the multiplicative scale. Conclusion. CYP1A1*2A polymorphic genotypes are associated with an enhanced risk to UADT cancers, in particular, among the habitual tobacco smokers and chewers carrying mutant genotypes in the Tamilians of the Indian population. © 2008 Wiley Periodicals, Inc. Head Neck, 2008

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.

In the past decade, substantial advances in pharmacogenomics have increasingly unveiled the genetic basis of interindividual differences in drug responses. Majority of these advances have been made in the field of oncology which revolutionized cancer therapy. Pharmacogenomics plays a significant role in the pharmacotherapy of cancer as narrow therapeutic indices, variable response rates, rapid and severe systemic toxicity, and unpredictable efficacy are all hallmarks of cancer therapies. Personalized oncologic care is becoming a trend because of the constant advancement in the clinical molecular diagnostics and biomarker discoveries. In addition, the intricate molecular mechanisms that provoke neoplastic cell transformation along with the dysregulation of alternative complementary pathways have been increasingly understood. This review discusses the current commonly used predictive and prognostic biomarkers in clinical oncology molecular testing.

Hepatitis C viral infection is a major cause of progressive liver disease. HCV genotype is one of the most significant baseline predictors of response to HCV antiviral therapy. The objective was to evaluate an HCV genotyping method that targets the 5'-untranslated region (UTR) to detect genotypes/subtypes using the GenMark eSensor® XT-8 system. The HCV amplicon of major genotypes/subtypes from the Roche TaqMan® HCV assay served as a template for the nested PCR followed by a direct analysis on the XT-8 detection system. The assay was validated for limit of detection (LOD), specificity, accuracy and precision. The LOD determined was below 175 IU/ml for all the subtypes except 6ab. The genotypes detected using this assay were in concordance with the LiPA assay. The high performance characteristics (LOD, specificity, intra- and inter-assay precision, and accuracy), make this assay particularly well suited for clinical HCV genotyping in order to guide antiviral therapy.

Background: The combined genetic effects of single nucleotide polymorphisms may additively or synergistically contribute to the increased cancer risk. The interactions associated with xenobiotic metabolizing enzymes and transporter protein involved in the biotransformation and transport of xenobiotics could determine the functional outcomes over the independent effects of a single susceptibility gene in the risk of upper aerodigestive tract cancers. Methods: The hospital-based case–control study evaluated CYP1A1 ( *2A and *2C ), CYP2E1 ( *1B , *5B , and *6 ), GST ( M1 , T1 , and P1 ) and ABCB1 3435C> T polymorphisms among 408 histopathologically confirmed cases and 220 controls using polymerase chain reaction based methods in an Indian population. Results: The multivariate logistic regression analyses demonstrated potentially high risk gene–gene interactions with the concurrent deletions of the GSTT1 and GSTM1 genes and GSTP1 variant genotypes (OR 5.81; 95% CI 1.01–40.28), the deletions of GSTT1 and GSTM1 genotypes with variant genotypes of CYP1A1*2A (OR 8.21; 95% CI 1.91–49.48), GSTT1 and GSTM1 deficient genotypes along with CYP2E1*1B variant genotypes (OR 6.73; 95% CI 1.32–22.81), the polymorphic genotypes of ABCB1 and deficient GSTT1 (OR 6.08; 95% CI 2.21–16.76) and an enhanced risk with the combined variant genotypes of CYP1A1*2A , GSTT1 and ABCB1 (OR 11.14; 95% CI 2.70–46.02). Conclusion: The findings indicate that the interactions associated with various drug metabolizing enzymes and transporter protein exhibit high risk for UADT cancers than that ascribed to a single susceptible gene. This was particularly established among the polymorphic carriers of CYP1A1 *2A, GSTT1 and ABCB1 genes in the population investigated.

The aim of the study was to establish the frequencies of CYP2C9*1, *2, *3 and CYP2C19*1, *2 and *3 in the south Indian population and to compare them with the inter-racial distribution of the CYP2C9 and CYP2C19 genetic polymorphisms. Genotyping analyses of CYP2C9 and CYP2C19 were conducted in unrelated, healthy volunteers from the three south Indian states of Andhra Pradesh, Karnataka and Kerala, by the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). The allele frequencies of the populations of these three states were then pooled with our previous genotyping data of Tamilians (also in south India), to arrive at the distribution of CYP2C9 and CYP2C19 alleles in the south Indian population. Frequencies of CYP2C9 and CYP2C19 alleles and genotypes among various populations were compared using the two-tailed Fisher's exact test. The frequencies of CYP2C9*1, *2 and *3 in the south Indian population were 0.88 (95% CI 0.85-0.91), 0.04 (95% CI 0.02-0.06) and 0.08 (95% CI 0.06-0.11), respectively. The frequencies of CYP2C9 genotypes *1/*1, *1/*2, *1/*3, *2/*2, *2/*3 and *3/*3 were 0.78 (95% CI 0.74-0.82), 0.05 (95% CI 0.03-0.07), 0.15 (95% CI 0.12-0.18), 0.01 (95% CI 0.0-0.02), 0.01 (95% CI 0.0-0.02) and 0.0, respectively. CYP2C19*1, *2 and *3 frequencies were 0.64 (95% CI 0.60-0.68), 0.35 (95% CI 0.31-0.39) and 0.01 (95% CI 0.0-0.03), respectively. As a result of a significant heterogeneity, the data on CYP2C19 genotype frequencies were not pooled. The frequency of CYP2C9*2 mutant alleles in south Indians was higher than in Chinese and Caucasians, while CYP2C9*3 was similar to Caucasians. CYP2C19*2 was higher than in other major populations reported so far. The relatively high CYP2C19 poor-metabolizer genotype frequency of 12.6% indicates that over 28 million south Indians are poor metabolizers of CYP2C19 substrates.