Antiretroviral Treatment Monitoring Research Articles

Comparison of the performance of Aptima HIV-1 Quant Dx Assay with Abbott RealTime HIV Assay for viral load monitoring using plasma and Dried Blood Spots collected in Kenya.

HIV-1 viral Load (VL) testing is recommended for the monitoring of antiretroviral treatment. Dried Blood Spots (DBS) are an effective sample type in resource limited settings, where safe phlebotomy and reliable shipping are hard to guarantee. In HIV high burden countries, high throughput assays can improve access to testing services. The Hologic Aptima HIV-1 Quant Dx Assay (Aptima Assay) is a high throughput assay that runs on the CE-IVD approved Panther platform. The objectives of this study were to assess the performance characteristics of Aptima for VL monitoring using plasma and venous DBS specimens and to determine the stability of HIV-1 RNA in DBS. This was a cross-sectional study of 2227 HIV infected adults visiting health facilities in Nairobi and Busia, Kenya. Each provided a venous blood sample; plasma was prepared from 1312 samples while paired DBS samples and plasma were prepared from the remaining 915 samples. The agreement between the Aptima assay and the Abbott RealTime HIV-1 Assay (Abbott RT) was analysed by comparing the HIV-1 VL in both assays at the medical decision point of 1000 copies/mL. To assess stability of HIV-1 RNA in DBS, VL in DBS spotted on day 0 were compared with that from the same DBS card after 21 days of storage at room temperature. Overall, 436 plasma samples had quantifiable results in both Aptima and Abbott RT. The agreement between the two assays at 1000 copies/mL was 97.48% with a Pearson's correlation coefficient (r) of 0.9589 and gave a mean bias of 0.33 log copies/mL on Bland-Altman analysis. For fresh DBS, the agreement in both assays was 94.64% at 1000 copies/mL, with an r of 0.8692 and a mean bias of 0.35 log copies/mL. The overall agreement between DBS tested in Aptima on day 0 versus day 21 was 95.71%, with a mean bias of -0.154. The Aptima HIV-1 Quant Dx assay is an accurate test for VL monitoring of HIV-1 using DBS and plasma sample types in Kenya.

HIV-1 drug resistance genotyping success rates and correlates of Dried-blood spots and plasma specimen genotyping failure in a resource-limited setting

BackgroundHIV-1 drug resistance genotyping is critical to the monitoring of antiretroviral treatment. Data on HIV-1 genotyping success rates of different laboratory specimen types from multiple sources is still scarce.MethodsIn this cross-sectional study, we determined the laboratory genotyping success rates (GSR) and assessed the correlates of genotyping failure of 6837 unpaired dried blood spot (DBS) and plasma specimens. Specimens from multiple studies in a resource-constrained setting were analysed in our laboratory between 2016 and 2019.ResultsWe noted an overall GSR of 65.7% and specific overall GSR for DBS and plasma of 49.8% and 85.9% respectively. The correlates of genotyping failure were viral load (VL) < 10,000 copies/mL (aOR 0.3 95% CI: 0.24–0.38; p < 0.0001), lack of viral load testing prior to genotyping (OR 0.85 95% CI: 0.77–0.94; p = 0.002), use of DBS specimens (aOR 0.10 95% CI: 0.08–0.14; p < 0.0001) and specimens from routine clinical diagnosis (aOR 1.4 95% CI: 1.10–1.75; p = 0.005).ConclusionsWe report rapidly decreasing HIV-1 genotyping success rates between 2016 and 2019 with increased use of DBS specimens for genotyping and note decreasing median viral loads over the years. We recommend improvement in DBS handling, pre-genotyping viral load testing to screen samples to enhance genotyping success and the development of more sensitive assays with well-designed primers to genotype specimens with low or undetectable viral load, especially in this era where virological suppression rates are rising due to increased antiretroviral therapy roll-out.

HIV-1 viral load testing in resource-limited settings: Challenges and solutions for specimen integrity.

HIV-1 viral load (VL) testing is a crucial element in providing an antiretroviral treatment monitoring program. The success of these programs depends on the availability and quality of the VL testing services. There are several pre-analytic factors which can affect the quality of VL testing. Many of the challenges faced by resource-limited countries result in a compromise of specimen integrity, thus limiting widespread access to VL monitoring. The various logistic and financial challenges that exist are not insurmountable and several innovative solutions currently exist to overcome these barriers to providing widespread VL testing. This review summarizes the VL testing challenges in resource-limited settings and provides an overview of potential solutions including testing dried blood spots, dried plasma spots, plasma separation cards and the use of point of care tests.

Purifying selection in HIV-1 pol gene in perinatally HIV-1-infected children harboring discordant immunological response and virological nonresponse to long-term antiretroviral therapy

Background: Biological monitoring of antiretroviral treatment (ART) in human immunodeficiency virus (HIV)-infected pediatric population remains challenging. The aim of the present study was to assess the long-term HIV-1 genetic diversity in pol gene in HIV-1-infected children in virological failure under antiretroviral regimen adapted according to the successive World Health Organization (WHO) guidelines for resource-constrained settings. Methods: HIV-1 diversity in pol gene was assessed in HIV-1-infected children and adolescents born from HIV-infected mothers (median age at follow-up: 13.8 years) in virological failure (VF + ) despite long-term regimen recommended by the WHO. The numbers of nonsynonymous substitutions per potential nonsynonymous site (dN) and of synonymous substitutions at potential synonymous sites (dS) in HIV-1 pol gene and the dN/dS ratios were used to estimate the selective pressure on circulating HIV-1. Results: The immunological responses to ART basically corresponded to: 1) Full therapeutic failure with immunological (I - ) and virological nonresponses in one-quarter (24.6%) of study children ((I - , VF + ) subgroup); 2) Discordant immunovirological responses with paradoxical high CD4 T cell counts (I + ) and high HIV-1 RNA load in the remaining cohort patients (75.4%) ((I + , VF + ) subgroup). The mean dS was 1.8-fold higher in (I + , VF + ) than (I - , VF + ) subgroup (25.9 ± 18.4 vs. 14.3 ± 10.8). In the (I + , VF + ) subgroup, the mean dS was 1.6-fold higher than the mean dN. Finally, the mean dN/dS ratio was 2.1-fold lower in (I + , VF + ) than (I - , VF + ) subgroup (0.6 ± 0.3 vs. 1.3 ± 0.7), indicating purifying selection in the immunovirological discordant (I + , VF + ) subgroup and positive selection in the immunovirological failure (I - , VF + ) subgroup. Conclusions: Children and adolescents in immunovirological therapeutic failure harbor positive selection of HIV-1 strains favoring diversifying in pol -encoded amino acids. In contrast, children with persistent discordant immunovirological responses show accumulation of mutations and purifying selection in pol gene sequences, indicating limited genetic evolution and likely suggesting genetic adaptation of viruses to host functional constraints. J Clin Med Res. 2020;12(6):369-376 doi: https://doi.org/10.14740/jocmr4157

Brief Report: Interferon-γ-Inducible Protein 10-A Potential Marker for Targeted Viral Load Monitoring of Antiretroviral Treatment?

The use of surrogate markers for targeting viral load (VL) testing could be an alternative to universal VL testing during antiretroviral treatment (ART) and would allow for more effective resource allocation. We investigated the correlation between levels of HIV RNA and interferon-γ-inducible protein 10 (IP-10) in Ethiopian adults at 12 months after ART initiation. In addition, we specifically investigated differences in IP-10 levels between patients with and without virological suppression. Cohort of HIV-positive adults receiving ART at Ethiopian health centers. Using a nested case-control design, individuals without virological suppression (HIV RNA ≥ 150 copies/mL) at 12 months after ART initiation were gender-matched with virologically suppressed controls (1:2 ratio). IP-10 levels were correlated with HIV RNA, and the distribution of IP-10 was compared for 3 VL strata: <150 copies/mL (VL < 150), 150-999 copies/mL (VL150-999), and ≥1000 copies/mL (VL ≥ 1000). At 12 months after ART initiation, the following VL distribution was found among 192 individuals (50% women): VL < 150, 122/192 (63.5%); VL150-999, 23/192 (12.0%); and VL ≥ 1000 47/192 (24.5%). IP-10 and HIV RNA levels were positively correlated (r = 0.481; P < 0.0001). Median IP-10 levels for the VL strata were VL < 150: 159 pg/mL [interquartile range (IQR) 121-246], VL150-999: 174 pg/mL (IQR 131-276), and VL ≥ 1000: 343 pg/mL (IQR 190-529), respectively. These differences were statistically significant for VL ≥ 1000 versus VL < 150 (adjusted P < 0.001) and VL150-999 (adjusted P = 0.004), respectively. IP-10 and HIV RNA levels during ART showed significant correlations, with significantly higher IP-10 concentration in ART recipients with VL ≥ 1000 copies/mL compared to those with suppressed or undetectable VL.

Three Decades and Counting: HIV Service Provision in Outpatient Mental Health Settings.

People with serious mental illness in the United States have higher human immunodeficiency virus (HIV) infection rates than the general U.S. population. This study aimed to assess delivery of HIV services in New York State's outpatient mental health programs. Greater access would enhance efforts to improve HIV prevention and care outcomes. The authors surveyed directors of licensed outpatient mental health care programs statewide to investigate their HIV service delivery. Data were compared with surveys conducted in 1997 and 2004 in order to examine differences in services between geographic regions and time periods. Outpatient mental health programs have improved in the volume and range of HIV services offered, but their provision of preexposure prophylaxis, condoms, HIV testing, and HIV antiretroviral treatment monitoring has lagged. New York's initiative to end the HIV epidemic is not optimized to reach people with serious mental illness in settings designed for their care.

Purifying Selection in Human Immunodeficiency Virus-1 pol Gene in Perinatally Human Immunodeficiency Virus-1-Infected Children Harboring Discordant Immunological Response and Virological Nonresponse to Long-Term Antiretroviral Therapy.

BackgroundBiological monitoring of antiretroviral treatment (ART) in human immunodeficiency virus (HIV)-infected pediatric population remains challenging. The aim of the present study was to assess the long-term HIV-1 genetic diversity in pol gene in HIV-1-infected children in virological failure under antiretroviral regimen adapted according to the successive World Health Organization (WHO) guidelines for resource-constrained settings.MethodsHIV-1 diversity in pol gene was assessed in HIV-1-infected children and adolescents born from HIV-infected mothers (median age at follow-up: 13.8 years) in virological failure (VF+) despite long-term regimen recommended by the WHO. The numbers of nonsynonymous substitutions per potential nonsynonymous site (dN) and of synonymous substitutions at potential synonymous sites (dS) in HIV-1 pol gene and the dN/dS ratios were used to estimate the selective pressure on circulating HIV-1.ResultsThe immunological responses to ART basically corresponded to: 1) Full therapeutic failure with immunological (I-) and virological nonresponses in one-quarter (24.6%) of study children ((I-, VF+) subgroup); 2) Discordant immunovirological responses with paradoxical high CD4 T cell counts (I+) and high HIV-1 RNA load in the remaining cohort patients (75.4%) ((I+, VF+) subgroup). The mean dS was 1.8-fold higher in (I+, VF+) than (I-, VF+) subgroup (25.9 ± 18.4 vs. 14.3 ± 10.8). In the (I+, VF+) subgroup, the mean dS was 1.6-fold higher than the mean dN. Finally, the mean dN/dS ratio was 2.1-fold lower in (I+, VF+) than (I-, VF+) subgroup (0.6 ± 0.3 vs. 1.3 ± 0.7), indicating purifying selection in the immunovirological discordant (I+, VF+) subgroup and positive selection in the immunovirological failure (I-, VF+) subgroup.ConclusionsChildren and adolescents in immunovirological therapeutic failure harbor positive selection of HIV-1 strains favoring diversifying in pol-encoded amino acids. In contrast, children with persistent discordant immunovirological responses show accumulation of mutations and purifying selection in pol gene sequences, indicating limited genetic evolution and likely suggesting genetic adaptation of viruses to host functional constraints.

Quantification of HIV-2 DNA in Whole Blood.

Time to AIDS infection is longer with HIV-2, compared to HIV-1, but without antiretroviral therapy both infections will cause AIDS-related mortality. In HIV-2 infection, monitoring of antiretroviral treatment (ART) efficacy is challenging since a large proportion of HIV-2-infected individuals displays low or undetectable plasma RNA levels. Hence, quantification of cellular DNA load may constitute an alternative method for monitoring ART efficacy. Moreover, sensitive HIV-2 DNA quantification protocols are also important for the characterization of the HIV-2 reservoirs, and ultimately for the development of HIV-2 cure strategies. We have developed a sensitive and robust HIV-2 DNA quantification protocol based on whole blood as DNA source, including normalization of leukocyte cell numbers using parallel quantification of the single copy porphobilinogen deaminase gene. The specificity and sensitivity of the assay was 100%. The limit of detection was 1 copy and limit of quantification was 5 copies. When applying this protocol to HIV-2 infected, it was found that HIV-2 viral DNA was detectable in individuals in whom viral RNA was undetectable or under quantification level. Thus, this method provides a sensitive approach to HIV-2 DNA viral quantification from whole blood of HIV-2 infected patients.

Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

BackgroundHIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures.Methods and FindingsUsing a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (<3,000 copies/mL) environments.ConclusionsWhole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in field conditions are needed.

Challenges of Antiretroviral Treatment Monitoring in Rural and Remote-Access Regions in Africa

Challenges of Antiretroviral Treatment Monitoring in Rural and Remote-Access Regions in Africa

Utilidad de los dried blood spots para monitorizar la infección por virus de la inmunodeficiencia humana en los programas de salud pública de países en desarrollo

As access to antiretroviral treatment increases in the developing countries, efforts towards making it easier and less costly to collect, store, and deliver the biological samples to reference laboratories, where the serological and genetic diagnosis techniques are performed, have become a high priority. Blood sampling on filter papers is an inexpensive and practical alternative to plasma for antiretroviral treatment monitoring in countries with limited resources and no access to cold chains or refrigeration. The main clinical applications and uses of blood-sampling onto filter papers (dried blood spots [DBS]) are reviewed, focusing on how these can be applied in monitoring HIV infection, particularly for use in National Health Programs in developing countries, or in resource-limited settings. A review is presented of studies that have used the DBS technique for quantifying viral load, analysis of antiretroviral drug-resistance mutations, early infant diagnosis, adult serological diagnosis, detection of viral p24 antigen, and molecular epidemiology of HIV-1, in different geographical locations. Those variables that could affect the use of DBS, particularly in the HIV field, as well as explaining how these procedures can be optimised to increase their sensitivity are also reviewed. The aim of this study was to review the advantages of implementing the DBS technique in the HIV field, especially in resource-constrained regions.

Dried Blood Spots Perform Well in Viral Load Monitoring of Patients Who Receive Antiretroviral Treatment in Rural Tanzania

Monitoring of antiretroviral treatment (ART) with human immunodeficiency virus (HIV) viral loads, as recommended in industrialized countries, is rarely available in resource-limited settings because of the high costs and stringent requirements for storage and transport of plasma. Dried blood spots (DBS) can be an alternative to plasma, but the use of DBS has not been assessed under field conditions in rural Africa. The present study investigates the performance of DBS in HIV viral load monitoring of patients who received ART in rural Tanzania. From November 2007 through June 2008, parallel plasma and DBS specimens were obtained from patients who received ART at Haydom Lutheran Hospital in rural Tanzania. DBS specimens were stored at tropical room temperature for 3 weeks before testing with the NucliSENS EasyQ HIV-1 v1.2 assay. Results obtained with DBS were compared with results obtained with use of a gold-standard plasma assay. Ninety-eight plasma-DBS pairs were compared, and plasma viral loads ranged from <40 to >1,000,000 copies/mL. The correlation between plasma and DBS viral load was strong (R(2) = 0.75). The mean difference (+/- standard deviation) was 0.04 +/ 0.57 log(10) copies/mL, and only 8 samples showed >1 log(10) copies/mL difference. HIV type 1 RNA was detected in 7%, 60%, and 100% of DBS specimens with corresponding plasma viral loads of 40-999, 1000-2999, and 3000 copies/mL, respectively. DBS, in combination with the NucliSENS EasyQ HIV-1 v1.2 asay, performed well in monitoring HIV viral loads in patients who received ART in rural Tanzania, although the sensitivity was reduced when viral burden was low. The use of DBS can simplify virological monitoring in resource-limited settings.

Body Shape and Metabolic Abnormalities in Thai HIV-Infected Patients

Fat and metabolic abnormalities and their associated factors in HIV-infected patients in Thailand were examined. Body fat and fasting lipids (total cholesterol, TC; triglyceride, TG; and HDL-cholesterol, HDL-c) were evaluated in 247 HIV-infected Thais. Body fat was evaluated by subjects and blinded observers, and measured using dual-energy X-ray absorptiometry. Descriptive statistics and logistic regression were used for analyses. Antiretroviral (ARV)-treated Thais were significantly older, more likely to be male, and had higher education and income compared to untreated subjects. The prevalence of lipoatrophy was 10.3% in ARV-naive patients, 36.7% in patients receiving non-protease inhibitor (PI)-based ARV, and 78.7% in PI-based ARV-treated patients (p < 0.001). Excess abdominal or neck fat was found in 0.8%, 6.7%, and 24.6% of the naive, non-PI-treated, and PI-treated, respectively (p < 0.001). Hypercholesterolemia (TC > or = 240 mg/dl) was found in 4.8%, 26.6%, and 42.6%; hypertriglyceridemia (TG > or = 150 mg/dl) in 8.2%, 48.3%, and 75.4%; and low HDL-c (HDL-c < 40 mg/dl) in 42.9%, 20.0%, and 31.2% of the naive, non-PI treated, and PI-treated, respectively (p < 0.05 for all). Central to peripheral fat ratios were 1.11 +/- 0.03, 1.45 +/- 0.06, and 1.93 +/- 0.08 for the naive, non-PI, and PI-treated, respectively (p < 0.001). Treatment was associated with abnormal fat. The adjusted ORs (95% CI) of lipoatrophy for excess fat were 4.6 (2.0-10.7); 6.3 (0.6-71.1) for ARV-naive vs. non-PI; 5.6 (3.4-9.1); 10.7 (3.4-33.8) for ARV-naive vs. PI, and 5.7 (2.4-13.9); 5.3 (1.2-11.4-13.9) for ARV-naive vs. PI. ARV-associated metabolic abnormalities are common in this non-Western population. Appropriate selection and monitoring of ARV treatment are critical to minimize the risk of long-term complications.

Antiretroviral treatment in resource-poor settings: A view from India

The introduction of highly effective generic antiretroviral drugs at reduced cost has transformed the face of HIV/AIDS epidemic in developing countries like India. However, there is an urgent emphasis on developing and implementing guidelines for antiretroviral treatment monitoring by laboratory methods utilizing the available technologies in resource-limited settings. We studied the efficacy of antiretroviral treatment, adherence to therapy and motivation of patients for regular treatment monitoring by CD4 counts. A longitudinal cohort study on an established cohort of 166 HIV-1-infected Indian individuals. Study subjects were followed up for the period from January 2002 to November 2006. Their clinical status and treatment regimen were recorded and CD4 counts were performed at each visit. Repeated-measures ANOVA was used to compute changes in median CD4 counts at each visit in the different treatment groups. We observed a growing awareness and motivation for regular HIV disease monitoring among patients, accompanied by a trend of increasing median CD4 counts at all subsequent follow-up visits after initiation of antiretroviral treatment. The study gives an insight into the institutional efforts for the establishment of cohorts for longitudinal studies, which will help in designing effective treatment guidelines, thus providing impetus to the free public sector antiretroviral therapy program in India. Such formative research aims to fill the lacunae in the limited available data for the formulation of treatment-monitoring guidelines in resource-poor settings of developing countries like India.

Evaluation of p24-based Antiretroviral Treatment Monitoring in Pediatric HIV-1 Infection

Worldwide, 700,000 infants are infected annually by HIV-1, most of them in resource-limited settings. Care for these children requires simple, inexpensive tests. We have evaluated HIV-1 p24 antigen for antiretroviral treatment (ART) monitoring in children. p24 by boosted enzyme-linked immunosorbent assay of heated plasma and HIV-1 RNA were measured prospectively in 24 HIV-1-infected children receiving ART. p24 and HIV-1 RNA concentrations and their changes between consecutive visits were related to the respective CD4+ changes. Age at study entry was 7.6 years; follow-up was 47.2 months, yielding 18 visits at an interval of 2.8 months (medians). There were 399 complete visit data sets and 375 interval data sets. Controlling for variation between individuals, there was a positive relationship between concentrations of HIV-1 RNA and p24 (P < 0.0001). While controlling for initial CD4+ count, age, sex, days since start of ART, and days between visits, the relative change in CD4+ count between 2 successive visits was negatively related to the corresponding relative change in HIV-1 RNA (P = 0.009), but not to the initial HIV-1 RNA concentration (P = 0.94). Similarly, we found a negative relationship with the relative change in p24 over the interval (P < 0.0001), whereas the initial p24 concentration showed a trend (P = 0.08). Statistical support for the p24 model and the HIV-1 RNA model was similar. p24 may be an accurate low-cost alternative to monitor ART in pediatric HIV-1 infection.

Recent advances in antiretroviral therapy and HIV infection monitoring.

Potent new antiretroviral drugs combined with updated treatment strategies have now achieved efficient inhibition of HIV replication in most patients. The major targets for antiretroviral therapy are the reverse transcriptase and the protease of HIV. Up to date, 11 antiretroviral drugs have been licensed in the US. New nucleoside reverse transcriptase inhibitors (NRTI), i.e. abacavir, and adefovir dipivoxil proved to be effective in clinical trials. The antiretroviral activity and impact on clinical outcome of nonnucleoside reverse transcriptase inhibitors (NNRTI) are mostly short lived. Compared to NRTIs, protease inhibitors (PIs) have the highest antiretroviral activity. New PIs that conserve activity against mutant HIV strains or derivatives that have a simpler biosynthesis are being developed. A major drawback of highly active antiretroviral therapy is the selection of resistant mutants under suboptimal dosage, in advanced stages of disease or after pre-treatment with mono- or double-combination regimens. Monitoring of antiretroviral therapy is achieved by measurement of viral load using nucleic acid amplification techniques. The goal of antiretroviral therapy should be to reduce viral load below the detection limit of current assays in order to delay the emergence of resistant mutants and to maximally reduce disease progression. Recommendations for antiretroviral therapy and monitoring are evolving constantly due to the rapid progress in the development of active compounds and new insights into HIV pathogenesis. Resistance determination of HIV patient isolates seems to play a progressively increasing role in monitoring of antiretroviral treatment since treatment-naive patients may already harbour drug-resistant virus. Due to cross-resistance between different compounds, the choice of the treatment regimen should be guided by resistance patterns of HIV in order to warrant maximal and long-lasting efficiency.