Durable Suppression of HIV-1 after Virologic Monitoring-Based Antiretroviral Adherence Counseling in Rakai,Uganda

ObjectivesHIV viral load is recommended for monitoring antiretroviral treatment and identifying treatment failure. We assessed the durability of viral suppression after viral load-triggered adherence counseling among patients with HIV viremia 6 months after ART initiation.DesignObservational cohort enrolled in an antiretroviral treatment program in rural Uganda.MethodsParticipants who underwent routine viral load determination every 24 weeks and had at least 48 weeks of follow-up were included in this analysis. Patients with viral loads >400 copies/ml at 24 weeks of treatment were given additional adherence counseling, and all patients were followed to assess the duration of viral suppression and development of virologic failure.Results1,841 participants initiating antiretroviral therapy were enrolled in the Rakai Health Sciences Program between June 2005 and June 2011 and were followed with viral load monitoring every 24 weeks. 148 (8%) of patients did not achieve viral suppression at 24 weeks and were given additional adherence counseling. 85 (60%) of these patients had undetectable viral loads at 48 weeks, with a median duration of viral suppression of 240 weeks (IQR 193-288 weeks). Failure to achieve an undetectable viral load at 48 weeks was associated with age <30 years and 24 week viral load >2,000 copies/ml in multivariate logistic regression analysis.ConclusionsThe majority of patients with persistent viremia who were provided adherence counseling achieved robust viral suppression for a median 4.6 years. Access to virologic monitoring and adherence counseling is a priority in resource-limited settings.     

Dry Blood Spots a Reliable Method for Measurement of Hepatitis B Viral Load in Resource-Limited Settings

MethodsThe performance of DBS in HBV quantification was investigated using a modified commercial test (Abbott RealTime HBV assay). Paired DBS and plasma samples were collected from an HBV positive cohort in Addis Ababa, Ethiopia. DBS were stored at ambient temperature for 4–39 days before shipment to the laboratory.ResultsTwenty-six paired samples were selected covering the total range of quantification, from 2.14 log IU/ml to >7 log IU/ml. HBV was detected in 21 of 21 (100%) DBS from patients with a corresponding plasma viral load above 2.70 log IU/ml. The mean difference between plasma and DBS was 0.59 log IU/ml, and the correlation was strong (R2 = 0.92). In stability studies there was no significant change in DBS viral load after storage at room temperature for up to 12 weeks.ConclusionsThis study suggests that DBS can be a feasible and reliable alternative to plasma for quantification of HBV in resource-limited settings. DBS can expand access to antiviral treatment for patients in low- and middle-income countries.     

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background

WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda.

Methods

We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VL_ref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml).

Results

496 paired VFA and VL_ref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VL_ref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively.

Conclusions

VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.     

Monitoring HIV Viral Load in Resource Limited Settings: Still a Matter of Debate?

Introduction

Consequences of lack of viral monitoring in predicting the effects of development of HIV drug resistance mutations during HAART in resource-limited settings (RLS) is still a matter of debate.

Design

To assess, among HIV+ patients receiving their first-line HAART, prevalence of virological failure and genotypic resistance mutations pattern in a Médécins Sans Frontières/Ministry of Health programme in Busia District (Kenya).

Methods

Patients with HAART treatment for ≥12 months were eligible for the study and those with HIV-RNA ≥5000 copies/ml underwent genotypic study. Total HIV-1 RNA from Dried Blood Spots was extracted using Nuclisens method.

Results

926 patients were included. Among 274 (29.6%) patients with detectable viral load, 55 (5.9%) experienced treatment failure (viral load >5.000 copies/ml); 61.8% were female and 10 (18.2%) had clinical failure. Median CD4 cell count was 116 cell/mm3 (IQR: 54–189). Median HIV-RNA was 32,000 copies/ml (IQR: 11000–68000). Eighteen out of 55 (33%) samples could be sequenced on PR and RT genes, with resistance associated mutations (RAMs) in 15 out of 18 samples (83%). Among patients carrying RAMs, 12/15 (81%) harboured RAMs associated to thymidine analogues (TAMs). All of them (100%) showed M184V resistance associated mutation to lamivudine as well as NNRTI''s RAMS.

Conclusions

Virological failure rate in resource-limited settings are similar to those observed in developed countries. Resistance mutation patterns were concordant with HAART received by failing patients. Long term detectable viral load confers greater probability of developing resistance and as a consequence, making difficult to find out a cost-effective subsequent treatment regimen.     

Cytomegalovirus Viral Load Kinetics in Patients with HIV/AIDS Admitted to a Medical Intensive Care Unit: A Case for Pre-Emptive Therapy

Background

Cytomegalovirus (CMV) infection is associated with severe diseases in immunosuppressed patients; however, there is a lack of data for pre-emptive therapy in patients with HIV/AIDS.

Method

This was a retrospective study, which enrolled patients diagnosed with HIV/AIDS (CD4<200 cells/μl), who had detectable CMV viral load (VL) during their stay in an adult medical intensive care unit between 2009–2012.

Results

After screening 82 patients’ records, 41 patients met the enrolment criteria. Their median age was 37 (interquartile range [IQR]: 31–46), and median CD4 count was 29 cells/μl (IQR: 5–55). Sixteen patients (39%) had serial measurements of CMV VL before treatment with ganciclovir. Patients whose baseline CMV VL values were between 1,000–3,000 copies/ml had significantly higher values (median of 14,650 copies/ml) on follow-up testing done 4–12 days later. Those with undetectable VLs at baseline testing had detectable VLs (median of 1,590 copies/ml) mostly within 20 days of follow-up testing. Patients who had VLs >1,000 copies/ml at baseline testing had significantly higher mortality compared to those who had <1,000 copies/ml {hazard ratio of 3.46, p = 0.003 [95% confidence interval (CI): 1.55–7.71]}. Analysis of the highest CMV VL per patient showed that patients who had VLs of >5,100 copies/ml and did not receive ganciclovir had 100% mortality compared to 58% mortality in those who received ganciclovir at VLs of >5,100 copies/ml, 50% mortality in those who were not treated and had low VLs of <5,100 copies/ml, and 44% mortality in those who had ganciclovir treatment at VLs of <5,100 copies/ml (p = 0.084, 0.046, 0.037, respectively).

Conclusion

This study showed a significantly increased mortality in patients with HIV/AIDS who had high CMV VLs, and suggests that a threshold value of 1,000 copies/ml may be appropriate for pre-emptive treatment in this group.     

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.     

Drug resistance testing in PBMCs of HIV infected patients.

HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.     

Virologic Response to Tipranavir-Ritonavir or Darunavir-Ritonavir Based Regimens in Antiretroviral Therapy Experienced HIV-1 Patients: A Meta-Analysis and Meta-Regression of Randomized Controlled Clinical Trials

Background

The development of tipranavir and darunavir, second generation non-peptidic HIV protease inhibitors, with marked improved resistance profiles, has opened a new perspective on the treatment of antiretroviral therapy (ART) experienced HIV patients with poor viral load control. The aim of this study was to determine the virologic response in ART experienced patients to tipranavir-ritonavir and darunavir-ritonavir based regimens.

Methods and Findings

A computer based literature search was conducted in the databases of HINARI (Health InterNetwork Access to Research Initiative), Medline and Cochrane library. Meta-analysis was performed by including randomized controlled studies that were conducted in ART experienced patients with plasma viral load above 1,000 copies HIV RNA/ml. The odds ratios and 95% confidence intervals (CI) for viral loads of <50 copies and <400 copies HIV RNA/ml at the end of the intervention were determined by the random effects model. Meta-regression, sensitivity analysis and funnel plots were done. The number of HIV-1 patients who were on either a tipranavir-ritonavir or darunavir-ritonavir based regimen and achieved viral load less than 50 copies HIV RNA/ml was significantly higher (overall OR = 3.4; 95% CI, 2.61– 4.52) than the number of HIV-1 patients who were on investigator selected boosted comparator HIV-1 protease inhibitors (CPIs-ritonavir). Similarly, the number of patients with viral load less than 400 copies HIV RNA/ml was significantly higher in either the tipranavir-ritonavir or darunavir-ritonavir based regimen treated group (overall OR = 3.0; 95% CI, 2.15 – 4.11). Meta-regression showed that the viral load reduction was independent of baseline viral load, baseline CD4 count and duration of tipranavir-ritonavir or darunavir-ritonavir based regimen.

Conclusions

Tipranavir and darunavir based regimens were more effective in patients who were ART experienced and had poor viral load control. Further studies are required to determine their consistent viral load suppression effect as the duration of treatment is more prolonged.     

Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

Introduction

To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods.

Methodology/Principal Findings

We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads ≥1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds'' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods.

Discussion

These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results.     

The use of a method for the quantitative determination of HIV-1 RNA for assessing the severity and prognosis of the development of the disease

The informatory role of a new marker of HIV infection, characterizing the content of HIV-1 RNA in the biological fluids of the patient's body, is evaluated. The quantitative determination of HIV-1 RNA, carried out in a single assay, was made in the blood of 25 HIV-infected patients. These studies confirmed that the determination of the level of RNA in the plasma (viral load) was a reliable criterion indicating the severity and progress of the disease. The viral load of more than 100,000 copies/ml was a sign prognosticating the future pronounced progress of the disease in spite of moderate clinical manifestations and relatively high values of CD4 cells in the patient's blood at the moment of testing.     

High levels of viral replication during primary simian immunodeficiency virus SIVagm infection are rapidly and strongly controlled in African green monkeys

In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (10(4) to 10(5) DNA copies/10(6) peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 x 10(6) to 2 x 10(8) RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 x 10(5) to 3 x 10(6) RNA copies/10(6) LN cell [LNC] and 10(3) to 10(4) DNA copies/10(6) LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p. i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (10(2) to 10(3) DNA copies/10(6) PBMC and 2 x 10(3) to 2 x 10(5) RNA copies/ml of plasma). In LNs, viral loads declined to 5 x 10(1) to 10(3) DNA copies and 10(4) to 3 x 10(5) RNA copies per 10(6) LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 x 10(4) RNA copies/10(6) LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8(+) cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.     

Quantitative viral load measurement for BKV infection in renal transplant recipients as a predictive tool for BKVAN

Infection by polyomavirus BK (BKV) is an emerging problem in the clinical management of renal transplant patients because it is responsible for nephropathy and consequently can cause loss of the transplanted organ (BKV associated nephropathy, BKVAN). Aim of this study was to evaluate the use of blood viral load measurement as a screening tool for diagnosis of BKV infection and to identify a threshold value for the management of patients. A total of 75 kidney transplant patients, corresponding to 338 consecutive plasma samples, were analyzed by an automatic system for nucleic acid extraction and quantitative real-time polymerase chain reaction (PCR) for detection of BKV. BKV was detected in 170 samples (26 patients) with a median viral load of 4.1 log10 copies/mL; among these 26 patients, seven (34.7%) were found to have BKVAN on allograft biopsy together with a median viral load of 5 log10 copies/mL. The ROC curve analysis identified a viral load equal to 4.1 log10 copies/mL as the best discriminant cut-off value to predict the disease and to identify patients at risk of developing BKVAN.     

Failure time analysis of HIV vaccine effects on viral load and antiretroviral therapy initiation

The world's first efficacy trial of a preventive HIV vaccine was completed in 2003. Study participants who became HIV infected were followed for 2 years and monitored for HIV viral load and initiation of antiretroviral therapy (ART). In order to determine if vaccination may have altered HIV progression in persons who acquired HIV, a pre-specified objective was to compare the time until a composite endpoint between the vaccine and placebo arms, where the composite endpoint is the first event of ART initiation or viral failure (HIV viral load exceeds a threshold x(vl) copies/ml). Specifically, with vaccine efficacy, VE(tau, x(vl)), defined as one minus the ratio (vaccine/placebo) of the cumulative probability of the composite endpoint (with failure threshold x(vl)) occurring by tau months, the aim was to estimate the four parameters {VE(tau, x(vl)): x(vl) is an element of {1500, 10,000, 20,000, 55,000} copies/ml} with simultaneous 95% confidence bands. A Gaussian multipliers simulation method is devised for constructing confidence bands for VE(tau, x(vl)) with x(vl) spanning multiple discrete values or a continuous range. The new method is evaluated in simulations and is applied to the vaccine trial data set.     

Dried Blood Spots for Viral Load Monitoring in Malawi: Feasible and Effective

Objectives

To evaluate the feasibility and effectiveness of dried blood spots (DBS) use for viral load (VL) monitoring, describing patient outcomes and programmatic challenges that are relevant for DBS implementation in sub-Saharan Africa.

Methods

We recruited adult antiretroviral therapy (ART) patients from five district hospitals in Malawi. Eligibility reflected anticipated Ministry of Health VL monitoring criteria. Testing was conducted at a central laboratory. Virological failure was defined as >5000 copies/ml. Primary outcomes were program feasibility (timely result availability and patient receipt) and effectiveness (second-line therapy initiation).

Results

We enrolled 1,498 participants; 5.9% were failing at baseline. Median time from enrollment to receipt of results was 42 days; 79.6% of participants received results within 3 months. Among participants with confirmed elevated VL, 92.6% initiated second-line therapy; 90.7% were switched within 365 days of VL testing. Nearly one-third (30.8%) of participants with elevated baseline VL had suppressed (<5,000 copies/ml) on confirmatory testing. Median period between enrollment and specimen testing was 23 days. Adjusting for relevant covariates, participants on ART >4 years were more likely to be failing than participants on therapy 1–4 years (RR 1.7, 95% CI 1.0-2.8); older participants were less likely to be failing (RR 0.95, 95% CI 0.92-0.98). There was no difference in likelihood of failure based on clinical symptoms (RR 1.17, 95% CI 0.65-2.11).

Conclusions

DBS for VL monitoring is feasible and effective in real-world clinical settings. Centralized DBS testing may increase access to VL monitoring in remote settings. Programmatic outcomes are encouraging, especially proportion of eligible participants switched to second-line therapy.     

Increased frequency of regulatory T cells accompanies increased immune activation in rectal mucosae of HIV-positive noncontrollers

Gut-associated lymphoid tissue (GALT) is a major site of HIV replication and CD4(+) T cell depletion. Furthermore, microbial translocation facilitated by mucosal damage likely contributes to the generalized immune activation observed in HIV infection. Regulatory T cells (Treg) help maintain homeostasis and suppress harmful immune activation during infection; however, in the case of persistent viral infections such as HIV, their role is less clear. Although a number of studies have examined Treg in blood during chronic infection, few have explored Treg in the gastrointestinal mucosa. For this study, paired blood and rectal biopsy samples were obtained from 12 HIV noncontrollers (viral load of >10,000 copies/ml plasma), 10 HIV controllers (viral load of <500 copies/ml plasma for more than 5 years), and 12 HIV seronegative control subjects. Noncontrollers had significantly higher percentages of Treg in rectal mononuclear cells (RMNC), but not in blood, compared to seronegative subjects (P = 0.001) or HIV controllers (P = 0.002). Mucosal Treg positively correlated with viral load (P = 0.01) and expression of immune activation markers by CD4(+) (P = 0.01) and CD8(+) (P = 0.07) T cells. Suppression assays indicated that mucosal and peripheral Treg of noncontrollers and controllers maintained their capacity to suppress non-Treg proliferation to a similar extent as Treg from seronegative subjects. Together, these findings reveal that rather than experiencing depletion, mucosal Treg frequency is enhanced during chronic HIV infection and is positively correlated with viral load and immune activation. Moreover, mucosal Treg maintain their suppressive ability during chronic HIV infection, potentially contributing to diminished HIV-specific T cell responses and viral persistence.