HIV Drug Resistance (HIVDR) in Antiretroviral Therapy-Naïve Patients in Tanzania Not Eligible for WHO Threshold HIVDR Survey Is Dramatically High

The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population. HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25-63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072-0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095-0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma. ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population

Effects of Prednisolone on Disease Progression in Antiretroviral-Untreated HIV Infection: A 2-Year Randomized, Double-Blind Placebo-Controlled Clinical Trial

Background HIV-disease progression correlates with immune activation. Here we investigated whether corticosteroid treatment can attenuate HIV disease progression in antiretroviral-untreated patients. Methods Double-blind, placebo-controlled randomized clinical trial including 326 HIV-patients in a resource-limited setting in Tanzania (clinicaltrials.gov NCT01299948). Inclusion criteria were a CD4 count above 300 cells/μl, the absence of AIDS-defining symptoms and an ART-naïve therapy status. Study participants received 5 mg prednisolone per day or placebo for 2 years. Primary endpoint was time to progression to an AIDS-defining condition or to a CD4-count below 200 cells/μl. Results No significant change in progression towards the primary endpoint was observed in the intent-to-treat (ITT) analysis (19 cases with prednisolone versus 28 cases with placebo, p = 0.1407). In a per-protocol (PP)-analysis, 13 versus 24 study participants progressed to the primary study endpoint (p = 0.0741). Secondary endpoints: Prednisolone-treatment decreased immune activation (sCD14, suPAR, CD38/HLA-DR/CD8+) and increased CD4-counts (+77.42 ± 5.70 cells/μl compared to -37.42 ± 10.77 cells/μl under placebo, p < 0.0001). Treatment with prednisolone was associated with a 3.2-fold increase in HIV viral load (p < 0.0001). In a post-hoc analysis stratifying for sex, females treated with prednisolone progressed significantly slower to the primary study endpoint than females treated with placebo (ITT-analysis: 11 versus 21 cases, p = 0.0567; PP-analysis: 5 versus 18 cases, p = 0.0051): No changes in disease progression were observed in men. Conclusions This study could not detect any significant effects of prednisolone on disease progression in antiretroviral-untreated HIV infection within the intent-to-treat population. However, significant effects were observed on CD4 counts, immune activation and HIV viral load. This study contributes to a better understanding of the role of immune activation in the pathogenesis of HIV infection

Phylogenetic analysis of RT and PR sequences from the Tanzanian cohort.

<p>A neighbour-joining phylogenetic tree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Saitou1" target="_blank">[30]</a> was constructed from the 88 patient derived HIV-1 sequences from the Tanzania cohort and the two partner-derived sequences. Reference sequences were obtained from the Los Alamos HIV sequence database. The analyzed 1302 bp region includes the complete Protease and Reverse Transcriptase coding region. The tree was constructed using Mega software version 4, and the evolutionary distances were calculated using the Kimura 2-parameter method. The bootstrap consensus tree was inferred from 50000 replicates and values greater than 70% are indicated on the branch lengths. The scale at the bottom left indicates the calculated genetic distances between the branches of the phylogenetic tree. Circles represent the 88 samples from our cohort. Black-dotted circles are without RAM, open-circled sequences are with RAM, open triangles are sequences with RAM from HIV-infected partners of two study subjects, which were not included in the determination of HIVDR as these patients received ART. Sequences without symbols are subtype reference sequences derived from Los Alamos database. The subtype is indicated at the end of each sequence name. Relative subtype frequency: A1: 34%, A1D: 7%, C: 26%, CRF10_CD: 4%, D: 28%, B: 1%. Sequences isolated from two couples (couple I, couple II) with NVP resistances.</p

NVP resistance and PMTCT.

<p>Patients with NVP resistance mutations were analyzed regarding PMTCT (with NVP monotherapy) as a possible cause for the emergence of the mutation. PMTCT as a cause for the mutation's appearance is discussed as “possible” if the mother received PMTCT; it is discussed as “unclear” if the PMTCT status and the dates of birth of the children are unknown; for plausibility reasons PMTCT was excluded (“no”) as a trigger of the NVP mutation if the patient is either male or if a female patient presented with unknown PMTCT history combined with the HIV infection being diagnosed only since the date of birth of the youngest child.</p

HIVDR affects efficacy of local first- and second-line ART regimens.

<p>Effects of HIVDR (high- and low-level resistances) on drugs included in the local antiretroviral regimens. A: Effects on local first-line ART regimen. HIVDR that affects at least one of the drugs included in first-line therapy (AZT/D4T plus 3TC plus NVP/EFV) scored positive. B: Effects on local second-line ART regimen. HIVDR that affects at least one of the drugs included in second-line therapy (ddI/ABC plus LPV/SQV plus RTV) scored positive. C: Proportion of patients with HIVDR that affects first-line ART regimen who also carry HIVDR that affects second-line ART regimen.</p

The frequency of HIVDR is age-dependent.

<p>HIVDR was determined by bulk sequencing from ART-naïve patients. A.: Frequency of WHO-defined HIVDR in patients aged under 25 years (n = 20, left bar), patients aged over 25 years (n = 68, middle bar), and in the total study population (n = 88, right white bar). B: Frequency of WHO-defined HIVDR peaks in different age groups. Numbers in brackets indicate the number of individuals tested in each age group. Data as means ± S.E.M.. For statistical analysis, Fisher's exact test was performed. Differences with a P<0.05 were regarded as statistically significant. C: Number of WHO-defined RAM per individual (“RAM burden”). D: Number of affected antiretroviral drug classes (NRTI, NNRTI, PI) per individual in relation to the number of WHO-defined RAM per individual.</p

Demographic patient characteristics of the study population.

<p>The study population consisted of 120 ART-naive HIV-1 infected adults. PBMC- or plasma samples from 88 patients yielded amplicons for the bulk sequencing reaction. Data are expressed as means ± S.D. and range in parentheses. Patients with CD4 counts <200/ml at sample date initiated ART if CD4 counts remained below 350/ml four weeks later.</p

HIVDR in the Mwanza cohort.

<p>RAM according to the Stanford HIV Drug Resistance Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Stanford1" target="_blank">[17]</a> in 16/88 baseline samples. Mutations associated with a score of 60 were attributed as high-level resistance-associated mutations (RAM) and mutations with a score of 10–35 were attributed as low-level RAM. Mutations listed for WHO HIVDR surveillance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Bennett3" target="_blank">[18]</a> are indicated in bold. Superscripted numbers identify the reasons for excluding the respected mutations from the HIVDR list: 1) nonpolymorphic, but at highly polymorphic position; 2) polymorphic position in subtypes B, F, CRF01_AE; 3) polymorphic in multiple subtypes. ABC: Abacavir; ATV: Atazanavir; AZT: Zidovudine; ddI: Didanosine; DLV: Delaviridine; D4T: Stavudine; EFV: Efavirenz; FTC: Emtricitabine; LPV: Lopinavir; NFV: Nelfinavir; NVP: Nevirapine; SQV: Saquinavir; 3TC:Lamivudine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Joint1" target="_blank">[29]</a>. <u>Underlined</u> drugs are part of the local first line regimens, double line underlined drugs are part of local second line regimens. Data derived from 88 sequenced samples. Therapeutic drug monitoring (TDM) was performed from plasma samples collected at baseline for NNRTIs (Efavirenz and Nevirapine) and PIs (Nelfinavir, Saquinavir, Atazanavir and Lopinavir).</p

Long-term persistence of RAM.

<p>Of the 16 patients who presented with RAM at baseline, we analyzed plasma specimens collected at later time points (“follow-up 1”, “follow-up 2”) for the presence of RAM. We detected three different scenarios, including persistent RAM, disappearing RAM, and newly emerging RAM (some samples appear in more than one scenario). Time to follow-up sample is indicated in months. In some cases, no PCR product from plasma samples could be generated. The investigation for long-term stability of RAM is insofar incomplete as plasma specimens collected at later time points were not available (“n.a.”) for all patients, referred to as “n.d.” (not determined).</p

Study drug adherence and loss to follow-up.

<p><b>A, B</b>: Study drug adherence calculated from pill counts of returned, unused study medication in female (A) and male (B) study participants. P-values were calculated by 2way ANOVA <b>C</b>: Loss to follow-up during the two-year study. P value was calculated by log-rank (Mantel-Cox) analysis.</p