The role of HIV-1 RNA in the emergence of resistance to antiretroviral therapies (ARTs) is well documented while less is known about the role of historical viruses stored in the proviral DNA. The primary focus of this work was to characterize the genetic diversity and evolution of HIV drug resistant variants in an individual’s provirus during antiretroviral therapy using next generation sequencing.Blood samples were collected prior to antiretroviral therapy exposure and during the course of treatment from five patients in whom drug resistance mutations had previously been identified using consensus sequencing. The spectrum of viral variants present in the provirus at each sampling time-point were characterized using 454 pyrosequencing from multiple combined PCR products. The prevalence of viral variants containing drug resistant mutations (DRMs) was characterized at each time-point.Low abundance drug resistant viruses were identified in 14 of 15 sampling time-points from the five patients. In all individuals DRMs against current therapy were identified at one or more of the sampling time-points. In two of the five individuals studied these DRMs were present prior to treatment exposure and were present at high prevalence within the amplified and sequenced viral population. DRMs to drugs other than those being currently used were identified in four of the five individuals.The presence of DRMs in the provirus, regardless of their observed prevalence did not appear to have an effect on clinical outcomes in the short term suggesting that the drug resistant viral variants present in the proviral DNA do not appear to play a role in the short term in facilitating the emergence of drug resistance. While the role of HIV-1 RNA in the emergence of resistance to highly active antiretroviral therapy (HAART) has been widely documented, less is known about the role of historical viruses stored in the proviral DNA. Following treatment interruption, latent viruses stored in the provirus have been shown to be responsible for the rapid rebound of viral load following treatment interruption . HoweverRecent research on HIV-1 in Karonga District, Malawi has shown that of the 40 HIV-1 subtype C infected individuals on HAART that we tested, 14% contained drug resistance mutations (DRMs) in the reverse transcriptase (RT) gene of viruses stored in the provirus  with cloSamples were collected from five patients recruited to an antiretroviral cohort study between 2007 and 2009 from an ART clinic in northern Malawi. Antiretrovirals have been available in the District since 2005 and in this clinic since 2006. All patients have been started on a fixed dose combination of stavudine, lamivudine and nevirapine on the basis of clinical staging or CD4 count. Previous work identified discrepancies or ambiguities in the presence/absence of drug resistant mutations in viral sequences from samples collected from these five individuals at sequential time-points during treatment [DNA extraction and nested PCR was performed as previously described  with theSequence reads from each individual sample were separated based on their MIDs and subsequent quality control and analysis was performed independently for the reads corresponding to each sample. Next, the data were trimmed using the modified-Mott algorithm implemented in Geneious 5.4.3  with a 0The prevalence of drug resistant mutations (DRMs) was determined for each sample with three categories of DRMS identified. Firstly, DRMs detected in less than 1% of the reads sequenced from the amplified viral population for that position were discounted to account for potential errors due to the error rate of PCR and UDPS. The second category of mutations consisted of those with a prevalence greater than 1% and less than 20%, corresponding to those that cannot be determined using consensus sequencing. The final category consisted of those DRMs with prevalence greater than 20%, comprising mutations that can, in theory, be observed using traditional consensus sequence genotyping. Given that the number of reads that were sequenced at each timepoint in a patient were unlikely to be exactly the same, we implemented a statistical approach for prevalence calculations as opposed to using a rigid fixed cutoff. For every drug resistant mutation, the 95% confidence interval from the binomial sampling distribution was calculated for the observed frequency taking into account the number of reads sequenced at that timepoint. The upper 95% confidence interval was used as a cutoff for the allocation of DRMs into both the greater than 1% and less than 20% category and the greater than 20% category.Recent work has described HIV drug resistance mutations that are located within, or adjacent to, homopolymers in the HIV Pol gene . Given tPermission for the study was received from the National Health Sciences Research Committee, Malawi, and the Ethics Committee of the London School of Hygiene and Tropical Medicine, UK. Written informed consent for participation in the study was obtained from all participants.nd baseline sample from patient 42, the amplification was very weak compared to secondary amplifications from other patients. Similar results were obtained from 16th month from Patient 32 and 13th month sample from patient 45.Despite considerable effort, PCR amplification was not uniformly successful for all DNA samples. Secondary PCR was successful from ten primary PCRs for six of the 15 samples with all 20 secondary PCR reactions being successful in four of these  or due to low identity to the template sequence. Between 677 and 3007 reads per patient were excluded based upon the read length cutoff  of drug resistance mutations identified by deep sequencing were detected at prevalence levels less than 1% of the sequenced viral population Figure .UDPS identified an additional 32 DRMs (17 against current therapy) that had not been observed by consensus sequencing  to investigate the presence of low abundance drug resistance mutations present within the proviral DNA of individuals on ART. Previous studies have suggested that the mutational archive stored in proviral DNA remains unchanged during HAART  with higBulk sequencing approaches can only detect viral variants present in greater than 20% of the viral population -25 and wst line therapy. In particular K65R, strongly associated with the development of virologic failure in subtype C infected individuals [Resistant viruses that make up as little as 1% of the viral population within an individual have been suggested to be clinically important as they can expand rapidly under the selective pressure exerted by exposure to HAART -16. The ividuals -37, was ividuals  we did u3 after at least 12 months on ART) with all remaining patients showing satisfactory treatment response. While the CD4 counts of two of these individuals, patients 45 and 76, were sufficiently high to avoid being interpreted as treatment failures, their semi-borderline CD4 counts (258 and 268 CD4 T cells cells/mm3) coupled with the presence of multiple minor variant DRMs in their provirus could suggest that these patients may be at high risk for imminent failure. Nevertheless, the CD4 counts of all patients rose over the course of the study despite the detection of low abundance variants at early timepoints and all of the participants remained on 1st line therapy until their final sampling timepoint up to as much as 24 months after treatment initiation. Thus, it appears that the presence of low abundance DRMs in the provirus of these individuals has little effect on treatment outcome in the short-term. Previous work suggests that HIV rebounds from latently infected cells rather than as a result of continuing low-level replication [Despite the observation of low abundance drug resistant variants in all individuals, these viruses appear to have had minimal effect on measured treatment outcome. Due to the geographically isolated setting resulting in logistical difficulties viral loads are not routinely assessed and treatment success, to date, has been measured using WHO stages and CD4 counts. Thus, at the final sampling timepoint one (patient 2) is defined as having immune failure  The total number of sequenced reads obtained for each sampling time-point is shown as the total size of the bar representing each time-point. The number of reads mapped to the reference sequence and removed as a result of length and identity cutoffs are also shown. (B) The mean and range of read lengths observed for each sequenced sample are shown both before and after quality trimming. In all cases the mean and range of read lengths decreases significantly following trimming.454 sequence quality control. (Click here for fileObserved DRMs against ARVs not present in the patients’ current regimen at all sequenced sampling time-points for each patient. DRMs with observed prevalence less than 1% of the amplified and sequenced viral population are not included. For each prevalence point, the upper and lower 95% confidence intervals from the binomial sampling distribution are shown as error bars.Click here for file


Adherence to treatment is the key to the success of combination antiretroviral therapy (cART) for HIV infection. It is generally assumed that simplification of cART will lead to better adherence, higher treatment satisfaction, and quality of life (QoL), but randomized studies demonstrating such advantages of simplified regimens are scarce.®) were randomly assigned to either continue LPV/r/Combivir® or switch to simplified therapy with bid fixed-dose AZT/3TC/abacavir (Trizivir®). Both arms yielded similar antiviral efficacy after 48 weeks as reported previously [1]. Patients completed standardized questionnaires on adherence (Simplified Medication Adherence Questionnaire (SMAQ)), treatment satisfaction (HIVTSQ) and QoL (MOS-HIV) at randomization and at weeks 48, 72 and 96. Adherence data were analyzed using generalized estimating equations, and satisfaction and QoL using mixed linear models.Antiretroviral-naïve patients who achieved viral load <50 c/ml in two consecutive samples between 12–24 weeks after commencing induction therapy with bid lopinavir/ritonavir (LPV/r) and fixed-dose combination AZT/3TC (Combivir®-group (n=30) tended to skip fewer doses both during the preceding 7 days (p=0.055) and during the preceding weekend (p=0.09) than patients in the LPV/r-group (n=20). Moreover, patients in the Trizivir®-group found their regimen significantly more convenient (p=0.022) than patients in the LPV/r-group. However, patients in the LPV/r-group reported a better QoL than patients in the Trizivir®-group in the domains measuring cognitive functioning (p=0.003), energy/fatigue (p=0.046) and social functioning (p=0.074).Patients in the Trizivir® was perceived to be more convenient, and tended to result in improved adherence, but at the expense of a lower level of QoL. Our findings suggest that the choice for simplified regimens should be individualized and may involve a trade-off between convenience and QoL.In this randomized trial simplification of therapy to fixed-dose Trizivir


The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay.Between October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40–250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy .underestimated by TaqMan due to poor binding of the proprietary TaqMan primers.TaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40–250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36–283] copies/mL). In addition, in ∼2.4% of samples pVL was dramatically The replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure. However, the manufacture of the Amplicor v1.5 assay has been discontinued.Highly Active Antiretroviral Therapy (HAART) has resulted in dramatic improvements in HIV outcomes The Roche COBAS AmpliPrep/COBAS TaqMan v1 HIV-1 Test  Sudden unexpected HIV detectability leads to additional pVL testing, and medical visits for re-evaluation of adherence, concomitant medications, drug resistance and comorbidities, resulting in significant additional costs and stress for patients, their support network, and health care providers underestimate pVL in a minority of cases due to imperfect binding of the primers and/or probes.We undertook the present study to compare the results of the previous Amplicor v1.5 and the replacement TaqMan v1 assays in cases of low-level viremia. First, we evaluate the concordance of the two assays when low-level pVL (40–250 copies/mL) are reported by TaqMan v1. Second, we evaluate short-term virological and antiretroviral resistance outcomes in patients with low-level detectable TaqMan v1 pVL receiving stable HAART. Third, we show that the TaqMan v1 assay may also systematically The TaqMan v1 assay was introduced in British Columbia (BC), Canada in February 2008. After observing a high number of patients with newly detectable viral loads by the new assay and poor concordance of pVL results <100 copies/mL from both assays Subsequent short-term virological and resistance outcomes were followed in patients who started HAART at least 6 months prior and who did not change therapy regimens during follow-up (N = 279 patients). HIV RNA detectability by TaqMan v1 and Amplicor v1.5 were compared at the latest follow-up timepoint up until April 2010. Antiretroviral drug resistance genotyping was performed for all follow-up samples with viral loads >250 copies/mL by TaqMan v1, as described elsewhere underestimated viral loads by TaqMan v1 compared to Amplicor v1.5. For these patients, archived samples were re-tested with the Roche TaqMan version 2 (“TaqMan v2”) m2000 RealTime HIV-1 assay . Where sufficient plasma was available, HIV gag was also sequenced to assess potential TaqMan v1 primer incompatibility.Re-testing samples with Amplicor v1.5 revealed a subset of samples (N = 29) with systematically Ethical approval for this study was granted by the Providence Health Care/University of British Columbia Research Ethics Board (H10-01778). The requirement for individual consent was waived by the Research Ethics Board as the study involved no more than minimal risk to subjects.Between October 2009 and April 2010 a total of 1198 samples (from 950 patients) with pVL 40–250 copies/mL by TaqMan v1 were re-tested with the Amplicor v1.5 assay. This comprised approximately 10% of all tests performed in British Columbia during this period. In BC ∼97% of antiretroviral-treated patients are infected with subtype-B HIV-1 The concordance between the two viral load assays was poor at low viral load strata . The medDespite the low concordance between the two assays, there was a stepwise increase in detectability by Amplicor v1.5 with increasing pVL by TaqMan v1 . SamplesTo determine whether low-level TaqMan v1 pVL is a predictor of emergent virologic failure, we followed a subset of patients treated with stable HAART. A total of 279 of 950 (29%) eligible patients with low-level viremia by TaqMan v1 and remaining on a constant regimen were followed for a median of 3.2 months (IQR: 2.0–4.2 months). A median of one (IQR: 1–2) follow-up pVL test per patient was performed. Note that during this period, the Amplicor v1.5 pVL results were used to guide therapy decisions. Baseline patient characteristics are shown in Follow-up samples collected prior to September 2010 with viral load >250 copies/mL by TaqMan v1 were also tested for antiretroviral resistance using standard genotypic methods . A median of one genotype per patient (IQR 1–2) was obtained over a median of 4.4 (IQR: 0.9–6.9) months. New resistance mutations in protease/reverse-transcriptase (PR-RT) were observed in eight patients (12%) compared to pre-therapy sequences, suggesting that, in general, low-level pVL by TaqMan v1 may not be indicative of emerging resistant variants. It is notable that only three patients with newly-detected resistance mutations had undetectable viral loads by Amplicor v1.5 at their baseline visit.10lower than by Amplicor v1.5  (N = 15) if sample volume permitted. Of 18 samples with undetectable pVL by TaqMan v1, five (28%) were detectable by TaqMan v2 . In 13 samples with low but detectable pVL by TaqMan v1, TaqMan v2 results were a median of 1.1 log10 copies/mL higher (IQR: 0.87–1.34 log10 copies/mL), consistent with a systematic underestimation of the pVL levels by TaqMan v1 in a specific subset of patients  had pVL levels by TaqMan v1 >0.5 logcor v1.5 . For thepatients . This obgag assay primers. To explore this issue further, we sequenced HIV-1 gag in samples exhibiting low TaqMan v1, but high Amplicor v1.5 pVL. As the sequences of the TaqMan v1 assay primers are unpublished, gag sequences were sent to Roche  for interpretation. Of the nine gag sequences shared with Roche, seven (78%) apparently had mutations potentially incompatible with the TaqMan v1 primers: two (22%) and five (56%) showed incompatibilities with the “upstream” and “downstream” primers, respectively. Since the TaqMan v1 primer and probe sequences remain proprietary, we cannot report the specific mutations involved.We hypothesized that the underestimation of pVL results by the TaqMan v1 assay was due to inefficient binding of the HIV 10 copies/mL as compared to TaqMan v2.New resistance mutations were detected in one patient sample out of seven with longitudinal PR-RT sequences available. In this sample, viral load was underestimated by 2.5 logThe introduction of a new HIV viral load assay (“TaqMan v1”) in February 2008 resulted in an unexpected increase in the prevalence of detectable HIV loads in British Columbia, Canada The clinical consequences of low-level viremia were unclear even before the TaqMan v1 assay was instituted. Some studies have indicated that intermittent low-level viremia is associated with a higher risk of virological failure or drug resistance New resistance mutations were observed in only 12% of tested patients with pVL >250 copies/mL by TaqMan v1 over the course of follow-up. However, it should be noted that uncertainty and stochastic variation in testing samples with low viremia could result in resistance mutations being missed or detected by chance in some cases. Nevertheless, these results suggest that low but detectable pVL <250 copies/mL by TaqMan v1 do not correlate with detectability by Amplicor v1.5, and are not indicative of impending short-term virological failure or drug resistance. Rather, we estimate that on average a pVL value of 150 copies/mL by TaqMan v1 is approximately equivalent to a pVL value of 50 copies/mL by Amplicor v1.5. However, it is important to note that the reverse may not be true – an Amplicor v1.5 pVL value of 50 copies/mL may not be equivalent to a TaqMan v1 value of 150 copies/mL as this scenario was not evaluated. While this is not sufficient to entirely repudiate the validity of the TaqMan v1 assay, there exists no positive evidence to support that TaqMan v1 pVL levels of 40–250 copies/mL are clinically relevant. Given the change in assays, the definition of “virological failure” should be re-validated against clinically meaningful parameters; the British Columbia HIV treatment guidelines have revised the definition of treatment failure as two consecutive pVL measurements >250 copies/mL by TaqMan 10 copies/mL as confirmed by re-testing by Amplicor v1.5, TaqMan v2, and/or the Abbott RealTime assays . Due to the limited availability of Amplicor v1.5 kits in Canada following its discontinuation only a one-way analysis could be performed.This study also confirmed an unrelated but potentially more serious shortcoming of the TaqMan v1 assay. The TaqMan v1 primers were unable to efficiently amplify their targets in a subset of ∼2.4% of samples tested leading to the systematic underestimation of viral loads by up to 2.5 logsee also . When HI6 copies/mL may have obfuscated clinically meaningful differences between the two assays. Therefore, we recommend that in the future new or “upgraded” pVL assays be validated with a particular focus on the clinically-relevant range before commercial approval.The discrepancies between Amplicor v1.5 and TaqMan v1 pVL at low-level pVL may have arisen due to the methods used to validate the TaqMan v1 assay. Validation was performed by testing TaqMan v1 results against the Amplicor v1.5 assay over a very wide range of pVL. No special attention was placed on pVL in the clinically-relevant range (<1000 copies/mL), where the test results are directly used to guide therapeutic decisions An updated version of the TaqMan v1 assay (version 2.0) is now available. While the assay upgrade appears to have corrected the systematic underestimation of pVL occasionally seen in TaqMan v1 Figure S1Bland-Altman plot of results from parallel testing of viral load samples by TaqMan v1 and Amplicor v1.5. Visual inspection of the plot suggests a bias towards higher pVL results obtained by TaqMan v1 at low pVL (40–250 copies/mL). When attempting to interpret these results it is important to note that 1) Amplicor v1.5 pVL values below the limit of quantification (<50 copies/mL) were coded as 49 copies/mL, and 2) the plotted results are restricted to samples with TaqMan v1 results 40–250 copies/mL.(TIF)Click here for additional data file.


Environmental enteropathy (EE) is an asymptomatic abnormality of small bowel structure and function, which may underlie vaccine inefficacy in the developing world. HIV infection co-exists in many of these populations. There is currently no effective treatment. We conducted a secondary analysis of a randomised controlled trial of high dose multiple micronutrient (MM) supplementation on small bowel architecture in EE in participants with or without HIV infection.In a double-blind parallel-group trial of the effect of MM on innate immune responses to oral vaccines, consenting Zambian adults were randomised to receive 6 weeks of 24 micronutrients as a daily capsule or placebo. HIV status was established after randomisation. Proximal jejunal biopsies were obtained after the supplementation period. Villous height, crypt depth, villous width, villous perimeter per 100 μm muscularis mucosa , and villous cross sectional area per 100 μm muscularis mucosa (a measure of villous compartment volume) were measured in orientated biopsy sections using semi-automated image analysis. Analysis was by intention to treat.2/100 μm; 95% CI of difference 818–11130 μm2/100 μm; P = 0.03), and median villous perimeter was 29.7% greater . There was no significant effect on crypt depth or villous width. No effect was observed in HIV positive patients. There were no adverse events attributable to MM.18 patients received MM and 20 placebo. 6/18 MM and 9/20 placebo patients had HIV. In HIV negative patients given MM compared to placebo, mean villous height was 24.0% greater , mean villous area was 27.6% greater (27623 v. 21650 μmMM improved small bowel villous height and absorptive area, but not crypt depth, in adults with EE without HIV. Nutritional intervention may therefore selectively influence villous compartment remodelling. In this small study, there was a clear difference in response depending on HIV status, suggesting that EE with superimposed HIV enteropathy may be a distinct pathophysiological condition. Salmonella Typhi/Paratyphi provide high rates of effective immunity in Western populations, their efficacy in low income countries is markedly reduced [Environmental enteropathy (EE) is an asymptomatic disorder which is highly prevalent throughout the developing world. It is thought to be the result of recurrent exposure to gastrointestinal pathogens , but cou reduced  which co reduced ,6. From  reduced .HIV co-exists with EE in many populations, particularly in sub-Saharan Africa. We have observed that this results in an asymptomatic HIV-associated enteropathy that has subtle histological and functional differences from EE in HIV negative patients. For example compared to HIV negative patients, EE with HIV is characterised by more marked crypt hypertrophy and intestinal permeability. Villous height is also correlated with CD4 count ,6. This There is no established therapy to reverse the changes of EE. Antibiotics , probiotThere are few data on MM interventions for EE, but in our own previously published trials, long-term lower-dose MM supplementation had a modest impact on diarrhoea and nutritional outcomes, and some effect on antimicrobial peptide expression ,15. SimiWe have also shown that lower micronutrient doses did not reliably increase the concentrations of the micronutrients in blood . While tThis was a secondary study of endoscopic small bowel biopsies obtained during the course of a single centre, randomised, double-blind, placebo-controlled, parallel-group trial investigating the effect of high dose MM supplementation on innate immune effects of oral vaccines. This was carried out in adult volunteers living in a poor community in Lusaka, Zambia between June and October 2009, where we have demonstrated that EE is virtually universal [Volunteers were recruited from among residents of Misisi suburb, where we have carried out several previous studies of intestinal inflammation. Age between 18 and 60 years was the only inclusion criterion. The only absolute exclusion criterion was helminth infection (diagnosed by stool microscopy at screening). Participants who were pregnant, lactating, had had vaccination within 6 months, had taken antibiotics or non-steroidal anti-inflammatory drugs within two weeks, or who had had diarrhoea within one month were not included in any part of the study until their temporary exclusion criterion no longer applied. Informed consent followed a three stage process: door-to-door notification, focus group discussions, and individual counselling leading to fully informed written consent.The composition of the multiple micronutrient supplement  is shown in Table Participants were provided with a six week supply of once-daily trial medication and given instructions on how to take it days 1 – 42). After the supplementation period they underwent enteroscopy with proximal jejunal biopsy under conscious sedation (performed by PK) on day 43. Patients were then vaccinated with a full course of Ty21a oral typhoid vaccine , and were re-biopsied 14 days later  using a Zeiss KS400 v.33.0 image analysis system . Maximal villous height (VH), maximal villous width perpendicular to the VH axis (VW), and crypt depth (CD) were measured, along with villous perimeter (VP) as a measure of epithelial surface area, and villous cross sectional area (VA) as a measure of villous compartment volume  in villous height .Based on the morphometry results from our previous study of enteropathy in the same patient population , we estiAlthough VH, CD and VA were normally distributed, VP and VW were not (Shapiro-Wilk test). Two-tailed t- or paired t-tests were used for normally distributed data. Wilcoxon signed rank (for paired observations) or Kruskal-Wallis (for independent observations) tests were used for non-parametric data. Spearman correlation coefficients were used where required. Where both pre- and post-vaccination data were available, analysis was performed using either only pre- or only post-vaccination biopsy measurements. The most conservative results are presented here.Statistics were calculated using SPSS v.21  and Stata v.12 .This study was a secondary analysis. The primary comparison for this study was the difference in morphometric variables after 6 weeks of either high-dose MM supplementation or placebo depending on HIV status.Approval was obtained from the University of Zambia Biomedical Research Ethics Committee (007-10-07). The trial was registered as ISRCTN68751738.P = 0.52, Fisher's exact test). Baseline characteristics in MM and placebo groups were comparable , mean VA per 100 μm of mucosa was 27.6% greater , and median VP per 100 μm of mucosa was 29.7% greater  . There was no significant difference in V:C ratio between treatment groups in HIV positive patients.In HIV negative patients given MM compared to placebo, mean VH was 24.0% greater  is more of a pharmacological approach, whereas combined nutrient supplements could be thought of as restoration of nutritional health. In the supplement used in this trial, most micronutrients were at doses of at least twice the recommended nutrient intake, as previous work demonstrated that lower doses did not reliably increase the concentrations of the micronutrients in blood , possiblAlthough pre-supplementation biopsies were not obtained, the changes observed are most easily explained by the supplement given, rather than by unidentified differeces in the treatment arms at baseline. Firstly, EE is virtually universal in this population ,3. SeconS. Typhi for the Peyer patches of the distal ileum. Finally, histological analysis of enteric fever in humans has confirmed that proximal small bowel morphometry is unaffected in the course of illness [Similarly, we found no effect of vaccination on morphometry. We therefore included these measurements so that we were able to analyse an almost complete data set. Again, a small effect of vaccination on morphometry may have been masked by the small sample size, but we think this is unlikely. Firstly, our results were significant with datasets using either pre- or post-vaccination data. Secondly, the lack of vaccine effect on upper small intestinal morphometry is consistent with the predilection of  illness .Approximately 2 billion people in the developing world live in conditions where safe water is difficult to obtain, food is neither secure nor diverse, and sanitation is suboptimal. These populations have a high prevalence of environmental enteropathy. Our data suggest that high dose MM may provide an effective and affordable treatment option to reverse the histological abnormalities observed in EE in HIV negative patients, which will hopefully result in enhanced intestinal absorption, permeability, and perhaps even vaccine responses. Larger randomised controlled trials with physiological and clinical endpoints are therefore urgently needed.ART: Anti-retroviral therapy; CD: Crypt depth; EE: Environmental enteropathy; HIV: Human immunodeficiency virus; MM: Multiple micronutrient; VA: Villous area; VH: Villous height; VP: Villous perimeter.The authors declare that they have no competing interest.JL-A conducted the research, analysed data, performed statistical analysis, and wrote the paper. SG wrote the morphometry program, provided materials, and revised the paper. MS designed and conducted the research and revised the paper. RS conducted the research and revised the paper. RB conducted the research and revised the paper. PK designed and conducted the research, provided materials, analysed data, performed statistical analysis, wrote the paper, and has primary responsibility for final content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/14/15/prepubMacro used for morphometric analysis.Click here for fileComparison of significant results in HIV negative patients, using either pre- or post-vaccination datasets. Comparison of significant results in HIV negative patients, using a dataset including either pre- or post-vaccination data in 14/38 patients where data was available for both time points. Results show differences and P values comparing HIV negative patients given MM versus placebo. There remained no significant differences in crypt depth or villous width, or in any variables in HIV positive patients. MM, multiple micronutrient supplementation; VA, villous area; VH, villous height; VP, villous perimeter.Click here for fileAdverse events during period of supplementation. aP values given for Fisher's exact test. bSevere diarrhoea defined as diarrhoea resulting in time off work or usual activities, use of antibiotics, use of oral rehydration solution, or healthcare assessment . MM, multiple micronutrient supplementation.Click here for fileComparison of morphometric studies of EE and/or HIV enteropathy in Zambian adults. *Significant difference (P <0.05) between patient groups. Mucosal thickness in the Kelly study was not stated and has been calculated by adding mean VH and CD. VH, CD, VW and MT expressed in μm ; VA and VP expressed in μm/100 μm mucosal length . CD, crypt depth; EE, environmental enteropathy; IQR, interquartile range; MM, multiple micronutrient supplementation; MT, mucosal thickness; VA, villous area; VH, villous height; VP, villous perimeter; VW, villous width.Click here for file


Multi-nucleos(t)ide resistance (MNR) mutations including Q151M, K65R mutations, and insertion at codon 69 of HIV-1 reverse transcriptase coding region may confer resistance to all molecules of nucleos(t)ide reverse transcriptase inhibitors (NRTI). The presence of these mutations is an emerging problem compromising non-nucleoside reverse transcriptase inhibitors and protease inhibitors-based therapies. Furthermore, factors associated with selection of these mutations are still not well defined. The current study aimed to evaluate the frequency and to characterize factors associated with the occurrence of multi-nucleos(t)ide resistance mutations among HIV-1 infected patients failing recommended first-line antiretroviral regimens in Cambodia.This is a retrospective analysis of HIV-1 drug resistance genotyping of 520 HIV-1 infected patients in virological failure  while on first-line antiretroviral therapy in Cambodia with at least one reverse transcriptase inhibitor resistance associated mutation. Among these 520 patients, a total of 66 subjects  presented ≥1 MNR mutation, including Q151M, K65R, and Insert69 for 59 (11.3%), 29 (5.6%), and 2 (0.4%) patients, respectively. In multivariate analysis, both Q151M (p = 0.039) and K65R (p = 0.029) mutations were independently associated with current stavudine- compared to zidovudine-use.Such selection of mutations by stavudine drastically limits the choice of antiretroviral molecules available for second-line therapy in resource-limited settings. This finding supports the World Health Organization’s recommendation for stavudine phase-out. Q151M, K65R substitutions, and insertions at codon 69 (Insert69) in the reverse transcriptase encoding region of HIV-1 genome confer resistance to a large range of nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and are thus called multi-nucleos(t)ide resistance (MNR) mutations. Q151M confers resistance to all NRTIs, except tenofovir (TDF) + T-cell count, high HIV RNA viral load (VL) and experience of more than two ARV regimens before resistance genotyping testing Factors associated with selection of these mutations are still not well defined. d4T/ddI dual therapy has been associated with the occurrence of Q151M and K65R The current study was a retrospective and anonymous analysis of data collected in the context of routine care of HIV infected patients. Data was stored in a database of HIV/Hepatitis Laboratory, Institut Pasteur in Cambodia situated in Phnom Penh . This kind of analysis complies with the Cambodian ethical guidelines for exemption from ethical approval requirement We conducted a retrospective and observational study using data routinely collected between December 2004 and January 2011 from 9018 HIV-1 infected patients, on first-line ARV regimen, and in the context of routine care. All samples of these 9018 individuals were referred from many parts of Cambodia .+ T-cell count every six months whereas HIV-1 RNA VL and drug resistance genotyping were carried out to confirm clinical and/or immunological failure In agreement with the World Health Organization (WHO) recommendation, the Cambodian national guidelines have recommended d4T, 3TC, and nevirapine (NVP) as the standard first-line ARV regimen and zidovudine (AZT), TDF, ABC, efavirenz (EFV), and/or protease inhibitors in case of drug side-effect or interaction. Laboratory monitoring included CD4All specimens were assessed for HIV-1 RNA VL testing and drug resistance genotyping in HIV/Hepatitis Laboratory of Institute Pasteur in Cambodia which was (at the time of the study) centralizing all virological tests for HAART monitoring.HIV-1 RNA VL was performed on stored (−80°C) plasma specimens using the G2 Generic HIV-1 VL ANRS kit  Of 9018 patients referred to our laboratory, 8304 (92.1%) had undetectable HIV-1 RNA VL. This rate represented a good virological outcome and was consistent with studies previously conducted in Cambodia amongst HIV-1 infected patients after one to three years of first-line ARV regimen 10 copies/mL [IQR: 4.0-5.1]. Three hundred and twenty eight patients (63%) (212 children and 116 adults) were receiving d4T-containing regimens and the remaining was under an AZT-containing regimen.Their median age was 28 years [Inter Quartile Range (IQR): 9-38] at the time of sampling, and 233 (44.8%) patients were children (≤15 years) followed-up at the National Pediatric Hospital . Median According to the ANRS algorithm (version 19) 10 copies/mL), current first-line regimen (d4T- or AZT-containing) and duration (≤ or >30 months) at sampling time.To identify factors associated with the presence of Q151M and K65R mutations, univariate and multivariate logistic regression analysis were performed using the following variables: gender, age group (≤ or >15 years), HIV-1 RNA VL level  than in those on AZT-containing regimens (6.3%) (p = 0.005). A high HIV-1 RNA level was also significantly associated with Q151M (p<0.0001). Q151M mutation tended to be associated with age ≤15 years (p = 0.069) and duration >30 months under current regimen (p = 0.071). In multivariate analysis, independent risk factors for Q151M were d4T-containing regimen (Odds ratio (OR) [95% confidence interval (CI)]: 2.21 [1.05−4.65]), high HIV-1 RNA level (OR [95% CI]: 4.02 [2.13−7.59]), and duration >30 months under current first-line regimen (OR [95% CI]: 2.11 [1.18−3.76]) .The occurrence of K65R was significantly associated with d4T-containing regimens (p = 0.017) and high HIV-1 RNA level (p = 0.003), while there was a trend with duration >30 months under current first-line regimen (p = 0.054). In multivariate analysis, independent risk factors for K65R were d4T-containing regimen (OR [95% CI]: 3.02 [1.12−8.13]), high HIV-RNA level (OR [95% CI]: 3.97 [1.64−9.61]) and duration >30 months under current first-line regimen (OR [95% CI]: 2.6 [1.16−5.83]) .2 test, p<0.001). This result is consistent with previous study conducted in Italy In multivariate analysis, besides higher HIV-1 RNA VL level, probably associated with longer duration of failure, we found that d4T-use remained independently associated with the presence of MNR mutations (either Q151M or K65R). Finally, the Q151M and K65R mutations were positively associated to each other. Indeed, K65R was detected in 12/59 (20.3%) patients harboring Q151M compared to 3.9% of patients without Q151M  at the time of genotyping. As reported recently (2012) by Phan et al., in a follow-up conducted between 2003 and 2010 in the Sihanouk-Hospital-Center-of-Hope in Phnom Penh, Cambodia, d4T was discontinued and switched to AZT in around 48% of patients due to d4T-toxicity per se is associated with the emergence of both Q151M and K65R MNR mutations which confer resistance to a large range of NRTIs. It is important to note that the effect of d4T is observed even after adjusting for HIV-1 RNA VL. Selection of such mutations is a critical issue in resources-limited settings where NRTI molecules available for second-line regimen are still limited. Although the primary reasons for the 2010 WHO’s recommendation to reduce or abandon the use of d4T as part of first-line ARV regimen in resource-limited settings were related to excessive toxicity In conclusion, our data suggests that the administration of d4T Five hundred and twenty nucleotide sequence of CRF01_AE HIV-1 reverse transcriptase coding region included in the current analysis have been submitted to Genkbank under the accession numbers KF292310 to KF292829.


Stavudine continues to be used in antiretroviral treatment (ART) regimens inmany resource-limited settings. The use of zidovudine instead of stavudinein higher-risk patients to reduce the likelihood of lactic acidosis andhyperlactatemia (LAHL) has not been examined.Antiretroviral-naïve, HIV-infected adults initiating ART between 2004and 2007 were divided into cohorts of those initiated on stavudine- orzidovudine-containing therapy. We evaluated stavudine or zidovudine use,age, sex, body mass index (BMI), baseline CD4 cell count, creatinine,hemoglobin, alanine aminotransferase, and albumin as predictors of time toLAHL with Cox Proportional Hazards (PH) regression models.Among 2062 patients contributing 2747 patient years (PY), the combinedincidence of LAHL was 3.2/100 PY in those initiating stavudine- and 0.34/100PY in those initiating zidovudine-containing ART . In multivariable Cox PH analysis, stavudine exposure , female sex , higher BMI ,higher creatinine , higher albumin , and lower CD4 cell count  at baseline were associated with higher LAHLrates. Among participants who started on stavudine, switching to zidovudinewas associated with lower LAHL rates . Subgroup analysis limited to women with higher BMI≥25kg/m2 initiated on stavudine also showed that switch to zidovudine wasprotective when controlling for other risk factors .Stavudine exposure, female sex, and higher BMI are strong, independentpredictors for developing LAHL. Patients with risk factors for lacticacidosis have less LAHL while on zidovudine- rather thanstavudine-containing ART. Switching patients from stavudine to zidovudine isprotective. Countries continuing to use stavudine should avoid this drug inwomen and patients with higher BMI. Lactic acidosis is a potentially fatal side effect of nucleoside analog reversetranscriptase inhibitors (NRTIs) Of the NRTIs, the dideoxynucleosides (stavudine and didanosine) confer the highestrisk of lactic acidosis Observational studies suggest that specific risk factors associated with thedevelopment of hyperlactatemia include female sex 2, in August 2005 the HIV Clinic at McCord Hospital inDurban, South Africa substituted zidovudine for stavudine in initial ART forpatients with these two risk factors Until April 2010, first-line therapy in South Africa included stavudine, lamivudine,and either efavirenz or nevirapine. Based on observational findings from asite-specific study that identified a high incidence of lactic acidosis in womenwith BMI≥28 kg/mTo evaluate the impact of risk factor-guided selection of initial therapy, wecompared the combined incidence of lactic acidosis and hyperlactatemia amongtreatment-naive patients initiating stavudine-containing therapy with those startingzidovudine-containing therapy. We hypothesized that risk-factor-guided ART(initiating women with BMI≥28 kg/m2 on zidovudine rather than stavudine) would beassociated with decreased incidence of lactic acidosis and hyperlactatemia. We alsoassessed predictors of lactic acidosis and hyperlactatemia.Ethics approvals were obtained from the McCord Hospital Medical Ethics ResearchCommittee and from the Partners Healthcare Institutional Review Board . Given the nature of the study (retrospective chart review), the requirementfor informed consent was waived by the ethics committees.Patient data were collected from the outpatient HIV clinic at McCord Hospital inDurban, South Africa which has initiated over 8000 patients on ART. During thestudy period, initial ART included two NRTIs and one NNRTI: stavudine  or zidovudine pluslamivudine and either efavirenz or nevirapine.The study population included antiretroviral (ARV)-naïve, HIV-infectedadults (age ≥18 years) with baseline laboratory data and at least onefollow-up visit after ART initiation. Two retrospective cohorts were identified.The first cohort included patients who initiated stavudine-containing therapybetween July 2004 and March 2007. The second cohort included patients whoinitiated zidovudine-containing ART between July 2004 and March 2007. Bothcohorts included patients who initiated ART between August 2005 and March 2007when the clinic made women with BMI≥28 kg/m2 eligible for initiation ofzidovudine -containing therapy or for regimen switch from stavudine tozidovudine.The primary outcome was event-free survival defined as the time from treatmentinitiation to development of lactic acidosis (symptomatic or asymptomatic) orhyperlactatemia (symptomatic or asymptomatic) . Lactic Blood was drawn for lactate levels without use of a tourniquet and specimens weretransported on ice and processed within four hours . A handheld lactate detection device, a reliable proxyfor serum samples, was introduced in 2006 (Accutrend model #3012522) Outcomes were classified from a review of the medical records of patientsinitiating ART during the study period. This review was facilitated by therequirement that clinicians record the reason for any change or discontinuationin ARV regimen from an electronic pull down menu. For patients who had a regimenchange noted in the electronic record, paper charts were reviewed for thefollowing: 1) documentation of a regimen change due to lactic acidosis; 2)documentation of a regimen change and signs or symptoms that could be consistentwith lactic acidosis or hyperlactatemia ; 3) documentation of a regimen change without specific reason listed;4) death. Serum lactate test results for all study patients were reviewed. Alactate value above 3 mmol/L prompted review of the medical record for symptomsof hyperlactatemia, all available lactate values, other possible causes forsymptoms or elevated lactate levels, and clinical outcome. Data were abstractedusing standardized abstraction forms . For a subset of patients(n = 20), two physicians carried out the abstractions with100% agreement on outcome classification . Cases with unclearoutcomes were adjudicated by a senior clinician (RG).We also identified patients for whom clinicians had changed ART due to peripheralneuropathy, lipodystrophy, high BMI, and drug resistance, as indicated in theelectronic medical record. We identified patients with regimen switch forclinical suspicion of LA or HL but who did not meet criteria. These subjectswere not censored at change in regimen but followed out to a total two years offollow-up from treatment initiation. In addition, we identified subjects whochanged clinic site, stopped ART, died or were lost to follow up.2) for all subjectsin whom height and baseline weight were available. Sex, date of birth andbaseline  CD4 count,creatinine, hemoglobin, alanine aminotransferase, and albumin were extractedfrom the electronic record or the paper chart. All specimens were processedusing standardized methods at laboratories in Durban.Covariates were obtained from paper chart abstractions and included weight attreatment initiation (within 3 months) and height. Weights obtained duringpregnancy were excluded. BMI was calculated  defined as the time fromtreatment initiation up to development of lactic acidosis or hyperlactatemia.Patients were also censored for loss to follow-up, change in clinic site,termination of treatment, death, or at study end. All others were followed fortwo years or until the primary outcome. Time on stavudine and zidovudine wascalculated from start and stop dates entered by clinicians in the medicalrecord.We calculated crude incidence rates for the combined primary outcome (LAHL), thecombined incidence of peripheral neuropathy and lipodystrophy, death, andloss-to-follow-up. Confidence intervals for event rates based on initial therapywith stavudine or zidovudine were estimated using methods for exact binomialconfidence intervals and compared using Chi-square tests Two-thousand-sixty-two patients contributing 2747 person years of follow-up wereincluded in the study. The median age was 34.7 years  and60% were women. Eighty-nine percent initiated therapy with astavudine-containing regimen. One-hundred sixty one (77%) of those whowere initiated on a zidovudine-containing regimen were started because of higherBMI or other perceived lactic acidosis risk factors. The remaining patients wereinitiated on zidovudine because of pre-existing lipodystrophy (<1%),peripheral neuropathy (<1%), pregnancy (10%), or unknown reason(10%).Median CD4 count at entry was 80 cells/mm3 (IQR 29–142). Median BMI forsubjects with complete data  was 22 kg/m2 . Compared with thoseinitiated on a stavudine-containing regimen, patients started on zidovudine wereolder, more likely to be female, had a higher BMI, higher CD4 cell count, higheralbumin and higher hemoglobin. Other characteristics are described in In intention to treat analysis, combined incidence of LAHL was 3.2/100 PY in thestavudine- and 0.34/100 PY in the zidovudine-initiated group . There were 36lactic acidosis and 43 hyperlactatemia events in the stavudine group. Incontrast, there was 1 lactic acidosis event in the zidovudine group: thisoccurred in a woman who initiated zidovudine-based therapy because of high BMI(31 kg/m2); one year later, she was switched to stavudine because of anemia;after eight months on stavudine-containing ART, she was diagnosed with lacticacidosis. Mortality due to causes other than LAHL was 8.3% and2.8% in stavudine- and zidovudine-initiated patients, respectively. The combined incidence of physician-reportedperipheral neuropathy and lipodystrophy was 16.8/100 PY in stavudine- and0.34/100 PY in zidovudine-initiated groups . Loss to follow-up was equivalentbetween the two groups . In univariate Cox proportional hazards analysis, stavudine in the initialtreatment regimen, female sex, higher BMI, and higher baseline albumin were eachassociated with increased risk of LAHL . The Kapa priori knowledge suggested an association withlactic acidosis In multivariable Cox PH regression to assess predictors of event-free survival,hemoglobin and ALT were removed but age and CD4 cell count were included becauseprior data and Women with BMI greater than or equal to 25 kg/m2 comprised 326 patients with 434years of follow-up. The 194 women initiated on stavudine were younger; withlower BMI, baseline CD4 cell count, creatinine, albumin, and hemoglobin comparedwith 132 women initiated on zidovudine . Obese wOf the 194 women with higher BMI who initiated stavudine-inclusive therapy, 137were switched to zidovudine for reasons other than LAHL. Baselinecharacteristics were not significantly different from women with higher BMI initiated onstavudine-treatment who did not switch treatment arms. Women were switched forhigh BMI ; lipodystrophy, peripheral neuropathy, or these pluselevated BMI ; lab values and/or symptoms suggestive ofhyperlactatemia that did not meet criteria for LAHL ; and theremainder were switched for anemia, pregnancy, rash or other reasons. Theseparticipants subsequently contributed an additional 131.7 woman-years offollow-up (mean 1.1 years ±0.5) during which there were 5 LAHL events(3.8/100 woman years). All but one event occurred within 2–8 weeks ofswitching off stavudine after an average of 0.6±0.4 years on stavudine,suggesting that the recent and cumulative stavudine exposure contributed to thetoxicity. When controlling for other LAHL risk factors, switch to zidovudineconferred 80% lower hazards of LAHL for this subgroup . The remainder ofwomen in this subgroup of obese women, initiated on stavudine and switched tozidovudine, included two who subsequently had anemia and two who died; the restwere followed until the end of the study, change in service provider, or amaximum of two years of follow-up without adverse events.In our study of 2062 HIV-positive patients who initiated ART, stavudine use confers afourteen-fold increased risk of developing hyperlactatemia or lactic acidosis whencontrolling for other risk factors . Otherrisk factors for the primary outcome of LAHL were female sex, higher baseline BMI,higher baseline creatinine or albumin, and lower initial CD4 cell count. Forpatients who started a stavudine-containing regimen, switching to zidovudine wasassociated with 85% lower hazards of developing LAHL . For the high-risk subgroup of women with BMI≥25 kg/m2 whoinitiated therapy on stavudine-containing ART, switch to zidovudine was alsoprotective when controlling for other risk factors .2 or 24 to 31kg/m2), we observed a three-fold increase in the LAHL rate. For the fulldataset, the effect was only significant in the first year of follow-up which mayreflect increased risk earlier in treatment or insufficient power to detect anassociation after the first year. During the study, patients with weight >60 kgreceived 80 mg of stavudine daily, which has been linked to worse mitochondrialtoxicities compared to use of 60 mg Our study adds to the literature by demonstrating that female sex is a strongindependent risk factor for developing LAHL Higher creatinine was associated with increased hazards of LAHL, about 25% per1 mg/dL unit increase in creatinine. This risk factor has not been reported in priorunivariate analyses and has not been included in studies that control for other riskfactors, but is not unexpected given the kidney's role in lactate metabolismThe Lactic Acidosis International Study group showed an association of older age (age>40 years) with the development of LAHL. This was not seen in our cohort nor inother studies based in Southern Africa For the subgroup of women with higher BMI, stavudine use, when controlling for otherrisk factors, remained associated with a significant increase in risk of LAHL.Switching these women to zidovudine conferred an 80% reduction in hazards ofLAHL. These data suggest that higher-risk individuals should be switched offstavudine-based therapy in order to reduce adverse events.There are several limitations to this study. The two treatment groups were quitedifferent as demonstrated in As of April 2010, first-line ART in South Africa includes tenofovir, lamivudine, andeither efavirenz or nevirapine


To determine the frequency and patterns of acquired antiretroviral drug resistance in a rural primary health care programme in South Africa.Cross-sectional study nested within HIV treatment programme.Adult (≥18 years) HIV-infected individuals initially treated with a first-line stavudine- or zidovudine-based antiretroviral therapy (ART) regimen and with evidence of virological failure  were enrolled from 17 rural primary health care clinics. Genotypic resistance testing was performed using the in-house SATuRN/Life Technologies system. Sequences were analysed and genotypic susceptibility scores (GSS) for standard second-line regimens were calculated using the Stanford HIVDB 6.0.5 algorithms.A total of 222 adults were successfully genotyped for HIV drug resistance between December 2010 and March 2012. The most common regimens at time of genotype were stavudine, lamivudine and efavirenz (51%); and stavudine, lamivudine and nevirapine (24%). Median duration of ART was 42 months (interquartile range (IQR) 32–53) and median duration of antiretroviral failure was 27 months (IQR 17–40). One hundred and ninety one (86%) had at least one drug resistance mutation. For 34 individuals (15%), the GSS for the standard second-line regimen was <2, suggesting a significantly compromised regimen. In univariate analysis, individuals with a prior nucleoside reverse-transcriptase inhibitor (NRTI) substitution were more likely to have a GSS <2 than those on the same NRTIs throughout (odds ratio (OR) 5.70, 95% confidence interval (CI) 2.60–12.49).There are high levels of drug resistance in adults with failure of first-line antiretroviral therapy in this rural primary health care programme. Standard second-line regimens could potentially have had reduced efficacy in about one in seven adults involved. South Africa has the largest HIV burden in the world, with an estimated 5.6 million people living with HIV South Africa follows the public health approach to ART delivery with standardised drug regimens and simplified decision-making, with the inclusion of routine viral load monitoring for the detection of treatment failure The majority of studies from South Africa focused on acquired drug resistance  have been conducted in urban, hospital-based treatment programmes The study was approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (ref. BF052/10) and the Health Research Committee of the KwaZulu-Natal Department of Health (ref. HRKM 176/10). Written informed consent was obtained from all the study participants.The Hlabisa HIV Treatment and Care Programme is a decentralised, primary health care (PHC) programme in the predominantly rural Hlabisa health sub-district in northern KwaZulu-Natal. Details of the programme have been reported previously The programme adheres to the national antiretroviral treatment guidelines This was a cross-sectional study enrolling HIV-infected adults with virological failure on first-line antiretroviral therapy. Inclusion criteria were: adult (≥18 years); initiated on first-line d4T- or AZT-based regimen; received treatment for at least 12 months; and evidence of virological failure . Exclusion criteria were: prior use of nucleoside reverse transcriptase inhibitor (NRTI) monotherapy or dual therapy (not including regimens for the prevention of mother-to-child transmission (pMTCT)), prior protease inhibitor (PI) use, and initiated on first-line TDF-based regimen. The decision to include only those who commenced treatment with d4T- or AZT-based regimens, and exclude those who initiated TDF-based regimens, was primarily to allow formal comparison with other studies in the region.Individuals were recruited at all 17 PHC clinics between December 2010 and March 2012. There were two possible routes for enrolment: i) Routine clinic - adults with virological failure (latest VL >1000 copies/ml) were identified by clinic staff during routine visits and referred to the physician for review; ii) virological failure camp – eligible adults were proactively identified through the programme’s operational database, were contacted by programme staff, and were booked for physician review on a specific day at their regular clinic. In both models, the physician performed a clinical evaluation and obtained written informed consent for the study. A 5 ml EDTA whole blood sample for HIV drug resistance genotyping was collected during the clinical evaluation. Basic clinical and demographic data were collected on a standardised clinical form in parallel to the records in the Africa Centre’s ART Evaluation and Monitoring System (ARTemis), an operational database holding treatment and laboratory monitoring information. The clinical information was entered in anonymised form into a relational database, the SATuRN REGA database Specimens were transported daily from the clinics to the Africa Centre and the same day to the Africa Centre laboratory in Durban (200 km from site). At the laboratory, plasma was aliquoted and stored at −80°C until sequenced. Samples were sequenced within a week of collection. HIV RNA was extracted using the QIAMP RNA extraction kit (Invitrogen) modified to extract RNA from 200 µl of plasma instead of 140 µl, to concentrate the viral RNA for better amplification rates.st strand synthesis kit  followed by nested PCR using Platinum Taq polymerase . Successful PCR amplification was assessed using 1% agarose gel  electrophoresis run at a 100 V for 40 minutes. The PCR products were cleaned up using the PureLink QUICK PCR Purification Kit  and sequenced using the Big Dye Terminator kit ver3.1  and a set of four bidirectional primers. Capillary sequencing electrophoresis was done on 3130Xl Genetic Analyzer .A previously described in-house HIV-1 drug resistance genotyping method was used to genotype the samples n >1000) available from public HIV sequence databases. Phylogenies were constructed using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian methods. Reliability of the trees was assessed by bootstrap methods (1000 replicates) for NJ and ML. Posterior probabilities were calculated from a sample of 10 000 trees sampled over 1×107 generations to determine the reliability of Bayesian trees. Trees were visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree). All the sequences generated for this manuscript are available from Genbank with accession numbers KC951632–KC951853.The sequences covering all of the 99 protease codons and the first 300 codons of the reverse transcriptase region were assembled using Geneious Pro genetic analyzer The method was validated in using a panel of proficiency testing samples obtained from the French National Agencies for Research on AIDS and Viral Hepatitis (ANRS). The panel was analysed using the in-house method and the United States food and drugs Agency (FDA) approved Viroseq genotyping method in parallel. The laboratory participates in the Quality Control for Molecular Diagnostics (QCMD) proficiency testing programme, and receives one EQA panel per year. This validated method is an open access and discounted method, with reagent costs of about US$50, made available by a partnership between SATuRN and Life Technologies to laboratories in Africa. All resistance results were provided within 15 days to the physician and were used for clinical management.The sequence data were linked to the clinical and demographic data in the SATuRN REGA database. The interpretation of drug resistance data from the nucleotide sequences was done using the Stanford HIVSeq algorithm version 6.0.5 Age was defined as at the date of enrolment. Baseline CD4+ cell count was defined as the CD4+ cell count closest to but prior to the date of ART initiation. CD4+ cell count and viral load at time of genotype were the measurements closest to but prior to the date of genotype. Immunological failure was defined according to WHO guidelines: fall of CD4+ cell count to baseline or below; 50% fall from on-treatment peak value; or persistent CD4+ cell count below 100 cells/µl All statistical analyses were performed using STATA version 11.2 . Descriptive statistics were used to summarise the baseline demographic and clinical characteristics. Frequency distributions of specific mutations were calculated. Logistic regression was used to explore factors associated with a compromised second-line regimen (GSS<2). The variables used for regression analysis included demographic characteristics (age and sex), laboratory results , and treatment information .A total of 260 individuals with virological failure on first-line ART were enrolled between December 2010 and March 2012. Samples from 242 (93%) of the patients were successfully genotyped and 222 (85%) were included in the analysis . All theMedian duration of ART was 42 months (IQR 32–53) and median duration of antiretroviral failure was 27 months (IQR 17–40). One hundred and thirty four (60%) had achieved virological suppression (VL<1000 copies/ml) on at least one occasion prior to genotyping, but only 89 (40%) had achieved VL<50 copies/ml. Two hundred (90%) of the patients had two or more viral loads >1000 copies/ml before genotyping and the median number of viral loads >1000 copies/ml before genotyping was 3 (IQR 2–5). During therapy, 81 (36%) had one or more drug substitution, the majority of which were NRTI substitutions. At the time of genotype, 75 (34%) had evidence of immunological failure, according to WHO definitions.One hundred and ninety one (86%) individuals had at least one drug resistance mutation at the time of genotyping: 181 (82%) had NNRTI resistance mutations and 179 (81%) had NRTI resistance mutations  and 3. MThirty-four individuals (15%) had a calculated GSS <2 for the standard, guideline-recommended second-line regimen, suggesting a potentially compromised regimen. Five of these had a GSS of 1, suggesting that only the protease inhibitor would have full activity in the standard second-line regimen. The majority had a GSS of 2 . Logistic regression was used to identify factors associated with compromised second-line regimen, defined as GSS <2 . In univThis study assessed the levels of acquired drug resistance in adults with virological failure on first-line ART in a rural primary health care treatment programme. This is one of the largest adult drug resistance studies to date in South Africa and the first to focus on a single rural treatment programme. In common with other studies from South Africa, almost all individuals with virological failure on first-line ART had evidence of antiretroviral drug resistance. In the context of public health antiretroviral strategies based on standardised first- and second-line regimens, it is important to explore how drug resistance impacts not only on individual antiretroviral drugs but also on drug regimens. Of concern was the fact that one in seven of these adults had complex resistance patterns with the potential to limit the efficacy of the standard second-line ART regimen. Of equal concern were the long periods of time on failing regimens despite the use of viral load monitoring, which suggest critical deficiencies in programme quality.It has been well documented that prolonged failure on first-line regimens leads to the accumulation of drug resistance and to poorer outcomes on second-line therapy The K65R mutation was present in 6% of cases, although over half of these were on TDF at the time of genotyping, due to previous substitution from d4T or AZT. There is evidence that K65R develops more frequently in subtype C viruses, primarily due to a difference in the template nucleotide sequence Surprisingly, duration on ART and duration on failing regimen were not associated with more complex resistance patterns in this study population. This may partly be due to lack of statistical power but also potentially to the complex relationship between adherence and resistance In this high prevalence community, there has been rapid scale-up of ART over the past eight years and high levels of ART coverage have been achieved in the community The national antiretroviral treatment guidelines recommend routine viral load monitoring to identify virological failure. However, if the results are not appropriately acted on and individuals are maintained on failing regimens, there may be accumulation of further resistance and progression to immunological and clinical failure. In this study, the period of time spent on failing first-line regimens was excessively long. Similar findings of prolonged viraemia have recently been reported from a multi-centre programme in Johannesburg Health systems, particularly in rural areas, have been put under huge strain by the rapid scale-up of HIV testing, treatment and care. There is already evidence from South Africa that the quality of treatment programmes has declined as systems have become more stretched n = 930) of the adults active on first-line ART and with a viral load measurement beyond 12 months had a latest viral load >1000 copies/ml; the individuals included in this study represent around 30% of that total. We cannot therefore be certain that the enrolled individuals were representative of all adults with virological failure in the programme. There was a lack of reliable and appropriately measured indicators of individual-level adherence and also no information available from pharmacy records. The absence of baseline genotyping led to assumptions that all observed drug resistance were acquired during treatment, but this is likely to be a reasonable assumption given that we have found no evidence as yet of transmitted drug resistance in this community. The definition of compromised second-line regimen was based purely on the calculated GSS and we are prospectively following the cohort of individuals switched to second-line therapy to explore clinical outcomes on second-line therapy.Interpretation of these results is subject to a number of limitations. The study involved programme physicians recruiting cases as part of routine clinical care and constraints in the number of physicians meant that not all potentially eligible individuals could be recruited into the study. From programmatic data, as of 31 August 2012, approximately 10% (In summary, there are high levels of acquired drug resistance in adults with failure of first-line antiretroviral therapy in this rural programme. Whilst the levels of resistance are similar to those reported from other programmes in South Africa, the long periods of antiretroviral failure reported here give cause for concern. The transition from an emergency response to the HIV epidemic to a sustainable, long-term solution presents many challenges


Nevirapine (NVP) and Efavirenz (EFV) have generally comparable clinical and virologic efficacy. However, data comparing NVP durability to EFV are imprecise. We analyzed cohort data to compare durability of NVP to EFV among patients initiating ART in Mbabane, Swaziland. The primary outcome was poor regimen durability defined as any modification of NVP or EFV to the ART regimen. Multivariate Cox proportional hazards models were employed to estimate the risk of poor regimen durability  for the two regimens and also separately to estimate risk of drug-related toxicity. We analyzed records for 769 patients initiating ART in Mbabane, Swaziland from March 2006 to December 2007. 30 patients (3.9%) changed their NVP or EFV-based regimen during follow up. Cumulative incidence for poor regimen durability was 5.3% and 2.7% for NVP and EFV, respectively. Cumulative incidence for drug-related toxicity was 1.9% and 2.7% for NVP and EFV, respectively. Burden of TB was high and 14 (46.7%) modifications were due to patients substituting NVP due to beginning TB treatment. Though the estimates were imprecise, use of NVP - based regimens seemed to be associated with higher risk of modifications compared to use of EFV - based regimens (HR 2.03 95%CI 0.58 - 7.05) and NVP - based regimens had a small advantage over EFV - based regimens with regard to toxicity - related modifications (HR 0.87 95%CI 0.26 - 2.90). Due to the high burden of TB and a significant proportion of patients changing their ART regimen after starting TB treatment, use of EFV as the preferred NNRTI over NVP in high TB endemic settings may result in improved first-line regimen tolerance. Further studies comparing the cost-effectiveness of delivering these two NNRTIs in light of their different limitations are required. Non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART) has been the most affordable regimen for HIV-infected patients in resource limited settings. Combination ART with either efavirenz (EFV) or nevirapine (NVP), both NNRTIs, and two nucleoside reverse transcriptase inhibitors (NRTI) is recommended by the World Health Organization (WHO) as first-line therapy in resource-limited countries . NVP andWhere treatment options are limited, strategies to achieve maximum benefit from the available first-line regimens are vital and understanding which regimens are most tolerated by patients is therefore crucial. We set out to compare the durability of NVP to EFV in standard first-line regimens among HIV-infected treatment naïve patients initiating ART and followed up to a year at a large urban clinic in Mbabane, Swaziland.The Mbabane Government Hospital Antiretroviral Therapy Unit has previously been described . The hosThe primary outcome was poor regimen durability. We defined poor regimen durability as a physician initiated modification from either a NVP-based or EFV-based ART regimen due to any reason . Drug toxicity based modifications were based on treatment guideline recommendations and were also considered after patient complaints .Patient data was extracted from a computer-based system, patient files and pharmacy refill booklets. All patients were followed up until treatment modification, loss to follow-up, transferred out to another facility or administrative censoring after 12 months of follow-up. Since we did not have death data available for this cohort, a significant proportion of the patients classified as LTFU could have died.We calculated cumulative incidence of the NNRTI drug modifications by end of the 12 month follow up period. Multivariate Cox proportional hazards models were further employed to estimate the risk of poor regimen durability for the two regimens. Separate multivariate analyses were also performed to estimate risk of poor regimen durability caused by drug-related toxicity alone. All analyses were performed using STATA v.11 . Lastly, in order to understand how patients who were lost to follow up (LTFU) could have biased our data, we performed sensitivity analysis that assumed that all LTFU were failure events. Patients missing 3 consecutive clinic visits, a month apart and were unable to be contacted were defined as LTFU.Permission to access and use data for this study was granted by the ethics committees of the Swaziland Ministry of Health and Social Welfare and the Faculty of Health Sciences, University of Pretoria. This study drew data from a previous study that had assessed reasons for treatment change among patients initiating ART .Between 01 March 2006 and 31 December 2007, 824 patients fulfilled the inclusion criteria. Among these, 55 patients were excluded for missing data leaving a total of 769 patients for analysis. The majority of patients were initiated on to a NVP-based regimen  and patients on NVP were more likely to be initiated on a d4T/3TC NRTI backbone compared to those initiated on an EFV-based regimen . Patients initiating NVP-based regimens were more likely to be female . CD4 cell count, WHO clinical stage, weight and age were largely similar among the two groups at the time of ART initiation . At the TM. One patient switched treatment (NVP/3TC/d4T) to a second-line regimen due to confirmed treatment failure. All 14  discontinuations due to a drug contraindication were due to patients starting TB treatment whilst on a NVP- based regimen. A total of 13  patients experienced poor regimen durability caused by drug-related toxicity alone - this excludes 14 due to TB treatment, 2 due to drug shortage and 1 due to treatment failure. Cumulative incidence for NVP or EFV drug modification regardless of reason was 5.3% (26/488) and 2.7% (4/149) for NVP- and EFV- based regimens, respectively. The cumulative incidence for NVP or EFV drug modification due to drug-related toxicity alone by end of the 12 months was 1.9% (9/471) and 2.7% (4/149) for NVP- and EFV-based regimens, respectively. The reasons for these modifications are shown in A total of 30 patients (3.9%) had either a NVP- or EFV drug modification for any reason during this 12 month follow up period (NVP 4.5% (26/578), EFV 2.1% (4/191)). Two patients discontinued their regimen because of a stock out of NVP tablets as part of the fixed dose combination TriomuneIn Cox proportional hazards models adjusted for age, gender, baseline CD4 cell count and WHO stage, the estimates suggest that being on a NVP- based regimen may be associated with increased risk of poor regimen durability compared to a EFV - based regimen though the imprecise nature of the estimate does not allow us to infer the association with confidence . In analysis examining poor regimen durability due to drug related toxicity modifications, a NVP- based regimen seemed to have a small non-significant advantage over a EFV-based regimen. These estimates were also imprecise . Results are shown in In order to understand how the missing outcomes in the LTFU data could have affected our outcome estimates, we additionally conducted a sensitivity analysis in which we assumed that all LTFU patients actually had a NNRTI drug modification. In this analysis, there was no significant difference in outcome between the two regimens . This result may suggest that if LTFU did affect our main estimates, this bias was minimal.In this observational study in which patients were followed up to 12 months after initiating first-line ART in Mbabane, Swaziland, the hazard estimates suggest that being on a NVP- based regimen may be associated with increased risk of poor regimen durability compared to a EFV - based regimen though the imprecise nature of the estimate does not allow us to infer the association with confidence. In further analysis examining poor regimen durability due to drug related toxicity modifications, a NVP- based regimen seemed to have a non-significant small advantage over an EFV-based regimen though these estimates were also imprecise.It is important to note that all regimen changes due to drug contraindication were due to patients discontinuing NVP due to a tuberculosis (TB) diagnosis. This resulted in a far much higher proportion of discontinuations of NVP compared to EFV . According to local guidelines, concomitant use of NVP with rifampicin containing TB treatment is contraindicated as there is a potential of drug-drug interactions and increased toxicity . In accoOur study has a major strength of the use of routine clinical data collected from a real life clinical setting. However, the findings of this study should be considered alongside its limitations. We did not collect comprehensive information of concurrent non-HIV clinical conditions that may increase the risk of drug modification i.e. diabetes, epilepsy, resulting in unmeasured confounding of our findings. Additionally, because of the retrospective nature of this data, precautions could not have been taken to ensure that any chosen drug combinations at treatment initiation or modifications after initiation could have resulted from sub-optimal prescribing practices rather than genuine intolerance of the drug by patients. Furthermore, the small number of outcome events and small sample size may have compromised the power of the study in order to detect sizeable differences. Finally, the effect of loss to follow up which may have included deaths were not considered in the main analysis. While this may have introduced selection bias into the estimates presented, sensitivity analyses suggested minimal bias.In conclusion, we observed an increased risk of modifying NVP in initial first line regimens compared to that of EFV in the first 12 months of treatment initiation. A significant proportion of patients modifying ART did this due to starting TB treatment. Use of EFV as the preferred NNRTI over NVP in settings were TB is endemic may result in improved first-line regimen durability. Better screening methods for TB should also be made readily available in such settings so that better tolerated regimens are initiated. Additional studies should focus on comparing the cost-effectiveness of delivering these two NNRTIs in light of their different limitations. Improved initial ART regimen durability should be an additional vital treatment aim as this will increase the likelihood of achieving sustained viral response and, at the same time, preserving future treatment options.


In HIV-1 infected patients, increase of liver enzymes may be mainly due to viral coinfections, alcohol intake, hepatotoxic drugs or autoimmune diseases. Three cases of aminotransferase elevation occurred during a phase of uncontrolled viral replication combined with a severe immunodeficiency and resolved by an effective HAART are described, focusing on the etio-pathogenetic role possibly played by HIV-1 infection. Abnormal liver enzymes  are frequently seen in HIV-1 infected patients and may be due to a variety of factors, such as coinfection with hepatotropic viruses, i.e. hepatitis B (HBV) and C (HCV) viruses, Cytomegalovirus (CMV) and Epstein-Barr virus (EBV), opportunistic infections, cancers, autoimmune hepatitis (AIH), alcohol abuse, and exposure to hepatotoxic drugs, including highly active antiretroviral therapy (HAART). Identification and management of these factors is often difficult because of the coexistence of multiple causes [Human immunodeficiency virus type-1 (HIV-1) infection is commonly characterized by the presence of a progressive depletion of CD4+ T lymphocytes less than 200/cu.mm.) showing severe aminotransferase elevations  occurred during a period of uncontrolled viral replication, and in the absence of any other apparent cause of liver disease. AST and ALT values returned within the normal ranges in a few months after the onset of an effective HAART.We report three cases of HIV-1 infected patients with advanced immunodeficiency (CD4+ T lymphocytes 95 cells/cu.mm.). Not excluding a drug toxicity, HAART was discontinued and the patient underwent to a complete assessment of hepatic functions, including HAV, HBV and HCV antibodies, HBV-DNA, HCV-RNA, EBV-DNA and CMV-DNA, all resulted negative. A genetic test for hemochromatosis and the research of auto-antibodies for AIH were also negative. No diabetes or other metabolic disorders, with the exception of a mild increase in triglycerides, were present. A liver ultrasound showed hepatomegaly without parenchymal abnormalities. A liver biopsy revealed hepatosteatosis with multifocal lymphocytic lobular infiltrates. From August 2004 to August 2006, despite the presence of a multi-drug resistant virus, antiretroviral therapy was reintroduced, with the intent to delay the clinical evolution of HIV-1 disease  and a deterioration of AST and ALT values (400 UI/l and 600 UI/l respectively) were observed. On August 2006, a darunavir/ritonavir + enfuvirtide + tenofovir and lamivudine/zidovudine fixed-dose regimen was started, based on the results of a genotypic resistance test (GRT) and the availability of new drugs. After one month, HIV-RNA levels decreased to 100 copies/ml and AST and ALT values returned below 50 UI/ml. Three months later, plasma HIV-RNA was undetectable whereas LFTs reached normal levels. After one year, enfuvirtide was replaced with raltegravir, and lamivudine/zidovudine fixed-dose + tenofovir discontinued. Plasma viral load has remained on undetectable levels and CD4+ T lymphocytes have now reached 430 cells/cu.mm., with LFTs always within the normal ranges  was found to be HIV-1 positive (CDC A2) for a history of unprotected sexual intercourses. On September 1990 the patient has presented a CMV retinitis and a zidovudine-based antiretroviral therapy was started. From 1990 to 2002 he switched several antiretroviral treatments, due to side effects or development of drug resistance. During this period a partial immune recovery was observed and plasma HIV-RNA levels sometimes reached undetectable values, with liver function tests (LFTs) always within the normal ranges. On October 2003, increased levels of AST (135 UI/l) and ALT (89 UI/l) were present. Moreover, the patient showed a failure of the lopinavir/ritonavir-based therapy . He started antiretroviral therapy on November 1995 with zidovudine and didanosine, when his CD4+ T cell count was 32 cells/cu.mm. He reported a history of unprotected heterosexual intercourses. Up to September 2006, he switched several treatments for side effects or for the occurrence of drug resistance  with a worse in CD4+ T cell count (55 cells/cu.mm.) and an increase of LFTs (ALT 119 UI/l and AST 270 UI/l). No diabetes or other metabolic disorders were present. Antiretrovirals were discontinued and the patient underwent to laboratory and instrumental examinations to investigate the aetiology of the liver disease: HBV, HCV, CMV and EBV antibodies, and the relative DNA or RNA detections by polymerase chain reaction (PCR), were negative as well as the auto-antibody titres. A liver ultrasound showed only a mild hepatomegaly, whereas a liver biopsy revealed mild hepatosteatosis without fibrosis, and focal lymphocytic lobular infiltrates. The patient remained without antiretroviral therapy until October 2007, when, despite the persistence of abnormal LFTs, the treatment was restarted with a darunavir/ritonavir + enfuvirtide + raltegravir-based HAART, taking in account the results of a new GRT. After only one month of therapy, LFTs returned within the normal ranges and two months later plasma HIV-RNA values reached undetectable levels . Actually the patient is taking a simplified regimen with darunavir/ritonavir + raltegravir, his CD4+ T cells have reached 359/cu.mm., with plasma HIV RNA always below the limits of detection and LFTs within the normal ranges.A 31-years-old man on September 1995 was found to be HIV-1 positive during a hospitalization caused by + T lymphocytes raised from 155 to 461 cells/cu.mm. and plasma HIV RNA decreased from 251.000 copies/ml to undetectable levels. KS lesions disappeared and an amelioration of clinical conditions was observed. Up to April 2006, he continued HAART, switching from indinavir to nevirapine for simplification, and from stavudine to tenofovir for the onset of peripheral neuropathy. During this period plasma HIV RNA remained undetectable and CD4+ T lymphocytes were constantly over 500 cells/cu.mm. LFTs were always within the normal ranges or showed little abnormalities, clearly referred to the use of specific antiretroviral drugs . HBV and HCV serology were negative, whereas anti-CMV and anti-EBV IgG antibodies were present. From April 2006 the patient was no longer subjected to clinical and laboratory scheduled follow-up. He discontinued HAART for the onset of lipodistrophy signs, and on July 2009 he returned to our clinic, showing a worsening of clinical conditions . Laboratory tests revealed: HIV-RNA 153.465 copies/ml, CD4+ T lymphocytes 222 cells/cu.mm., ALT 131 UI/l and AST 282 UI/l. HBV and HCV serology remained negative as well as the relative DNA and RNA by PCR analysis; a liver ultrasound did not show signs of liver damage. Autoantibody research for AIH was negative. After the results of a resistance test, a new antiretroviral combination based on raltegravir plus tenofovir/emtricitabine fixed-dose was prescribed. A rapid amelioration of the clinical conditions was observed and after one month of therapy he showed an immune-virological recovery with a persistently improvement of LFTs  lesions with visceral involvement (CDC C3). HAART with indinavir, lamivudine and stavudine was started, shortly allowing an immune-virological recovery. After three months of therapy, CD4+ T cells < 200/cu.mm.) and a high level of viral replication, due to drug failure or voluntary HAART discontinuation. All patients showed plasma HIV RNA values > 100.000 copies/ml and the absence of other possible causes of liver injury, such as hepatotropic virus infections, hepatotoxic drugs administration, alcohol intake and/or other substances' abuse, or cancers. Furthermore, the search for other pathogens  resulted negative. Hepatic biopsy performed in two subjects was not of diagnostic value. A triggering role in the aminotransferase increase played by antiretroviral drugs (i.e. LPV/r and ATZ/r) at least in the first two subjects could not be excluded, being hepatotoxicity of these drugs frequently reported [Involvement of the liver during the course of HIV-1 disease may result from viral or other infections, or being secondary to cancers, toxic agents and drugs. The three patients described here showed an advanced HIV-1 disease with an unexpected increase of LFTs occurred in the presence of a severe immunodeficiency (CD4reported -7. In alreported -10. An areported . Only a reported ,13. Virareported . In the reported , such apIn conclusion, in HIV-1 infected patients presenting with an acute onset of abnormal LFTs, after the exclusion of the most common causes of liver disease, it is necessary to assess the effectiveness of the current HAART and strongly evaluate the opportunity of starting an alternative antiretroviral regimen, in the suspicion of a HIV-1 induced hepatic disease.Written informed consent was obtained from the patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.All authors read and approved the final manuscript, and significantly contributed to the work.


HIV-1 treatment-failure patients are increasingly being initiated on second-line antiretroviral therapy. The case we describe is of a treatment-failure patient who developed intractable chronic vulval ulceration presenting as immune reconstitution inflammatory syndrome (IRIS), following complete viral suppression with second-line highly active antiretroviral treatment (HAART). To the best of our knowledge, this is the first reported case of intractable vulval ulceration IRIS in an HIV-1 treatment-failure patient. IRIS manifesting as genital ulcer disease (GUD) has been documented in some reports , 2. GUD μL. She was initiated on HAART—with a fixed dose combination of lamivudine 150 mg/stavudine 40 mg/nevirapine 200 mg (Triomune-40) one tablet twice daily and cotrimoxazole prophylaxis to which she reported 100% adherence. After 1.5 years on HAART, she defaulted on therapy for a period of 4 months. She subsequently began receiving care from another HIV treatment centre where she had a dual drug switch to tenofovir, emtricitabine (Truvada), and nevirapine, to which she complied with 100% adherence. She continued treatment at that centre for 2 years. During this period she had an initial rise in CD4+ cell count to a peak of 389 cells/μL which subsequently started decreasing gradually both in absolute count and percentage. A 35-year-old HIV-positive woman presented to the Infectious Diseases Institute, Kampala, in February 2005 with World Health Organization (WHO) stage III disease having presented with a history of >10% weight loss and recurrent upper respiratory tract infections (URTIs). Her nadir CD4+ count was 9 cells/μL (2%). A decision was made to maintain her on truvada and nevirapine following adherence counseling. A repeat CD4+ count 8 months later showed a further decline in the absolute CD4+ count and percentage to 12 cells/μL (1%), while still on truvada and nevirapine. She had a detectable viral load of 36,795 copies/mL. She was then switched to abacavir, zidovudine/lamivudine (Combivir), and lopinavir/ritonavir (Alluvia) basing on possible development of K103N and M184V mutations. A resistance profile could not be done due to the high costs involved. A repeat laboratory profile four months later showed immunological improvement with a rise in CD4+ count and percentage to 75 cells/μL (4%) and an undetectable viral load of <400 copies/mL. At this juncture, she was referred back to our institute where she was found with a CD4+ count of 30 cells/μL (9%) six months later. Four months after commencing second-line HAART and achieving complete viral suppression, she presented with complaints of vulval ulcers. Genital examination revealed a 4 cm superficial right vulval ulcer. There were no other significant findings on systemic examination. Cryptococcal antigen and Syphilis serology  were negative. Herpes simplex virus-2 serology could not be done due to the high costs involved. Swabs from the vulval ulcer and cervix were unremarkable. Two biopsies revealed nonspecific chronic inflammation. The second-line HAART was well tolerated with sustained viral suppression of <400 copies/mL and immunological improvement to 209 cells/Treatment with oral acyclovir, famciclovir, ampiclox (ampicillin/cloxacillin), cloxacillin, ceftriaxone, fluconazole, metronidazole, ciprofloxacin, doxycycline, nonsteroidal anti-inflammatory drugs (NSAIDs), and topical hydrocortisone cream was unsuccessful. A routine of oral prednisolone 40 mg once daily given over ten days which was gradually tapered off over a period of 4 weeks was repeated quarterly with complete resolution approximately three years later. Chlorhexidine and cetrimide (Hibitane) soaks were also applied.tuberculosis, and M. avium complex have been implicated in chronic GUD-IRIS in addition to herpes simplex [GUD-IRIS has been shown to significantly peak in the first month after HAART commencement . Acute G simplex , 4, 5. IRIS may manifest as infectious or autoimmune clinical events predominantly occurring in the first 8 weeks following HAART initiation with the exception of Graves' disease which occurs at a median time of 2 years –9. ChronIn our patient, the cause of intractable chronic vulval ulceration for over three years was idiopathic, following extensive microbiological, histopathological investigations and extensive treatment—with the use of antibiotics, antivirals, NSAIDs, and steroids. It is therefore imperative that clinicians are aware of chronic intractable genital ulceration-IRIS that defies extensive treatment and investigations when HIV treatment-failure patients undergo immune reconstitution.