HIV-infection results in damage and dysfunction of the gastrointestinal system. HIV enteropathy includes pronounced CD4+ T-cell loss, increased intestinal permeability, and microbial translocation that promotes systemic immune activation, which is implicated in disease progression. A synbiotic is the combination of probiotics and prebiotics that could improve gut barrier function. Our study goal was to determine whether the use of a synbiotic, probiotics or a prebiotic can recover immunological parameters in HIV-infected subjects through of a reduction of microbial translocation and pro-inflammatory cytokine production.A randomized, double-blind controlled study was performed; twenty Antiretroviral treatment-naïve HIV-infected subjects were subgrouped and assigned to receive a synbiotic, probiotics, a prebiotic, or a placebo throughout 16 weeks.p = 0.048). Moreover, the probiotic and synbiotic groups demonstrated a decrease in total bacterial load in feces (p = 0.05). The probiotic group exhibited a significant increment of beneficial bacteria load  and a decrease in harmful bacteria load . In the synbiotic group, the CD4+ T-cells count increased  and the level of Interleukin 6 cytokine decreased significantly (p = 0.016).We had no reports of serious adverse-events. From baseline to week 16, the synbiotic group showed a reduction in bacterial DNA concentrations in plasma (Our study showed a significant increase in CD4+ T lymphocyte levels in the synbiotic group, which could delay the initiation of antiretroviral therapy and decrease costs in countries with limited resources. A huge Gastrointestinal (GI) pathology is observed in patients infected with HIV even during primary infection. Approximately 60% of total CD4+ T cells, reside in Gut-associated lymphoid tissue (GALT), and of all tissues, the latter is one of the most strongly affected during HIV infection . In 1984Once HIV enters the mucosa of the gut, it finds a large pool of resting Ki67-CD4+ T cells; up to 60% of these cells are infected and are capable of produce the virus, constituting a dense network of cells in the intestinal mucosa, which is capable of spreading the infection to uninfected cells through cell-to-cell contact. This spread allows the maintenance of a continuous chain of viral transmission and forms part of a large reservoir that is currently impossible to eradicate. On the other hand, activated CD4+ T cells (effector) produce high viral-load levels, generating high viral gradient in the mucous membrane tissues, resulting in infection amplification and spread over long distances .HIV exerts a direct effect on enterocytes. The viral tat protein inhibits glucose uptake by the enterocytes, altering their function; the gp120 protein increases enterocyte intracellular calcium and causes tubulin depolymerization, in turn causing cytoskeleton dysfunction, with destabilization of intercellular junctions and further increased intestinal permeability. There is a simultaneous occurrence of a decrease in the expression of genes involved in epithelial mucosal repair (such as trefoils) and increased immune activation genes, leading to high levels of pro-inflammatory cytokines as Tumor necrosis factor (TNF)-α, Interferon (INF)-γ, and Interleukin (IL)-6, -8, and −12 [The enteropathy associated with HIV-infection is characterized by villous atrophy, crypt hyperplasia, vitamin B12 and bile acid malabsorption, epithelial cell damage, with enterocyte apoptosis and disruption of tight and adherent junctions with an increase in intestinal permeability; moreover, depletion of Peyer’ s patches as well as of CD4+ T cells in lamina propria combined with a reduced ability to produce secretory IgA and defensins comprise conditions that favor persistent microbial overgrowth in the intestinal lumen, microbial translocation with increased plasma levels of Lipopolysaccharides (LPS), and 16S rRNA, which can provoke the development of chronic local and systemic immune stimulation that may lead to disease progression -11.Microbiota of intestinal tract constitute a complex ecosystem of microorganisms; there are from 10–100 trillion microorganisms that populate the adult intestine and these provide some benefits to the host . HIV inf7-1011 colony forming units (cfu)) and are classified by the US Food and Drug Administration (FDA) as “live biotherapeutics” when utilized in humans [Synbiotics are the combination of probiotics plus prebiotics. The definition of probiotics is live microbial feed supplements that confer health benefits when administered in adequate amounts  linkages, and some β(2–6) linkages, with branches and a glucose moiety [Lactobacillus rhamnosus HN001 plus Bifidobacterium lactis Bi-07 at 109 cfu/mL, and the synbiotic was the combination of probiotics plus prebiotic, the placebo was a product of Biogel without the two types of probiotics and without the prebiotic, but with the same flavor and characteristics. The Institutional Review Board at the study site approved the study, and all patients were enrolled immediately after providing written informed consent.Patients were randomly assigned to receive probiotics, a synbiotic, a prebiotic, or placebo during 16 weeks; all products (gel formulation) were made by biotechnology company called Biotek in Guadalajara city, they required refrigeration and were taken once daily. We used 10 g of agavins from e moiety . As prob3. Patients were not included in the study if they were pregnant, had hepatic or renal insufficiency, cancer, immunotherapy, use of antibacterial therapy (for >15 days), had knowledge of an allergy to bovine milk proteins or if the dose compliance with the gel formulation was <95%.Inclusion criteria included the following: documented HIV infection; age between 18 and 65 years; negative for Hepatitis B and C viruses; Antiretroviral (ARV) treatment-naïve, in stage A or B according to the U.S. Centers for Disease Control and Prevention (CDC) classification system, with a CD4+ count of >350 cells/mmParticipating patients were requested to check their body temperature, to report immediately to the researcher for possible symptoms of infections, and were examined by means of laboratory and physical testing at least once monthly.The Irritable Bowel Syndrome-Quality of life (IBS-QoL) questionnaire in a local language was utilized to evaluate the impact of gastrointestinal symptoms or treatment complications at baseline and at 16-week visits. The IBS-QoL is a self-administered instrument employed to derive an overall score with a 34-item, condition-specific questionnaire that has been approved for identifying GI-related QoL measures in HIV-infected patients ,26, in wAbsolute CD4+ T-cell counts were quantified as absolute concentration (cells per cubic mm) by means of flow cytometry at a state-certified laboratory.We measured IL-10 concentrations in plasma as anti-inflammatory cytokine, and pro-inflammatory cytokines IL-1β, IL-6, and TNF-α by ELISA using commercially available kits (R&D systems). Results were expressed in units of pg/mL.HIV-1 RNA was measured in plasma at baseline and at week 16, with the AMPLICOR HIV-1 MONITOR ultrasensitive assay  with a lower detection limit of 50 copies/mL at a state-certified laboratory.From 200 μL of plasma and from 200 g of frozen stool samples, bacterial DNA was extracted employing the DNeasy Blood and Tissue Kit and QIAamp DNA Stool Mini Kit (Qiagen), respectively, both according to the manufacturer’s instructions. DNA concentrations were determined with a DU 640 spectrophotometer.Bacterial translocation was measured by means of 16S rRNA levels in plasma, and 16S rRNA levels in feces were utilized to determinate total bacterial load in stool. We used a final volume of 10-μL amplification reaction consisting of 2 μL of 1X PCR buffer , 5 pmol forward and 5 pmol reverse primers and 2 ng of template plasma DNA or template feces DNA . We employed the following primer pairs: forward (8 F: 5′-AGT TTG ATC CTG GCT CAG-3) and reverse (515R: 5′-GWA TTA CCG CGG CKG CTG-3′) primers to amplify bacterial DNA templates encoding 16S rRNA . We amplBifidobacterium spp. Load, and as harmful bacteria, the Clostridium spp. load. The primer pairs used to detect Bifidobacterium spp. were the following: forward 5′-CGC GTC YGG TGT GAA AG-3′and reverse 5′-CCC CAC ATC CAG CAT CCA-3′ [Clostridium spp. were forward 5′-AAA TGA CGG TAC CTG ACT AA-3′ and reverse 5′-CTT TGA GTT TCA TTC TTG CGA A-3′ [Clostridium or Bifidobacterium, respectively) for 30 s, and at 72°C for 10 s. Additionally, melting temperature analysis was performed as follows: at 95°C for 10 s; at 60°C for 1 min, and by slowly heating the sample to 95°C at a temperature rate of 0.1°C/s.To detect changes in stool bacterial composition at baseline and at week 16, we measured the bacterial loads, dividing as beneficial bacteria the T CCA-3′ ,28. The CGA A-3′ ,29. All U tests. Correlations were analyzed utilizing Spearman rank-correlation for nonparametric data. Other statistical tests employed are indicated in the text. All statistical analysis was undertaken using SPSS version 14 software.Comparisons among all groups were carried out by performing the Kruskal-Wallis and the Mann–Whitney The study included 20 patients, with five participants for each of four groups. Characteristics of the patients in the four groups were similar and are depicted in Table In all groups, we observed an adherence upper than 98%, with good tolerance and we did not identify serious side effects that forced us to suspend the regimen.p = 0.048) . The prebiotic group showed a tendency toward higher bacterial load in feces (data non shown). On the other hand, some differences in bacterial stool composition were found between groups; the probiotic group exhibited a significant increase of microbial load of Bifidobacterium spp. (p = 0.049) and a decrease in Clostridium spp. (p = 0.063), while the synbiotic group demonstrated a reduction non-significant in beneficial and harmful bacterial loads   (data not shown).A reduction in bacterial DNA concentrations in plasma was observed in the probiotics, synbiotic, and prebiotic groups, but statistical significance was reached only in the synbiotic group  Figure . Additio p = 0.06, while tp = 0.05)   . The IL-6 cytokine level decreased significantly in the synbiotic group  Figure  and a neWithin the immunopathogenesis of HIV infection, there is a state of constant systemic immune activation that is attributed in part to the enteropathy caused by HIV itself and to the translocation of microbes and/or microbial products from the intestinal lumen into the circulation ,30.Lactobacillus rhamnosus HN001 plus Bifidobacterium lactis Bi-07 at 109 cfu/mL as probiotics, 10 g of agave inulin as prebiotic, and the combination of both as synbiotic in antiretroviral-naïve, HIV-infected subjects.We evaluated the efficacy and safety of There have been some studies in patients with HIV infection, using different probiotics in which mixed results have been obtained; this is perhaps secondary to the use of different concentrations and probiotic strains, which do not trigger the same immunostimulatory effect ,31,32.Bifidobacterium species, which have less potential for adhesion and translocation [Certain serious adverse effects have been reported with the use of probiotics, particularly endocarditis, liver abscess, bacteremia, and septicemia or septic shock, especially in immunocompromised patients, or in patients with chronic disease, or in those with ventilatory support or with central venous catheters -35. Durilocation . Furtherd-dimer and high-sensitivity C-reactive protein [ARV treatment has increased survival in patients with HIV infection; however, survival is at least 10 years less than in general population. In addition, cardiovascular risk is twice as high in HIV-positive subjects . In our  protein .Agave tequilana Weber var. azul are widely employed to produce the national drink, tequila, and could also be a potential source of prebiotics and part of synbiotic with accessible prices.ARV drugs can cause several adverse effects including dyslipidemia, insulin resistance, lactic acidosis, lipodystrophy, and diarrhea, among others ,40. Our On the other hand, we found no decrease in HIV-1 plasma viral load; therefore, the use of a synbiotic could not provide the benefit of maintaining an undetectable viral load as part of the primary prevention of HIV transmission.The authors have declared that no competing interests exist.Conceived and designed the experiments: LAG, MFM, JFAV; Performed the experiments: LAG, LFJS; Analyzed the data: LAG, JFAV, LFJS; Contributed reagents/materials/analysis tools: MFM, AECL, LGVG, KEMS, LFEG; Wrote the paper: LAG, JFAV. All authors read and approved the final manuscript.


CYP2C19*2 (poor metaboliser) genotype status and concomitant treatment with lansoprazole, tacrolimus, and antiretroviral (ARV) medications resulted in hepatic decompensation. This highlights the importance of careful interaction screening for all new drugs administered to patients with HIV who have complex treatment regimens as well as heightened clinical vigilance for unexpected toxicities.In general, kidney transplantation is safe and efficacious in patients receiving treatment for HIV. Although multiple drug interactions between antiviral and immunosuppressive treatments exist, few patients experience serious adverse reactions. We report a case of fatal lactic acidosis in a healthy kidney transplant recipient with stable HIV infection who had previously received treatment for and cleared hepatitis C virus infection. Death occurred less than one month following the initiation of tacrolimus therapy. Based on predicted drug interactions, appropriate tacrolimus dosing was calculated prior to its commencement, yet plasma tacrolimus levels were initially unexpectedly high. The patient subsequently developed lactic acidosis and hepatic steatosis, presumably due to mitochondrial toxicity from the antiretroviral regimen on which he had previously been stable. We suspect  Since the introduction of highly active antiretroviral therapy (HAART) in 1996, mortality in patients with human immunodeficiency virus (HIV) infection has decreased markedly . The preConcerns remain, however, regarding the concomitant use of antiretroviral medications and immunosuppressive agents that may be substrates for, or may induce or inhibit pharmacokinetic enzymes involved in drug handling, such as P-glycoprotein flux transporters or cytochrome P450 metabolizing enzymes found in the gut and liver. These interactions can lead to unexpected increases or decreases in drug plasma levels leading to drug toxicity, transplant rejection, or HIV disease breakthrough. Significant drug interactions have been observed depending on the class of antiretroviral agent . ClearlyμL) and undetectable HIV RNA viral load (<40 copies/mL). 11 years later, he developed end-stage kidney disease and started peritoneal dialysis. Later that year, he was diagnosed with acute HCV genotype 1 infection and was treated with sustained virological response. Liver function tests at baseline were normal , and the patient complained of tremor and weakness consistent with tacrolimus toxicity. Dosing was adjusted accordingly.Six months after engraftment, there was a rise in serum creatinine above baseline, and he underwent a biopsy of his transplanted kidney which showed acute transplant rejection. This was treated with three consecutive daily doses of intravenous methylprednisolone without immediate improvement in kidney function. A decision was therefore made to switch the base immunosuppressive therapy from ciclosporin to tacrolimus. Tacrolimus is primarily metabolized in the liver by cytochrome P450 (CYP3A), and nevirapine is a slight inducer of CYP3A , thus byTwo weeks later, the patient required hospital admission with malaise, metabolic acidosis, and deranged kidney and liver function . BacteriAt autopsy, the main pathology was in the liver. Weighing 1670 gm, it was soft, bright yellow, not fibrotic, and without focal lesions . HistopaCYP2C19*2) rendering this enzyme nonfunctional. Antiretroviral drug levels tested posthumously on stored serum samples lay within the therapeutic range . GenotypInteractions between immunosuppressive therapy and antiretroviral medications (ARV) are common . The mosCYP2C19*2 status. This mutation alters the mRNA reading frame, resulting in a premature stop codon, yielding a truncated nonfunctioning protein (and consequent low phenotypic CYP2C19 enzymatic activity). In Caucasians, homozygosity for the risk allele (A/A) is rare (5%). Posthumous genotyping revealed that our patient was homozygous for CYP2C19*2, rendering this enzyme nonfunctional. This resulted in competition between lansoprazole and tacrolimus for CYP3A4-mediated metabolism and accounted for the unexpectedly high tacrolimus levels.Proton pump inhibitors such as lansoprazole are metabolized by both CYP2C19 and CYP3A4 isoforms of cytochrome P450. This leads to potential drug interactions with other substrates of CYP3A4 (such as tacrolimus), particularly when CYP2C19 activity is impaired . Possessγ resulting in impaired synthesis of mitochondrial enzymes that generate adenosine triphosphate (ATP) by oxidative phosphorylation. Although 20–60% of patients on NRTI therapy have elevated lactate levels, only 0.1% to 0.4% develop lactic acidosis [n-acetylcysteine, uridine, and l-carnitine [Lactic acidosis is a rare but potentially life-threatening condition associated with exposure to nucleoside/tide reverse transcriptase inhibitors (NRTIs), particularly stavudine and didanosine, although reported with all NRTIs. Other reported risk factors include female gender, pregnancy, obesity, prolonged ARV duration, and use of ribavirin with didanosine in patients with hepatitis C coinfection . The cauacidosis , which macidosis . Treatmearnitine . The morarnitine . Other tDue to similar routes of transmission, coinfection of HIV with other sexual and blood-borne viruses such as hepatitis B virus (HBV) and HCV is relatively common. Of the 35 million people living with HIV worldwide, around 20% have chronic HCV infection. The mechanism of interaction between the two viruses and their impact on liver injury is not completely understood, but patients with HIV are less likely to achieve spontaneous HCV clearance and progress more rapidly to liver fibrosis and end-stage liver disease than HIV-negative individuals. Furthermore, HIV/HCV coinfected patients are more susceptible to liver toxicity and steatosis from ARV, more so with the use of NRTIs, and in such patients, hepatic steatosis is associated with more advanced hepatic fibrosis . Risk faIn the case we report here, although the patient had cleared HCV and remained HCV RNA negative with normal liver function tests, he had not had assessment of liver fibrosis by biopsy or transient elastography prior to kidney transplantation. Features which argue against a causal role of HCV in this case are sustained virological response to HCV eradication therapy; presence of Ishak stage 1-2 hepatic fibrosis on autopsy ; microvesicular steatosis, which is unrelated to HCV and consistent with a drug-induced lactic acidosis ; an AST-to-platelet ratio index (APRI) of 0.08, measured when the patient was stable, indicating haematological and biochemical absence of fibrosis.CYP2C19*2 status and concomitant lansoprazole, both of which affected tacrolimus metabolism by CYP3A4. However, this does not explain the mitochondrial uncoupling with lactic acidosis and steatosis that occurred shortly after tacrolimus initiation; there are no previous reports of tacrolimus-induced lactic acidosis. It is possible that a steatosis diathesis was created by existing treatment with NRTI, which, coupled with the presence of elevated tacrolimus levels, resulted in hepatic decompensation. Yet, serum levels of HAART were normal . An alternative hypothesis is that the unique combination of genetic variation and polypharmacy precipitated hepatic decompensation through unknown pathways.The close temporal association between the initiation of tacrolimus and the onset of progressive liver dysfunction suggests a causal relationship, but the mechanism for this remains unexplained. The unexpectedly high tacrolimus levels probably resulted from Organ transplantation for patients with HIV remains a novel therapy  and carr


Accurate interpretation of HIV drug resistance (HIVDR) testing is challenging, yet important for patient care. We compared genotyping interpretation, based on the Stanford University HIV Drug Resistance Database (Stanford HIVdb), and virtual phenotyping, based on the Janssen Diagnostics BVBA’s vircoTYPE™ HIV-1, and investigated their level of agreement in antiretroviral (ARV) naive patients in Asia, where non-B subtypes predominate.Sequences from 1301 ARV-naive patients enrolled in the TREAT Asia Studies to Evaluate Resistance – Monitoring Study (TASER-M) were analysed by both interpreting systems. Interpretations from both Stanford HIVdb and vircoTYPE™ HIV-1 were initially grouped into 2 levels: susceptible and non-susceptible. Discrepancy was defined as a discordant result between the susceptible and non-susceptible interpretations from the two systems for the same ARV. Further analysis was performed when interpretations from both systems were categorised into 3 levels: susceptible, intermediate and resistant; whereby discrepancies could be categorised as major discrepancies and minor discrepancies. Major discrepancy was defined as having a susceptible result from one system and resistant from the other. Minor discrepancy corresponded to having an intermediate interpretation in one system, with a susceptible or resistant result in the other. The level of agreement was analysed using the prevalence adjusted bias adjusted kappa (PABAK).Overall, the agreement was high, with each ARV being in “almost perfect agreement”, using Landis and Koch’s categorisation. Highest discordance was observed for efavirenz , all arising from susceptible Stanford HIVdb versus non-susceptible vircoTYPE™ HIV-1 predictions. Protease Inhibitors had highest level of concordance with PABAKs all above 0.99, followed by Nucleoside Reverse Transcriptase Inhibitors with PABAKs above 0.97 and non-NRTIs with the lowest PABAK of 0.88. The 68/75 patients with discordant efavirenz results harboured the V179D/E mutations compared to 7/1226 with no efavirenz discrepancy . In the 3-level comparison, all but one of the discrepancies was minor.The two systems agreed well with lowest concordance observed for efavirenz. When interpreting HIVDR, especially in non-B subtypes, clinical correlation is crucial, in particular when efavirenz resistance is interpreted based on V179D/E. In recent years, developing countries have experienced a rapid expansion of antiretroviral drugs (ARVs) in the treatment of HIV-1 infection-5, mainlThe aim of the Therapeutics, Research, Education and AIDS Training in Asia (TREAT Asia) Studies to Evaluate Resistance – Monitoring Study (TASER–M) is to monitor HIV-1 drug resistance (HIVDR) and its effects in HIV-infected patients in Asia. The TASER-M cohort consists mainly of ARV-naive Asian individuals infected with HIV-1 AE circulating recombinant form (CRF01_AE). This isThere are several software-based drug resistance interpretation systems currently available, which are used in patient care to design subsequent regimens. The two systems adopted in TASER-M are (i): the Stanford University HIV Drug Resistance Database (Stanford HIVdb) which has a publicly available tool that provides genotypic analysis and interpretation via web-based sequence submission. The Stanford HIVdb uses a rules based system in which all mutation scores are added to derive a level of predicted viral resistance to each ARV. These rules are pre-determined based on research findings of published studies,11; (ii)Because different interpretation algorithms use different rules to predict drug susceptibility, results may differ. Studies have shown that differences do exist with varying degree of discordances-21, and The objective of this study was to determine the yet unstudied level of agreement between genotype and virtual phenotype from the Stanford HIVdb and vircoTYPE™ HIV-1 resistance interpretation in the TASER-M cohort, where non-B subtypes predominate.TASER-M recruitment began in 2007, with a total of 12 participating sites from Thailand, Hong Kong, Malaysia, Philippines and Indonesia. The TASER-M cohort comprises of 95% treatment naive patients at time of enrolment with pre-treatment sequences contributing to more than 90% of total available sequences. Ethics approvals were obtained from the University of New South Wales Ethics Committee and institutional review boards at the participating clinical sites. Informed consent was obtained from participants prior to enrolment. ARV treatment-naive patients were required to have viral load and resistance testing performed prior to initiation of ARVs and at yearly intervals post initiation. GenotypSequences were included in this analysis if they were pre-treatment sequences derived from HIV-infected patients enrolled as treatment-naive in the TASER-M cohort. FASTA sequence files were analysed by both the Stanford HIVdb and vircoTYPE™ HIV-1. FASTA files were submitted to the Stanford HIVdb (Version 6.0.11) through Sierra – The Stanford HIV Web Service (Version 1.0) for genotypic interpretation. Subtyping was analysed using REGA HIV-1 Subtyping Tool - Version 2.0.For virtual phenotyping, FASTA files were submitted to Janssen Diagnostics. The vircoTYPE™ HIV-1 (VPT 4.3.01) results were then transferred to Advanced Biological Laboratories (ABL) TherapyEdge 3.9.2 web based software, and retrieved directly from TherapyEdge 3.9.2. The interpretation of the FC is made in reference to the clinical cut-offs (CCOs) or biological cut-offs (BCOs),27. If tA total of 15 ARV interpretations (7 Nucleoside Reverse Transcriptase Inhibitors (NRTIs), 3 non-NRTIs (NNRTIs) and 5 Protease Inhibitors (PIs)) and were compared. The Stanford HIVdb provides 5 levels of susceptibility prediction for each ARV . The vircoTYPE™ HIV-1 utilises either 2 or 3-level resistance prediction for any given ARV, depending on the cut-offs used.To be able to compare both systems effectively, the 5-level interpretations from the Stanford HIVdb needed to be combined in order to match the levels of interpretation provided by vircoTYPE™ HIV-1. For comparison of all ARVs, 2-level comparisons were performed to enable the inclusion of ARVs where virtual phenotyping only provided predictions at 2 levels. That is, the 3-level interpretations available from the Stanford’s Sierra database , initially grouped from the 5-level interpretation, were combined further into susceptible (susceptible) and non-susceptible (intermediate and resistant). The vircoTYPE™ HIV-1’s susceptible and maximal response were categorised as susceptible whilst reduced response, minimal response and resistant were grouped as non-susceptible. Discrepancy was defined as a discordant drug resistant interpretation between the Stanford HIVdb and the vircoTYPE™ HIV-1 report, using the 2-level interpretations, i.e. susceptible versus non-susceptible, and vice versa.The ARV that exhibited the highest discordances was chosen for further investigation into the mutation pattern and predicted FC values associated with the discrepancies. The mutations were analysed using the Stanford’s HIV Drug Resistance Mutations by Drug Class   was used to analyse the level of agreement between the two interpretation systems. The intMedian values were analysed using Wilcoxon rank sum test. Categorical data was analysed using Chi-square, Fisher’s exact test or McNemar’s test.Analyses were performed using SAS software version 9.2  and STATA software version 10.1 . Ordinal scale PABAK (PABAK-OS) for 3-level interpretation was analysed using PABAK-OS web-based tool.A total of 1301 eligible naive patients consented between 2007 and September 2010 were included. Out of the 1301 baseline sequence files analysed, most were from male patients (66%) with a median age of 36 years (IQR 31–44). The main ethnicity was Thai (71%) followed by Chinese (20%). Heterosexual contact was the predominant mode of exposure (72%). The median pre-treatment CD4 and RNA counts were 100 cells/μL (IQR 34–199) and 100,000 copies/mL , respectively. The main subtype was CRF01_AE (78%). Subtype B accounted for 15% and other non-B subtypes were 7%.The number of discrepant results, using the 2-level interpretation method, between Stanford HIVdb and the vircoTYPE™ HIV-1 for each ARV are illustrated in FigureThe PABAK values Table ranged fAs efavirenz had significantly higher number of discordances compared to other ARVs  and the lowest PABAK, it was chosen for further investigation. The relevant mutations were extracted from the Stanford HIVdb and compared against the Stanford HIVDR list only. 68/75 (91%) patients with discrepant efavirenz results harboured the V179D/E mutations (V179D (83%), V179E (17%)) compared to only 7/1226 (0.6%) of patients with no efavirenz discrepancy . Apart from V179D/E, these 68 patients did not present any other efavirenz RAMs. The other 7/75 (9%) patients who had the discrepancy did not harbour any efavirenz-associated mutations. These 7 discrepant patients may harbour other RAMs that were found to significantly affect susceptibility to efavirenz by the vircoTYPE™ HIV-1’s algorithm, which were not present in the Stanford HIVDR list. Of note, T39K, K43S, K122E, D123S, D177E, Q207A and R211S were the most common RT-mutations (86%) found in these 7 patients with respect to the reference wildtype virus. However no common RT-mutation was identified across all 7 patients. Out of the 75 patients with efavirenz discrepancy, 79% were non-B subtype compared to 85% in patients showing concordance (p=0.135). Among the 1226 patients with no discrepancy, the efavirenz-associated mutations were A98G (0.3%), K103N (0.8%), K103S (0.2%), V106M (0.2%), V179D (0.6%), Y181C (0.6%), G190A (0.3%), G190E (0.1%), P225H (0.2%), F227C (0.1%) and K238T (0.1%). RAMs found in discrepant patients for other NNRTIs were: etravirine (18 discordances): V179D (38.9%), V179E (11.1%), G190A (5.6%) and G190E (5.6%); nevirapine (12 discordances) V179D (75%); V179E (8.3%) and F227C (8.3%). Although G190A/E and F227 were found in a proportion of these patients, a similar pattern to efavirenz could be seen whereby discrepant patients with V179D/E did not harbour any additional NNRTI RAMs.In vircoTYPE™ HIV-1, the BCO was used to determine the level of susceptibility of efavirenz. Therefore only one cut-off point (BCO = 3.3) and two interpretations were available. There was no discrepancy found in patients with FC less than the assigned BCO of 3.3 for efavirenz. That is, all patients with the discrepancies had reported FC greater than 3.3, which resulted in a non-susceptible or resistant virtual phenotype interpretation.In light of 100% efavirenz discrepant patients possessing non-susceptible virtual phenotype results, median FC values were compared between the discordant and concordant group for those with non-susceptible vircoTYPE™ HIV-1 interpretation only. This is because by restricting the comparison to those that resulted in non-susceptible interpretation (FC>3.3), it was possible to make direct comparison without diluting the results with the low FC values whereby no discrepancy was found. There was a total of 102 patients with FC above the BCO of 3.3 for efavirenz. Out of these 102 patients, 27 had no discrepant result, with median FC of 31.90 and inter quartile range (IQR) (7.10-87.10). The median FC for those with discrepancies was 5.10 . This suggests that for patients with non-susceptible virtual phenotype results for efavirenz, the group with the discordances had a median FC that is lower and nearer to the cut-off point than those without discordances.Interpretations were regrouped into susceptible, intermediate and resistant in order to determine the number of minor and major discrepancies. Efavirenz, emtricitabine and nevirapine were excluded. FigureThe results of TableThe overall results suggest that both the Stanford HIVdb and the vircoTYPE™ HIV-1 provide comparable drug resistance interpretations. In the 2-level comparison, efavirenz showed the highest number of disagreements, all of which were Stanford HIVdb susceptible - vircoTYPE™ HIV-1 resistant pairs. The PABAK values for all ARVs considered remained in the “almost perfect agreement” category. The results from the 2-level comparison suggest that the lowest level of agreement belongs to the NNRTI drug class. This is in contrast to previous studies-19 whereThe RAMs associated with efavirenz discrepancy were V179D/E. These are likely to be natural polymorphisms in HIV-1 RT. V179D/E are often found in treatment-naive individuals infected with non-B subtypes,32,33. VSince the mutations were extracted and compared against the Stanford HIVDR list only, it should be noted that the RAMs found to be associated with the discrepancies are those based on the Stanford HIVDR list alone. This is one limitation of the study. The likely presence of additional mutations in vircoTYPE™ HIV-1 coupled with the differences in mutation weights or scores are the most likely causes of the discrepancies. Therefore, the significance of V179D/E found in this study should not be interpreted as the cause of the differences between the two interpreting systems, but merely reflects the mutation pattern based on the Stanford HIVDR list. However, it has been reported that natural polymorphisms found in non-B subtype have contributed to the majority of the discordances, although this was mainly associated with the protease region of the sequence.The 3-level comparison showed that most of the discrepancies found were minor in nature. These may have less clinical importance than the major discordances. The availability of only two virtual phenotype predictions for efavirenz, nevirapine and emtricitabine, another limitation of this study, did now allow for further break down of discordant groups. As such, discrepancies between intermediate and resistant categorisation for these three drugs could not be captured. Nevertheless, the minor discrepancies illustrated in this study suggest that the results did not differ greatly from one system to the other.The differences in median FC between those with and without efavirenz discrepancies suggest that one should take into consideration the actual predicted FC values in addition to the resistance interpretation when evaluating efavirenz resistance. Although discrepancies only occurred in sequences with resistant interpretation, those who were resistant with lower FC tended to result in discordant interpretation compared to those with high FC. Additionally, by simplifying resistance calls into categories, the true value of predicted phenotype is diminished. In the Stanford HIVdb, the 3 or 5-level classification should also be interpreted together with the resistance scores in order to take into account the variation of susceptibility within each classification. It would be of interest to re investigate when the CCOs become available for efavirenz, nevirapine and emtricitabine. It is likely that the FC values associated with efavirenz discrepancies would lie in the middle region between CCO1 and CCO2, resulting in minor discrepancies.This study has shown high concordance between the Stanford HIVdb and vircoTYPE™ HIV-1 in naive patients. The differences in the proportions of discordances from different patient groups reported in previous studies,22,23 anThe authors declare that they have no competing interests.AJ performed data manipulation, analysis plan, statistical analysis, interpretation of results and drafted the manuscript. SS originated the study concept, participated in the analysis plan, data collection, helped interpret results and edited the manuscript. RK participated in the analysis plan, helped interpret results and edited the manuscript. PL, SS, TS, PK, CL, AK, WR, RD, TS performed data collection and revised the final manuscript. All authors read and approved the final manuscript.· PCK Li† and MP Lee, Queen Elizabeth Hospital and KH Wong, Integrated Treatment Centre, Hong Kong, China;· N Kumarasamy and S Saghayam, YRG Centre for AIDS Research and Education, Chennai, India;· S Pujari and K Joshi, Institute of Infectious Diseases, Pune, India;· TP Merati‡ and F Yuliana, Faculty of Medicine, Udayana University & Sanglah Hospital, Bali, Indonesia;· A Kamarulzaman and LY Ong, University Malaya Medical Center, Kuala Lumpur, Malaysia;· C KC Lee and B HL Sim, Hospital Sungai Buloh, Sungai Buloh, Malaysia;· M Mustafa and N Nordin, Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia;· R Ditangco and RO Bantique, Research Institute for Tropical Medicine, Manila, Philippines;· YMA Chen, YJ Chen and YT Lin, Taipei Veterans General Hospital and AIDS Prevention and Research Centre, National Yang-Ming University, Taipei, Taiwan;· P Kantipong and P Kambua, Chiang Rai Regional Hospital, Chiang Rai, Thailand;· P Phanuphak and S Sirivichayakul, HIV-NAT/Thai Red Cross AIDS Research Centre, Bangkok, Thailand;· W Ratanasuwan and R Sriondee, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand;· T Sirisanthana and J Praparattanapan, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand;· S Sungkanuparph, S Kiertiburanakul, and L Chumla, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand;· R Kantor, Brown University, Rhode Island, U.S.A.;· AH Sohn, N Durier and T Singtoroj, TREAT Asia, amfAR -- The Foundation for AIDS Research, Bangkok, Thailand;· DA Cooper, MG Law, and A Jiamsakul, The Kirby Institute, University of New South Wales, Sydney, Australia.† Steering Committee Chair, ‡ Co-ChairThe content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above.


Drug-related toxicity has been one of the main causes of antiretroviral treatment discontinuation. However, its determinants are not fully understood. Aim of this study was to investigate predictors of first-line antiretroviral therapy discontinuation due to adverse events and their evolution in recent years.Patients starting first-line antiretroviral therapy were retrospectively selected. Primary end-point was the time to discontinuation of therapy due to adverse events, estimating incidence, fitting Kaplan-Meier and multivariable Cox regression models upon clinical/demographic/chemical baseline patients’ markers.1,096 patients were included: 302 discontinuations for adverse events were observed over 1,861 person years of follow-up between 1988 and 2010, corresponding to an incidence (95% CI) of 0.16 (0.14-0.18). By Kaplan-Meier estimation, the probabilities (95% CI) of being free from an adverse event at 90 days, 180 days, one year, two years, and five years were 0.88 (0.86-0.90), 0.85 (0.83-0.87), 0.79 (0.76-0.81), 0.70 (0.67-0.74), 0.55 (0.50-0.61), respectively. The most represented adverse events were gastrointestinal symptoms (28.5%), hematological (13.2%) or metabolic  (11.3%) toxicities and hypersensitivity reactions (9.3%). Factors associated with an increased hazard of adverse events were: older age, CDC stage C, female gender, homo/bisexual risk group , HBsAg-positivity. Among drugs, zidovudine, stavudine, zalcitabine, didanosine, full-dose ritonavir, indinavir but also efavirenz  were associated to an increased hazard of toxicity. Moreover, patients infected by HIV genotype F1 showed a trend for a higher risk of adverse events.After starting antiretroviral therapy, the probability of remaining free from adverse events seems to decrease over time. Among drugs associated with increased toxicity, only one is currently recommended for first-line regimens but with improved drug formulation. Older age, CDC stage, MSM risk factor and gender are also associated with an increased hazard of toxicity and should be considered when designing a first-line regimen. Combination antiretroviral therapy (cART) has markedly changed the prognosis of HIV-infected patients, reducing AIDS-related morbidity and mortality . Rates oTreatment-limiting cART toxicity has been associated with several factors such as demographical characteristics, drug-drug interactions, co-morbidities and recently genetic factors . HoweverThe aim of this study is to investigate predictors of first-line antiretroviral therapy discontinuation due to adverse events and their evolution in more recent years, characterized by an increased use of regimens with better tolerability profiles. In particular, the analysis is focused on the evaluation of baseline demographic and clinical characteristics, prescribed drugs and chemical parameters which could represent objective tools to tailor regimens on patients’ characteristics.HIV-infected patients followed up at the Infectious Diseases Clinic of the Catholic University of the Sacred Heart (CUSH) in Rome, Italy, starting a first-line anti-HIV antiretroviral therapy, were screened retrospectively via the electronic CUSH data base, which includes data on more than 4,300 HIV-infected patients, with the first available anti-HIV therapy record dated 1988. All patients included in the data base had previously signed an informed consent to be included in observational studies. Access and data analyses of the CUSH data base are regulated by an institutional internal ethics committee and conform to Italian and European privacy legislations. The latest available updated version (up to January 2011) of the CUSH data base was used.The baseline time was the start date of the anti-HIV antiretroviral therapy. The end-point of interest was the discontinuation date of the first-line anti-HIV antiretroviral therapy. Discontinuation was defined as stopping any antiretroviral drug for at least 2 weeks or switching (changing one or more drugs) to another regimen; the only exception was a substitution of lamivudine with emtricitabine or vice-versa, because it could simply reflect a shift to a fixed dose formulation of tenofovir-emtricitabine, abacavir-lamivudine or zidovudine-lamivudine. An adverse event was marked if the reason of discontinuation or switch  was an event corresponding to toxicity or allergy; otherwise data were censored at that time point or at the latest available time point if the patient did not stop that therapy, following a cause-specific approach. All discontinuation causes were ascertained by electronic and clinical reports , hepatitis B co-infection (HBsAg), anti-HCV interferon/ribavirin treatment, anti-Mycobacterium Tuberculosis (TB) therapy, anti-Pneumocystis jirovecii pneumonia (PCP) therapy, other antibiotic treatments, other concomitant (prescription or over-the-counter) drugs exposure, total bilirubin, total cholesterol, hemoglobin, glucose, glutamate pyruvate transaminases (GPT), gamma-glutamyltransferase (gammaGT), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TGL), and glomerular filtration rate (GFR) estimated by modification of diet in renal disease (MDRD) formula. Chemistry parameters were classified as normal, high or low according to established cut-offs [Covariates of interest, contemporary or the closest previous to the baseline date, were: calendar year, patient’s gender, age, nationality, risk group, first date of HIV-positive antibody test, CDC stage, viral subtype, anti-HIV antiretroviral therapy, HIV-RNA load, CD4cut-offs ,14  as a combination therapy of 2 nucleoside reverse transcriptase inhibitors (NRTI) + 1 non-nucleoside reverse transcriptase inhibitor (NNRTI), 2 NRTI + 1 protease inhibitor (PI), 2 NRTI + 1 PI boosted with ritonavir (PI/r), and other combinations; (ii) as single compounds, i.e. emtricitabine, lamivudine, abacavir, zidovudine, stavudine, zaltacibine, didanosine, tenofovir, nevirapine, efavirenz, atazanavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir full or boosting dose, and saquinavir; (iii) as a combination therapy of different brand names, including those combinations with a frequency of at least 10; (iv) as Truvada® + Sustiva®, Atripla®, Reyataz®, Kaletra®, any other NRTI+PI, any other NRTI+PI/r, any other NRTI+NNRTI, and other combinations. Other compounds including enfuvurtide, maraviroc, raltegravir, darunavir, and etravirine, were not included due to their low frequency.http://www.r-project.org).Statistical methods included descriptive summaries, calculation of incidence ratios and confidence intervals, Kaplan-Meier estimation for the probability of surviving an adverse event, multivariable proportional-hazard hypothesis testing, and multivariable Cox model fitting in order to identify factors associated to an increased/reduced hazard of an adverse event happening. Effect of covariate interactions and nested model comparison via ANOVA were also carried out. All statistical analyses were carried out using R software  was 4,388. Those with at least one anti-HIV therapy registered were 2,455, and 1,218 for which it was the actual first-line. Of these, 1,096 had a documented reason of discontinuation or did not stop the therapy and met the inclusion criteria, starting their first-line anti-HIV therapy between year 1988 and 2010 . Of these patients, 317 started the first-line anti-HIV therapy prior to 1999 (154 before 1997), whilst 779 afterwards  was 302, over 1,861 person years of follow up, corresponding to an incidence  of 0.16 (0.14-0.18) per person years of follow up (PYFU). Table Table Also, in order to analyze the possible role of the introduction in Italy of tenofovir (November 2002), Truvada® (September 2005), and Atripla® (October 2008), the calendar year was stratified into: 1988–1996, 1997–1998, 1999–2002, 2003–2005, 2006–2008, and 2009–2010. However, this did not lead to any appreciable difference across time periods.Of note, a trend toward an increased risk of adverse events was observed for HIV genotype F1 (when compared to genotype B). Moreover, unknown TGL showed a higher RH as compared to the corresponding “normal” (<150 mg/dL) category. We also formally tested for interactions between the calendar year and the efavirenz intake, using an ANOVA comparison on the nested models (i.e. with/without interaction), but the more complex model did not show a higher likelihood (p=0.5); the interaction term was not significant and the significant effect of efavirenz was canceled out.+ T cell counts, different Cox regression models were fit with either the sole CD4+ or log(CD4+) cell/mm3 count or including the full covariate set with the exception of the CDC stage. Only for the univariable log(CD4+) regression there was a significant (p<0.05) decreased hazard of an adverse event per log(CD4+) higher .In order to better assess the role of CD4As a sensitivity analysis, we repeated the multivariable Cox regression using the subset of therapies posterior to 1998 , including only cART regimens . The described relative hazards did not change, and in addition there was a significant reduced hazard of an adverse event for any other/unknown risk group as compared to the heterosexual category , and a trend of increased hazard for nevirapine , fosamprenavir , and nelfinavir . Moreover, in this model a significant higher risk of discontinuation for adverse events was demonstrated for HIV genotype F1 , while a trend was observed for genotype C .Since adverse events are the most frequent reason for first-line antiretroviral therapy discontinuation or switch, investigation of variables associated with their occurrence in a routine clinical practice setting is of increasing interest. Such an understanding is crucial to tailor antiretroviral regimens on patients’ characteristics in order to increase the probability of cART tolerability.In this study several demographic, clinical, laboratory and cART-related variables were investigated, providing useful information for the management of antiretroviral therapy. The prescribed antiretroviral drugs are main determinants of treatment discontinuation for toxicity. As expected, the use of older drugs such as zidovudine, stavudine, zalcitabine, didanosine and full-dose ritonavir was associated with increased risk of adverse events. However, these drugs are no longer recommended for first-line therapy because of their greater potential for toxicity . When coIn this study, female patients showed a higher risk of treatment discontinuation for drug-related side effects, in accordance with previous results ,21 . ThiAn interesting finding is the increased risk of discontinuation for adverse events observed in men who have sex with men. This could be ascribed to a different perception of side effects eventually related to sociocultural barriers, as described among vulnerable populations .+ cells count or viral load.Unlike previous studies ,3, in ouPatients harboring HIV genotype F1 demonstrated an higher risk of treatment discontinuation for adverse events and a trend toward an association was observed for genotype C. F1 subtypes have been mostly detected in Latin America , where tIn agreement with previous studies , we confPrevious studies suggested that prescription of concomitant medications used for the treatment of opportunistic infections could increase the risk of discontinuation owing to cumulative toxicities or drug interactions . In our Co-infection with HBV or HCV have been claimed as a predisposing factor for treatment discontinuation due to hepatic adverse events ; howeverThe calendar year was not showing an impact on the hazard of toxicity/allergy events. A similar observation has been previously described in other cohorts ,27. Thishttp://www.mastercohort.it) and Icona (http://www.fondazioneicona.org) foundations, and in Europe, including the Copenhagen HIV Programme and EuroSIDA (http://www.cphiv.dk) record such detailed information for all patients, besides the standard HIV serologic markers [The potential association of treatment discontinuation for side effects and baseline chemical parameters has not been investigated in previous studies. Chemistry characteristic can represent important tools on the basis of which clinicians can choose the appropriate antiretroviral regimens in order to limit the occurrence of toxicities. In our population, we did not observe any association with the explored parameters; however, the relevant proportion of missing data could have influenced such finding masking the real effects in terms of increased/reduced hazard of adverse events occurrence. Moreover, only baseline markers were analyzed, while the effect of current markers may be crucial, for instance the raise levels of bilirubin during atazanavir-containing antiretroviral therapy . We deci markers ,16.Some limitations should be recognized when interpreting the results of our study. First, cART toxicity was analyzed considering only treatment modification, but clinical events or alterations in laboratory parameters not leading to treatment discontinuation were not investigated, thus leading to an underestimation of overall drug-related side effects. There might be a population bias, since the first-line anti-HIV therapies could have been tuned appositely on the patients’ background, basing on the guidelines available at that time. This can be truer for drug combinations circulating for a longer time, whose adverse effect have been deeply disclosed. Since this study is retrospective, the bias cannot be ruled out even using strong selection criteria and a patient cohort with a high level of detail and follow up.Another possible limitation of this study is that we conducted a cause-specific analysis, ignoring the potential effects of competing events in the survival analysis; such events may drive the happening of adverse effects, and surely a design of a competing-risk analysis after this study is warranted.Given the current, fair, rate of virologic and immunologic successes of first-line anti-HIV antiretroviral therapies in the European Union and other developed countries -32, alonIn conclusion, several predictors of treatment discontinuation for drug-related adverse events were investigated. These findings could help clinicians to identify individuals at higher risk of developing toxicity, thus allowing an improved prescription of antiretroviral regimens tailored on patients’ characteristics. Moreover, these results suggest the importance of anticipating the probability of occurrence of adverse events in order to ensure close clinical and laboratory monitoring and adequate management of side effects which could improve the durability of cART.Pneumocystis jirovecii pneumonia; PI: Protease inhibitor; PI/r: PI boosted with ritonavir; PYFU: Person years of follow up; RH: Relative hazard; TB: Tuberculosis; TGL: Triglycerides; 95% CI: 95% Confidence Interval.cART: Combination antiretroviral therapy; CI: confidence intervals; CUSH: Catholic University of the Sacred Heart; gammaGT: gamma-glutamyltransferase; GPT: Glutamate pyruvate transaminases; GFR: Glomerular filtration rate; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; MDRD: Modification of diet in renal disease formula; NNRTI: Non-nucleoside reverse transcriptase inhibitor; NRTI: Nucleoside reverse transcriptase inhibitors; PCP: MF received speakers’ honoraria from Abbott Virology, Merck Sharp and Dohme and Janssen-Cilag. MZ received speakers’ honoraria from Abbott Virology, Merck Sharp and Dohme, Janssen-Cilag, Bristol-Myers Squibb and Gilead Science. MC was an employee of Bristol-Myers Squibb from May 2010 to February 2011 and resigned before starting the present work. RC was advisor for Gilead and Janssen-Cilag, received speakers’ honoraria from ViiV, Bristol-Myers Squibb, Merck Sharp and Dohme and Janssen-Cilag, and research support from “Fondazione Roma”. SDG received speakers’ honoraria from ViiV, Bristol-Myers Squibb, Merck Sharp and Dohme and Janssen-Cilag. All other authors: none to declare.MCFP contributed to study design, data analysis and interpretation, and article writing; MF and MZ contributed to interpretation of data and article writing; IF, MC, AM, ADA, AB contributed to data collection; RC and SDG coordinated the project and contributed to interpretation of data. All authors reviewed the manuscript during preparation, provided critical feedback and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/296/prepubDistribution of first-line anti-HIV therapies by calendar year.Click here for file


There is little information on adherence to antiretroviral therapy (ART) in the Eastern European region. This prospective study evaluated multiple measures of adherence and their association with viral suppression among HIV patients in Georgia.A prospective cohort study enrolled 100 consecutive antiretroviral-naïve adult (age ≥18 years) patients, who were followed for three months. Adherence was assessed by medication refill and three self-report measures . The VAS represented a line anchored by 0 and 100% corresponding to the percentage of prescribed doses taken. The rating task asked patients to rate their ability to take all medications as prescribed, with responses categorized into six levels of adherence: very poor (0%), poor (20%), fair (40%), good (60%), very good (80%) and excellent (100%). Patients with adherence of ≥95% by medication refill, ACTG and VAS, and ≥80% by rating task, were defined as adherent.Of 100 patients enrolled, eight had missing data and were excluded from analysis. Among the remaining 92 patients, the median age was 39 years, and 70% were men. Major modes of HIV acquisition were injection drug use  and heterosexual contact (44.1%). The proportions of adherent patients were as follows: 68% by medication refill, 90% by ACTG questionnaire, 38% by VAS and 42% by rating task. On average, four months after commencing ART, 52 (56.5%) patients had a viral load <400 copies/ml and 26 (28.3%) patients had a viral load <50 copies/ml. Of 43 persons with a history of IDU, 22 (51.2%) reached a viral load of <400 copies/ml. In multivariate analysis, only refill adherence was a statistically significant predictor of viral suppression of <400 copies/ml: the risk ratio was 1.7 (95% CI: 1.1–2.8). Refill adherence, VAS and rating task were associated with viral suppression of <50 copies/ml. Non-IDUs were twice as likely to achieve viral load <50 copies/ml compared to IDUs. Refill adherence had the largest area under the receiver-operating characteristic curve for predicting viral suppression.Medication refill adherence was the strongest predictor of viral suppression. IDUs can achieve optimal virologic outcomes, but may require additional adherence support. Access to antiretroviral therapy (ART) is rapidly expanding worldwide . People Adherence assessment methods commonly used in clinical trials and observational studies include electronic drug monitoring, prescription (medication) refills, pill counts and various self-reported methods. Electronic monitoring and unannounced pill counts were shown to be the best objective measures for real-time assessment of adherence . HoweverFormerly part of the Soviet Union, Georgia is an independent nation situated in the Caucasus region. Similar to other former Soviet Union (FSU) countries, the HIV epidemic in Georgia is closely tied to injection drug users (IDUs), who account for 54% of all reported HIV infections. As of 31 December 2012, a cumulative 3642 cases of HIV infection were reported in Georgia. Antiretroviral drugs have been available in the country since the 1990s, but the access was limited only to those able to afford them. However, beginning in 2004, through support from the Global Fund, Georgia became the only FSU country to initiate universal access to ART . IDU hasThe prospective cohort study was conducted at the Infectious Diseases, AIDS and Clinical Immunology Research Center (IDACIRC) in Tbilisi, Georgia, which is the country's referral institution for HIV diagnosis, treatment and care. The study included 100 consecutive antiretroviral-naïve adult (age ≥18 years) patients first initiating ART at the IDACIRC during the period from June 2010 to December 2010. Patients were approached after the first refill of their ART prescription at IDACIRC. Consenting patients were followed for three months after enrolment. The protocol included an enrolment interview and three follow-up adherence assessments at monthly intervals during drug refill visits. The main outcome measures were adherence to ART assessed by four different methods, and viral suppression was defined as plasma HIV-1 RNA levels  of <400 copies/ml and <50 copies/ml.The National ART Program is coordinated by the IDACIRC. HIV-related treatment and care services are provided at the IDACIRC clinic in the capital city of Tbilisi as well as at three affiliated regional centres in the cities of Kutaisi, Batumi and Zugdidi. All persons living with HIV in Georgia are referred to and followed by this centre and its affiliates.3 as well as those with hepatitis B or C and tuberculosis (TB) regardless of CD4 count.Provision of therapy is governed by the National HIV/AIDS Treatment and Care guidelines, which were developed based on the guidelines of the World Health Organization (WHO) European Region. At the time the study was conducted, ART was indicated in all patients with CD4 <200 cells/mmThe recommended initial regimen consists of two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI). A ritonavir (r)-boosted protease inhibitor (PI) is recommended in cases when an NNRTI cannot be prescribed. As per the Georgian national guidelines, the standard of ART monitoring relies upon laboratory monitoring of CD4 count, HIV-1 viral load and resistance assessment based on resistance genotype detection when indicated.Antiretroviral drugs are dispensed at four centres at monthly intervals. Monthly drug pick-up encounters serve as important points for adherence assessment by self-report, and refill adherence using pharmacy records is used to monitor drug pick-up practices. Patients demonstrating suboptimal adherence  receive additional home-based adherence support by mobile units operating countrywide.Adherence was treated as a continuous variable expressed as a percentage. Adherence was assessed by medication refill and three self-report measures. Provisions were made to maximize honest responses, including selecting interviewers who were not directly involved in ART provision and ensuring the confidentiality of data.Medication refill: Medication (or prescription) refill adherence was the objective measure of adherence used in the study. Adherence was defined as the number of days for which the supply of medication was dispensed divided by the days between prescription fills : 55–194), and the median viral load was 5.7 log10 copies/ml (IQR: 5.2–6.1). The majority of patients were started on an NNRTI-based regimen (88%), and the most commonly prescribed NRTI backbone was zidovudine (AZT)+lamivudine (3TC).Mean adherence as measured at each monthly assessment did not vary significantly over the study period. Therefore, the third-month adherence measurements were used in analyses. At the end of follow-up, the mean medication refill adherence was 94%. For self-reported measures, the mean adherence was 97% by four-day ACTG questionnaire, 89% by VAS and 67% by rating task. The proportion of patients showing ≥95% adherence was 68% by medication refill, 90% by four-day ACTG questionnaire and 38% by VAS. Overall, 85, 91 and 74% of patients showed adherence of at least 90% by medication refill, four-day ACTG questionnaire and VAS, respectively. Forty-two percent of patients showed ≥80% adherence by rating task .Response distribution to self-reported measures was as follows: 83 (90.2%) patients reported perfect (100%) adherence by four-day ACTG questionnaire; the most common response to rating task was “good” (48 [52.2%] patients), and none reported perfect adherence; and the most frequent response to VAS was 90% – for 33 (35.9%) patients – and only 6 (6.5%) patients rated their adherence as 100%.The mean medication refill adherence was similar when compared with sociodemographic characteristics, history of substance abuse or comorbidities. Exploring differences in the percentage of adherent patients showed that a greater proportion of females and non-substance abusers were ≥95% adherent by medication refill. There were no differences in the proportions of adherent patients when medication refill adherence was dichotomized at the 90% level . Females10 copies/ml (p<0.0001). Fifty-two (56.5%) patients had a viral load <400 copies/ml, and 26 (28.3%) patients had a viral load <50 copies/ml. Of 43 persons with a history of IDU, 22 (51.2%) reached a viral load of <400 copies/ml and six (13.9) completely suppressed a viral load below 50 copies/ml. There was a negative correlation between adherence and viral load. Correlations were significant for all studied measures .The association between adherence and plasma HIV viral load was also evaluated. The viral load was measured on average four (IQR: 3–5) months after commencing ART, and 11 (11.9%) patients had their viral load measured at least six months after initiating therapy. The mean viral load drop from baseline was 2.9 logp<0.0001) and rating task (p=0.04), but not compared to the area for VAS (p=0.43). There was also a statistically significant difference between areas generated by VAS and four-day ACTG (p=0.01). No difference was found between four-day ACTG and rating task.The area under the ROC curves for predicting undetectable viral load for both levels of suppression (<400 copies/ml and <50 copies/ml) was larger for refill adherence compared to other measures . Howeverp=0.04). Other significant predictors of viral suppression were a baseline viral load <100,000 copies/ml and an AZT-based regimen. Active IDUs showed a confounding effect only in the four-day ACTG model ; VAS, an RR of 2.9 ; and rating, an RR of 2.4 . Histories of IDU and AZT-based ART regimens were significantly associated with the outcome in all three models. Non-IDUs were more than twice as likely to achieve complete viral suppression compared to IDUs. Although gender was significantly associated with the outcome in bivariate analysis, it has lost significance in multivariate analysis, likely due to correlation with substance abuse variables.Predictors of complete viral suppression (<50 copies/ml) were assessed for refill adherence, VAS and rating task . None ofIn this study, we evaluated the performance of four measures of adherence, including a medication refill–based objective measure and three self-reported methods. The mean medication refill adherence was 94%, and 68% of patients demonstrated adherence of at least 95%, which is comparable to adherence reported elsewhere . MoreoveResponses to self-reported measures varied substantially. Findings of our study are consistent with previous reports, indicating that multi-item self-report methods with shorter recall periods (≤7 days) tend to overestimate actual adherence ,16,30–32Similar to the findings in other studies, substance abuse was associated with lower levels of adherence –36. LoweIn our cohort, women showed better adherence than men by all measures, except for the four-day ACTG model. This could be explained by the fact that none of the women had a history of substance abuse and the difference diminished after adjusting for IDU. This finding underlines the role that substance use plays in observed gender-specific differences .Our study design allowed us to evaluate adherence measures in relation to virological outcome. All adherence measures correlated with viral suppression. ROC statistics for predicting viral suppression at both the <400 and <50 copies/ml levels were larger for refill adherence, followed by VAS. Refill adherence and VAS yielded significantly larger areas under the curve compared to four-day ACTG and rating task at a viral suppression level of <50 copies/ml. In multivariate analysis, refill adherence was the strongest predictor of viral suppression, and it was the only adherence measure significantly associated with achievement of a viral load <400 copies/ml.Other factors associated with viral suppression included a lower baseline viral load  and the use of a zidovudine (AZT)-containing regimen. While the influence of baseline viral load is well described, the association found between the use of an AZT-containing regimen and viral suppression should be interpreted with caution. Regimens were selected by treating physicians based on patient characteristics and clinical conditions. Consequently, the observed difference could have resulted from these and other unmeasured confounding factors, and may not represent a real difference.Because of the small number of active substance abusers, the ability to evaluate associations with the outcome was limited. Patients with and without a history of IDU had similar rates of viral suppression at levels <400 copies/ml, but non-IDUs were more likely to achieve viral load levels <50 copies/ml. Overall, these findings suggest that undetectable viral load is achievable among IDUs, but this population may require additional adherence support to achieve complete viral suppression.Our study is subject to several limitations. We used medication refill data as an objective measure of adherence, which estimates maximum possible adherence without accounting for the possibility that patients may not take all refilled medications. Therefore, refill adherence is also prone to bias, but it is more accurate than self-reported methods. This study included ART-naïve patients who were followed for the first three months of therapy. In addition, the majority of patients had advanced diseases and such patients might bemore motivated, demonstrating better adherence early in the course of ART. High early adherence may wane over time, influencing virologic outcomes as well ,38. HoweAnother limitation is the small sample size, which limits the generalizability of our findings and also negatively influenced statistical power to assess associations in certain population subgroups, such as active substance abusers. In addition, of 100 initially enrolled patients, eight were excluded from analyses. Because of the small number of patients with missing data, we decided to exclude them rather than impute missing data. Also, the dramatic differences in the proportions of patients showing >95% adherence , as well as no 100% adherence by rating task, raise questions regarding whether patients had fully understood the self-reported measures. Interviewers were appropriately trained on how to instruct patients and correctly administer tools; however, the possibility of misunderstanding by patients cannot be ruled out.Our study has important clinical and public health implications. This study confirmed that medication refill adherence is a useful tool for predicting virological outcomes and can be used for monitoring adherence at both patient and programme levels ,13,41. BTo the best of our knowledge, this is the first prospective evaluation of multiple adherence measures reported from countries of the FSU. This region has the fastest growing HIV epidemic among IDUs and the lowest ART coverage . With th


The current expansion of antiretroviral treatment (ART) in the developing world without routine virological monitoring still raises concerns on the outcome of the strategy in terms of virological success and drug resistance burden. We assessed the virological outcome and drug resistance mutations in patients with 36 months’ ART experience, and monitored according to the WHO public health approach in Cameroon.We consecutively recruited between 2008 and 2009 patients attending a national reference clinic in Yaoundé – Cameroon, for their routine medical visits at month 36±2. Observance data and treatment histories were extracted from medical records. Blood samples were collected for viral load (VL) testing and genotyping of drug resistance when HIV-1 RNA≥1000 copies/ml.Overall, 376 HIV-1 infected adults were recruited during the study period. All, but four who received PMTCT, were ART-naïve at treatment initiation, and 371/376 (98.7%) started on a first-line regimen that included 3TC +d4T/AZT+NVP/EFV. Sixty-six (17.6%) patients experienced virological failure (VL≥1000 copies/ml) and 53 carried a resistant virus, thus representing 81.5% (53/65) of the patients who failed. Forty-two out of 53 were resistant to nucleoside and non-nucleoside reverse-transcriptase inhibitors (NRTIs+NNRTIs), one to protease inhibitors (PI) and NNRTIs, two to NRTIs only and eight to NNRTIs only. Among patients with NRTI resistance, 18/44 (40.9%) carried Thymidine Analog Mutations (TAMs), and 13/44 (29.5%) accumulated at least three NRTI resistance mutations. Observed NNRTI resistance mutations affected drugs of the regimen, essentially nevirapine and efavirenz, but several patients  accumulated mutations that may have compromised etravirine use.We observed a moderate level of virological failure after 36 months of treatment, but a high proportion of patients who failed developed drug resistance. Although we found that for the majority of patients, second-line regimens recommended in Cameroon would be still effective, accumulated resistance mutations are of concern and may compromise future treatment strategies, stressing the need for virological monitoring in resource-limited settings. Antiretroviral therapy (ART) has significantly reduced morbidity and mortality in human immunodeficiency virus type 1 (HIV-1)-positive patients in both industrialized and resource-poor countries. Because ART can fail as a result of toxicity, pretreatment HIV-1 drug resistance, insufficient patient adherence or incomplete suppression of viral replication leading to the emergence of drug-resistant viruses, adequate clinical and biological management can significantly improve treatment outcome and can prevent rapid failure . CurrentDespite these limitations in ART access and monitoring, recent studies assessing the outcome of ART in the developing world have shown significantly good results, with good virological success achieved after 12 and/or 24 months of ART, and even limited consequences of observed drug resistance mutations for second-line options . In addiSince the 2000s, ART access in Cameroon has been significantly improved through the implementation of the WHO simplify approach and the decentralization of ART services. The standard first-line therapy consists of two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-NRTI (NNRTI), and until 2010 when WHO recommended the replacement of stavudine with tenofovir, reference first-line antiretrovirals (ARVs) in Cameroon included zidovudine or stavudine plus lamivudine as NRTIs and nevirapine or efavirenz as NNRTIs. First- and second-line treatments have been freely provided to eligible patients since 2007, and treatment initiation and monitoring has been guided by clinical and/or immunological data.In this study, we evaluated the long-term virological outcome and implications for second-line regimens after 36 months’ ART in patients treated according to the WHO public health approach in Cameroon.From September 2008 to September 2009, we conducted a cross-sectional study among ARV-treated patients attending a reference treatment unit, the “Hôpital de Jour” of the Yaoundé Central Hospital. In this unit, patients received ART as per national recommendations, and the decision to treat or switch is mostly guided by clinical and/or immunological assessments. We consecutively enrolled 376 HIV-positive adults who attended the clinic for their follow-up visit after 36 months’ ART (±two months). Patients who agreed to participate were invited to sign an informed consent, and we administrated a standardized questionnaire to collect patients’ demographic, epidemiological and clinical data. We collected information on the last CD4 count test performed and the number of viral load (VL) done since ART start. The Cameroonian National Ethics Committee approved the study, and efforts were made to guarantee patients confidentiality throughout the study.http://www.hivfrenchresistance.org/).We collected from each patient 10 mL of whole blood from which plasma specimens were recovered after centrifugation and stored at −80°C. Because of the circulation of almost all identified HIV-1 groups in Cameroon and documentation of few HIV-2 strains, we performed an ELISA-based serotyping assay to differentiate between HIV-1 group M, N and O infections, as well as between HIV-1 and 2 infections . A virolThe newly reported protease and reverse transcriptase sequences are available in GenBank under the following accession numbers: JX982979 to JX983043.Within the study period, we recruited 376 HIV-1 infected adults, who met the criteria of having 36±2 months’ ART experience and who were attending the clinic for their routine monitoring visit. Two-thirds of these patients  were women, and the median age (interquartile range) was 40 (33–47) years. Almost all patients performed a CD4-T cells count within the last six months before recruitment, and the median CD4-T cells count (interquartile range) was relatively high: 373 (221–502) cells/µl. WHO clinical stages varied from stage 1 to stage 4, the majority of patients being at stage 3, 142 patients (37.8%), followed by stage 2, 4 and 1 at 90 (23.9%), 79 (21.0%) and 4 patients (1.1%), respectively . We founNone of the recruited patients reported previous exposure to ARVs before the initiation of ART, except for four women who received ARVs as prevention of mother to child transmission of HIV (PMTCT). PMTCT regimens included nevirapine only (one patient), zidovudine plus nevirapine (two patients) and stavudine plus lamivudine plus nevirapine (one patient). Three hundred and seventy one (98.7%) patients received lamivudine plus stavudine/zidovudine plus nevirapine/efavirenz, and only five (1.3%) patients received other regimens, including lamivudine plus didanosine plus efavirenz and indinavir plus lamivudine plus zidovudine . About hn=376) to evaluate virologic outcome after 36 months’ ART. All patients carried HIV-1 group M virus. Two hundred and ninety out of the recruited 376 (77.1%) patients had a VL level bellow the assay detection limit, 300 copies/ml, and the median HIV-1 RNA level for results>300 copies/ml was 4.79 (3.33–5.55) Log10 copies/ml (n=44), A (n=7), D (n=3), F2 (n=3), CRF11_cpx (n=3), C (n=1), G (n=1), H (n=1), CRF06_cpx (n=1) and other recombinant (n=1). Among the 65 patients with successful genotyping, 53 (81.5%) carried a virus with at least one major DRM. Up to 73.6% (39/53) were women, thus correlating with the gender ratio of our study population.We measured HIV-1 RNA level for all patients  were male and 30 (71.4%) were women. One patient was resistant to PIs and NNRTIs, two women to NRTIs only and eight  to NNRTIs only. Only one patient had a PI resistance mutation, L90M, and although this patient experienced several treatment substitutions, he received no PI. Almost all NRTI resistant patients carried the M184V mutation (43 out of 44), and 18 (40.9%) carried at least one Thymidine Analog Mutation (TAM); 13 (29.5%) accumulated at least three NRTI DRM, including several TAMs and, therefore, developed resistance to drugs of their regimen as lamivudine/emtricitabine or stavudine, but this accumulation also compromised other drugs that were not included in ART regimens as tenofovir and/or abacavir . Among tWe reported in this study the virological outcome and HIV drug resistance among patients followed-up in a national HIV treatment centre in Yaoundé – Cameroon, and who stayed under ART for 36±2 months. In the same clinic, we conducted a cross-sectional study in 2006–2007 to assess ART outcome and selection of drug-resistant viruses after 12 or 24 months of ART and found virological failures (VL≥1000 copies/ml) of 16.4% (41/249) and 22.5% (40/178), respectively, at months 12 and 24. We showed in that previous study that at month 24, up to 79% (30/38) of patients who failed, representing 16.9% of all patients recruited at month 24, carried at least one major DRM, while only 32.4% (11/34), 4.4% of all patients at month 12, developed drug resistance, thus illustrating significant adherence issues for these patients, since the majority of those failing treatment after 12 months had no resistant virus [However, in this study we found that patients failing ART with a resistant virus significantly accumulated DRMs, especially NRTI mutations and TAMs, probably because they stayed longer under failing regimens, correlating with similar studies in Africa –13. As aWe had one patient with a PI mutation, L90M, with an unclear origin since the patient did not receive any PI. Our analysis was limited by the lack of baseline data, including genotypic information before ART initiation, but threshold surveys conducted within the study period in Yaoundé revealed a low (<5%) level of transmitted PI-resistance mutations and low to moderate 5%–15%) rates of transmitted RTI-resistance mutations % rates o, or fromContrary to several recent reports assessing failure to stavudine-containing first-line regimens in developing countries, we found no K65R mutation or Q151M complex known as compromising almost all NRTIs ,17. SeveIn conclusion, studies evaluating virological outcome after 36 months of ARV treatment in patients routinely managed in national ART programmes from developing countries are currently rare. Despite the absence of routine virological management, we did not find a dramatic level of failure. Also, we showed that second-line ART will work well for the majority of patients who developed resistance, and although illustrating the situation of a specific clinic and not the entire national programme, these results indicate that even under the public health approach, long-term virological success could be achieved for the majority of patients. Nonetheless, significant challenges still exist and should be rapidly addressed. Rapid identification of failure to prevent accumulation of resistance mutations is essential, and availability of simple and affordable viral load tools is important. In addition, programme management should be improved to reduce unnecessary drug substitutions due to drug stock-outs, voluntary interruptions and ignorance as we reported here.