The levels of these markers decreased significantly after initiation of ART. In patients with CD4 >200 cells/μL, these markers are helpful in predicting disease progression.A prospective study was carried out to evaluate the efficacy of serum neopterin and soluble IL-2 receptor (sIL-2R) concentrations in comparison to CD4 count to study the progression of HIV disease and monitor response to ART in HIV cases. One hundred newly diagnosed HIV seropositive subjects were recruited. CD4 counts were determined by FACS system. Serum neopterin and sIL-2R levels were measured using enzyme immunoassay. In our study, levels of neopterin and sIL-2R were significantly higher in subjects with CD4 <200 cells/ HIV/AIDS continues to exact an enormous toll throughout the world, in both human and economic terms, posing a serious impediment to the growth and economic stability of many developing countries .Infection with HIV-1 produces a prolonged, gradually progressive disease which leads to opportunistic infections and eventually death. The likelihood and timing of development of clinical AIDS following seroconversion, for any particular individual, are not readily predictable; the use of nonclinical markers has become critically important to patient management. The various phases of HIV infection, including the early asymptomatic phase, are associated with quantifiable laboratory findings. Laboratory surrogate markers of HIV infection, are by definition, measurable traits that correlate with the development of clinical AIDS , 3.The cost of antiretroviral therapy has dramatically reduced and this has led to the increased use of antiretroviral therapy (ART) in developing countries. In view of that, inexpensive laboratory tests are also needed to monitor disease progression and treatment response in HIV infected individuals, living in resource-limited environments most heavily impacted by the epidemic. Currently the standard methods used to monitor HIV infection are clinical assessment, flow cytometry based CD4 T lymphocyte count (CD4 counts) measurement, and molecular assays to quantify plasma viral load (PVL). Though clinical assessment remains the most feasible approach, it lacks sensitivity in determining disease stage, progression, and therapy response; therefore, it is to be used in conjunction with laboratory measures. CD4 counts measurement and PVL quantification require expensive equipment and considerable skill, so few laboratories in resource limited countries can offer these tests free of cost. Therefore, alternative laboratory parameters are being investigated in the hope that these would reduce the economic burden of HIV/AIDS in the developing countries [Chronic immune activation is a characteristic of HIV disease progression. HIV triggers polyclonal B cell activation, increased T cell turnover, production of proinflammatory cytokines, and increased number of activated T cells . The levThe HIV community is now faced with myriad concerns: which measures serve as the best proxies for initiation of ART, monitoring HIV disease progression, and monitoring the effect of ART and to what extent they accurately reflect patient status, the ethics of providing a particular standard of care in one area of the world but providing an alternative or substandard of care in another .In view of the above, this study was undertaken to evaluate the potential role of immune activation markers including neopterin and sIL-2R levels in monitoring HIV disease among patients attending our ART clinic.One hundred newly diagnosed HIV seropositive subjects were enrolled for the study after obtaining an informed consent and proper counseling. The subjects were registered with the ART clinic of the hospital. Subjects were then referred to CD4 testing.μL , 200–500 cells/μL , and >500 cells/μL . Blood sample for PVL estimation was obtained for group A subjects at recruitment.At the time of recruitment, blood samples were collected for CD4 counts, serum IL-2R, and serum neopterin. All study subjects were categorized into three study groups: CD4 counts <200 cells/CD4 counts were determined by the FACS system (Becton Dickinson). The levels of serum neopterin were measured using the commercially available enzyme immunoassay (B.R.A.H.M.S). The levels of soluble interleukin 2 receptor (sIL-2R) were measured by the commercially available enzyme immunoassay (Immunotech). Plasma viral load (PVL) was estimated using Amplicor HIV-1 Monitor Test, version 1.5 (Roche Diagnostics), by the standard procedure.Group A cases were initiated on ART including 2 nucleoside reverse transcriptase inhibitors (NRTIs) plus 1 nonnucleoside reverse transcriptase inhibitors (NNRTI) as per National AIDS Control Organisation recommendations . These cGroups B and C cases were followed up after a period of six months to monitor disease progression by measuring the CD4 counts, levels of sIL2R, and serum neopterin.Forty-two age and sex matched HIV negative healthy controls were also included for comparing CD4 counts, levels of sIL2R, and serum neopterin with HIV positive individuals.To detect whether there was a significant difference in the levels of individual markers between baselines and followup wilcoxon signed rank test was applied. To analyze the correlation between different markers at baseline and at followup Spearman's rank correlation test was applied. SPSS software was used for analyzing the data. The diagnostic performance of different immunological tests was evaluated using receiver operating characteristic (ROC) curve analysis.Median age of HIV seropositive study subjects was 32.5 years. The predominant age group in males and females was 26–30 years. The majority (75%) of the study subjects enrolled were from urban areas. Thirty-one percent males and forty-two percent females were illiterate .P < 0.5) in the healthy control group as compared to HIV positive study subjects (805 cells/μL versus 279 cells/μL) at baseline. The values of neopterin (6.3 nmol/L versus 25.5 nmol/L) and sIL-2R (4.4 pM versus 63.0 pM) were significantly lower (P < 0.5) in the healthy control group as compared to the HIV seropositive study subjects  subjects died within six months and 5 (15.2%) were defaulters. Twenty two (66.6%) subjects reported for followup. Among group B subjects, one subject (2.9%) died within six months and 3 subjects (8.8%) were defaulters. Thirty (88.2%) subjects reported for followup. Among group C subjects, two subjects (6.1%) died within six months and 15 subjects (45.4%) were defaulters. Sixteen (48.5%) subjects reported for followup.Among group A, there was a statistically significant rise in the median CD4 counts after ART although two cases showed immunological failure as per NACO guidelines. There was statistically significant decrease in median neopterin levels and median level of sIL-2R after followup. At baseline, PVL ranged from 2636 to >750,000 copies/mL with a median PVL at the baseline of 165,000 copies/mL. At followup 90% of study subjects had undetectable level of viremia , while 2 cases showed 407 & 1100 copies/mL of HIV-1 RNA. Among group B, there was a fall in the level of CD4 counts at followup which was statistically insignificant but a statistically significant increase in median level of neopterin and median level of sIL-2R at followup was observed. Among group C, a statistically significant fall in the CD4 counts was observed at followup along with the statistically significant increase in median level of neopterin and median level of sIL-2R at followup .P value: 0.103) and sIL-2R  at baseline. Among group B, CD4 count had a negative correlation with serum neopterin and sIL-2R at baseline and followup both and it was found to be statistically significant only for neopterin at followup. Among group C, CD4 count had a negative correlation with neopterin and sIL-2R at baseline and followup both, but it was statistically insignificant  and sIL-2R  were significantly higher in subjects with CD4 counts <200 cells/μL as compared to subjects with CD4 counts >200 cells/μL.Median levels of serum neopterin  in the CD4 counts among subjects on ART, indicating a good immunological response which is supported by another Indian study, in which the median CD4 cell count after 3 months of ART rose to 304 cells/mm3, and at 6 months the median was 328 cells/mm3 [μL & 580 cells/μL, while at followup the median count was 274 cells/μL & 487 cells/μL, respectively. In a study conducted by Mehendale et al. on Indian seroconverters, between the two periods of 91–360 days after seroconversion and 361–720 days after seroconversion, median CD4 counts dropped from 631/mm3 to 497/mm3. The annual decline in CD4 cell count was 120 cells/year among patients who were not on ART [3 in the untreated group of HIV-1 infected patients with CD4 T-cell count above 500/mm3 [All group A subjects in our study showed good clinical improvement after six months of ART and no subject showed clinical failure. There was a statistically significant rise  after six months of ART (70.9% reduction). Our results correlate with a study conducted by Amirayan-Chevillard et al. which showed that ART significantly decreased the circulating levels of neopterin by about 30% (15.6 ± 1.5 ng/mL for treated versus 22.3 ± 3.7 ng/mL for naïve patients) . For gron levels .P value <0.5) from 96.1 pM to 45.5 pM after six months of ART. Our findings were in contrast to the findings of Bonnet et al., who in their study established that sIL-2R levels did not decrease significantly throughout the follow-up period in 46 therapy naïve patients  [Among group A, the median level of sIL-2R significantly decreased ( of ART) . There w of ART) .ρ = 0.517, P value <0.05). In one earlier study no correlation was found between evolutions of HIV RNA and sIL-2R from baseline to six months after ART (P > 0.05), but a significant positive correlation was noted between evolution of sIL-2R and HIV RNA  from baseline to month 12 [In our study, among group A, 90% of study subjects at followup had undetectable level of viremia (PVL of <400 copies/mL) which is supported by another study in which 95% of these patients had PVL less than 400 copies/mL after 6 months of ART [month 12 .μL as compared to subjects with CD4 counts >200 cells/μL . In our study, ROC curve analysis suggests S. neopterin levels of >25.1 nmol/L  and sIL-2R levels of >47.1 pM  as cutoff values for CD4 count of <200 cells/μL. This observation suggests that these markers can be utilized for initiation of antiretroviral therapy in HIV infected individuals. However, their cutoff values need to be calculated from further larger studies.Median levels of serum neopterin & sIL-2R were found to be significantly higher in subjects with CD4 counts <200 cells/μL, these markers are helpful in predicting disease progression; however, large scale studies are required to establish the utility of these markers in predicting the disease progression.Levels of these markers also showed significant decreases in response to ART at followup. However, larger studies are required to derive the levels of these markers that suggest an appropriate treatment response. In patients with CD4 counts >200 cells/


Data on suppression of HIV replication in the CNS and on the subsequent risk of neurocognitive impairment using monotherapy with boosted protease inhibitors are limited.Ours was an exploratory cross-sectional study in patients on lopinavir/ritonavir-based monotherapy (LPV/r-MT) or standard triple therapy (LPV/r-ART) for at least 96 weeks who maintained a plasma viral load <50 copies/mL. HIV-1 RNA in CSF was determined by HIV-1 SuperLow assay . Neurocognitive functioning was assessed using a recommended battery of neuropsychological tests covering 7 areas. Neurocognitive impairment (NCI) was determined and also a global deficit score (GDS) for study comparisons.Seventeen patients on LPV/r-MT and 17 on LPV/r-ART were included. Fourteen (82.4%) patients on LPV/r-MT and 16 (94.1%) on LPV/r-ART had HIV-1 RNA <1 copy/mL in CSF (p = 0.601). NCI was observed in 7 patients on LPV/r-MT and in 10 on LPV/r-ART . Mean (SD) GDS was 0.22 (0.20) in patients on LPV/r-MT and 0.47 (0.34) in those on LPV/r-ART (p = 0.012).Suppression of HIV in CSF is similar in individuals with durable plasma HIV-1 RNA suppression who are receiving LPV/r-MT or LPV/r-ART for at least 96 weeks. Findings for HIV-1 replication in CSF and neurocognitive status indicate that this strategy seems to be safe for CNS functioning. Standard-of-care antiretroviral therapy (ART) is based on a combination of at least 3 antiretroviral drugs including 2 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) plus 1 non-nucleoside reverse transcriptase inhibitor (NNRTI), 1 boosted-protease inhibitor (PI), or 1 integrase inhibitor Monotherapy with boosted PIs is particularly attractive as an NRTI-sparing strategy. Guidelines consider this approach in cases of NRTI-related toxicity or intolerance Nonetheless, consequences of PI monotherapy on the HIV-1 compartments have not been well studied. One of the concerns with monotherapy is whether it can maintain control of viral replication in different compartments, particularly in cerebrospinal fluid (CSF), where it has been associated with a higher risk of neurocognitive impairment by some authors The objective of this study was to explore the long-term virological efficacy of LPV/r monotherapy in CSF and neurocognitive function in patients with HIV-1 infection and sustained suppression of plasma viral load.We performed an exploratory, comparative, cross-sectional study of patients with HIV-1 infection and stable antiretroviral treatment with either LPV/r in monotherapy (LPV/r-MT) or LPV/r plus 2 NRTIs (LPV/r-ART) for at least 96 weeks, while maintaining a plasma viral load <50 copies/mL to assess a complete virological suppression in CSF .This study was conducted by the Lluita contra la Sida Foundation, Barcelona, Spain between August 2010 and June 2011. From a prospectively compiled database , we identified all consecutive patients who were receiving stable treatment with LPV/r at doses of 400/100 mg twice daily  in monotherapy or in combination with two NRTIs. The exclusion criteria were adherence <90%, temporary discontinuation of LPV/r for any reason, or medical contraindications for lumbar puncture during the current regimen. Patients with potential confounding comorbidities for the existence of neurocognitive impairment were included, not only to obtain a representative clinical sample of patients with HIV, but also to consider this variable in data comparisons. Confounding factors included self-reported past or current disease involving the CNS, diagnosis of a psychiatric disorder, psychopharmacologic treatment, or active/past drug use.The primary endpoint was the percentage of patients with complete virological suppression in CSF after 96 weeks on LPV/r-MT. Secondary endpoints were the percentage of patients with neurocognitive impairment, differences in neurocognitive status in terms of the global deficit score (GDS), and the assessment of LPV trough concentrations in CSF and plasma.The study was approved by the ethics committee of Hospital Germans Trias i Pujol (NCT 01116817), Badalona, Spain and performed according to the stipulations of the Declaration of Helsinki . All patients gave their written informed consent before enrollment.We recorded demographic and clinical variables including age, gender, ethnicity, nadir CD4+ T-cell count, current CD4+ T-cell count, CDC stage, hepatitis C virus co-infection, zenith of HIV-1 viral load, time since diagnosis of HIV infection, time with virological suppression (HIV-1 RNA <50 copies/mL), time on antiretroviral treatment, time on LPV/r-MT or LPV/r-ART, history of opportunistic infections and other comorbidities, and concomitant medication. Patients recorded the time they had taken the last LPV/r dose on the day before the visit.Paired CSF-plasma samples were obtained from each participant on the day of the study visit, and time of sampling was recorded. Plasma HIV-1 RNA, CD4+ T-cell count, and routine hematology and biochemistry tests were performed at the local laboratory.The method used for ultrasensitive measurement of HIV-1 RNA in CSF was the HIV-1 SuperLow assay. This method is based on a proprietary protocol and algorithm (bioMONTR®) 18 column (3 µm: 100 mm×2.1 mm) using a step-wise gradient mobile phase. LPV was quantified using a triple-quadrupole mass spectrometer . A 9-point linear CSF calibration curve  was validated using artificial CSF  with the addition of human serum albumin (0.2 g/L) . Accuracy ranged from 101.2–104% and imprecision was <8%. The recovery rate was high and reproducible.LPV concentrations in CSF were quantified using a validated protein precipitation method coupled with reverse-phase liquid chromatography-tandem mass spectrometry. CSF samples (100 µL) were extracted via protein precipitation (acetonitrile: 0.1% formic acid [50∶50] 200 µL) with the addition of an internal standard . LPV and QX were resolved on a reverse-phase Ascentis Cg, 15 minutes) and stored at –20°C until analysis. LPV concentrations were determined using high-performance liquid chromatography with a photo diode array detector  according to a validated method, which involved liquid-liquid extraction with tert-butyl methyl ether. The mobile phase consisted of gradient elution with phosphate buffer-acetonitrile. The method was linear over the range of 0.05–20 mg/L. Intra- and interday coefficients of variation were less than 10%. The assay was externally validated using the International Interlaboratory Quality Control Program for Therapeutic Drug Monitoring in HIV Infection  Blood samples for determination of LPV concentrations in plasma were collected in potassium EDTA tubes. Plasma was isolated by centrifugation  was used to describe non-normally distributed variables, which were compared using the Mann–Whitney test. Percentages were compared using the χ2 square test. Because this was an observational and cross-sectional investigation, the possibility of a lack of equivalence between groups was considered, and neuropsychological measures were adjusted using linear and logistic regressions when demographic or clinical variables were not balanced. All comparisons and statistical analyses were performed using SPSS version 15.0 . Differences were considered statistically significant at p<0.05.Patients receiving either LPV/r-MT or LPV/r-ART were matched according to the following characteristics: age, gender, CD4+ T-cell count at enrollment, nadir CD4+ T-cell count, and time on LPV/r-based treatment. Variables with a normal distribution were described as mean (standard deviation [SD]) and compared using the Thirty-seven patients agreed to participate in the study. Of these, 3 were excluded because they did not fulfill the inclusion criteria. Therefore, the final sample comprised 34 patients (17 on LPV/r-MT and 17 on LPV/r-ART): 29 (85.3%) were male, and the median (IQR) age was 47.3 (41.4–50.0) years. Overall, the median time on antiretroviral treatment was 8.1 (5.5–17.2) years and the median time on LVP/r-based treatment and with HIV-1 RNA <50 copies/mL was 3.8 (2.5–4.7) and 5.3 (3.5–7.4) years, respectively. The median number of previous antiretroviral regimens for patients receiving LPV/r-MT and patients receiving LPV/r-ART was 6 (2–10) and 2 (1–4), respectively (p = 0.018). Median time with virological suppression was 6.9 (5.5–8.9) years and 3.4 (2.3–5.1) years (p<0.001). The remaining demographic and clinical variables were similar for both groups .Three patients presented adverse events related to study procedures. Two patients experienced mild headache after lumbar puncture, and 1 patient had pain at the lumbar site during the 7 days after lumbar puncture.Fourteen (82.4%) patients on LPV/r-MT and 16 (94.1%) on LPV/r-ART had HIV-1 RNA <1 copy/mL in CSF (p = 0.601). Among patients who had a CSF HIV-1 RNA ≥1 copy/mL, 3 patients on LPV/r-MT had determinations of 1, 75, and 120 copies/mL, respectively, and the patient on LPV/r-ART (LPV/r plus abacavir/lamivudine co-formulation) had an HIV-1 RNA of 2 copies/mL.Neurocognitive impairment was observed in 7 patients on LPV/r-MT and 10 patients on LPV/r-ART . When patients with potential confounding comorbidities for neurocognitive impairment were excluded, the results were similar: 6/13 (46%) patients receiving LPV/r-MT and 8/13 (61%) patients receiving LPV/r-ART had neurocognitive impairment (p = 0.43) see . AccordiSignificant differences were observed between groups in neurocognitive functioning. The median (IQR) GDS was 0.2 (0.07–0.36) in the LPV/r-MT group and 0.47 (0.27–0.67) in the LPV/r-ART group (p = 0.012). When patients with potential comorbidities were not included in the analysis, differences were mostly similar: median (IQR) GDS was 0.27 (0.03–0.43) in the LPV/r-MT group and 0.47 (0.27–0.73) in the LPV/r-ART group (p = 0.022). Adjusted analyses with linear and logistic regressions confirmed that years of education, CDC stage, HCV co-infection, time since diagnosis of HIV, prior antiretroviral regimens and time with virological suppression were not associated with changes in neurocognitive impairment and GDS.50) for wild-type HIV-1  Median (IQR) time since the last dose was 12.8 (9.8–14.7) hours. Median plasma and CSF LPV concentrations were 5083 (4100–8562) ng/mL and 22.3 (10.7–31.4) ng/mL, respectively. Two plasma LPV samples and one CSF sample were below the lower limit of quantification , suggesting poor recent adherence. Overall, LPV concentrations in plasma and CSF were well correlated . The median (IQR) ratio of CSF to plasma concentration was 0.46% (0.34%–0.75%). LPV concentrations in CSF exceeded the 50% inhibitory concentration  of plasma HIV-1 RNA.50 for wild-type strains of HIV. This finding, together with the similar rates of complete CSF-virological suppression in patients receiving long-term LPV/r-MT and LPV/r-ART, support monotherapy LPV being able to maintain virological suppression in CSF, even taking into account that most of patients included in the group of LPV/r ART had TDF/FTC as backbone.Although up to 99% of LPV binds to proteins in plasma, LPV concentrations in CSF exceed the median inhibitory concentration for wild-type HIV-1 after boosting with ritonavir at standard doses CPE has been proposed as a clinical tool to evaluate the penetration of antiretroviral drug combinations in the CSF of patients with standard triple ART Our study is subject to a series of limitations. First, the sample was small, and the exploratory cross-sectional design could lead to bias or unmeasured confounding factors. Second, selection bias is implicit with the inclusion of only those patients who had treatment for at least 96 weeks with maintained HIV virological suppression. Therefore, we were not able to evaluate those patients who could have developed neurocognitive impairment before the study was performed. In addition, as mentioned above, selection bias could also have affected the quantitative difference observed in neurocognitive functioning between groups. Third, despite the fact that patients were matched for the main demographic and immunological characteristics, differences were observed. Patients on LPV/r monotherapy had previously used significantly more antiretroviral regimens and had more time with virological suppression, although both groups had similar time on antiretroviral treatments, partly because patients on LPV/r-MT had initiated monotherapy due to poor tolerance, toxicity, and simplification. Adjusted analyses, however, confirmed that there were no association between these variables and neurocognitive performance. In addition, patients on LPV/r-MT showed a non-significant trend to higher CD4+ T-cell count and more years of education at inclusion, which also suggests that monotherapy with LPV/r was used in more selected patients. Despite these limitations, ours is the first study to explore long-term virological efficacy in CSF and neurocognitive safety of LPV/r-MT as an NRTI-sparing strategy in patients with HIV-1 infection and sustained plasma virological suppression. Cohort and clinical trials with specific neurocognitive endpoints are ongoing, although results are not anticipated in the short or medium term In conclusion, the results of sensitive CSF examination and neurocognitive performance suggest that suppression of HIV-1 RNA is similar in patients with HIV-1 RNA suppression receiving monotherapy with LPV/r and patients receiving LPV/r-based ART for at least 96 weeks. However, our findings should be confirmed in prospective randomized trials.


In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients.A total of 35 blood samples from 16 patients who showed first-line ART failure  and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping.Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C.The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients.http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973The virtual slide(s) for this article can be found here:  Since the discovery of human immunodeficiency virus (HIV) as the causative agent in 1983, the HIV-1 pandemic is characterized by an extensive variability and the circulation of numerous genotypes and recombinant forms (CRFs) . SubtypeHIV-1 remains a crucial global health problem. Delivery of ART in low and middle income countries has required utilization of a public health approach in which standardized, rather than individualized, regimens are prescribed to very large numbers of HIV-1-infected individuals . At presThe introduction of antiretroviral (ARV) therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. Nevertheless, patients in routine clinical care experience sustain detectable viral replication even under potent combination therapy. Various factors underlie this failure to achieve viral suppression, but resistance to current medications has been increasingly recognized as an important factor. Rational choice of the NRTI component of second-line therapy should be based on patterns of resistance developed during first-line therapy .Djibouti is the smallest country in east Africa. The estimated prevalence of HIV-1 infection was around 2.5% (2009). Since the introduction of antiretroviral therapy in 2004, no data exist on the epidemiology of drug resistance mutations and little was known about HIV-1 genotypes circulating in Djibouti. Data on drug resistance in the routine care setting are urgently required to check ART efficacy. The present study aims to characterize circulating HIV-1 subtypes and to identify drug resistance mutations in patients who first-line therapy failed (>1000 copies/mL) and antiretroviral-naive patients from Djibouti.Between October and December 2009, the whole blood samples were collected in Djibouti city from 35 HIV-1 infected patients consulting at three health centers . EDTA (ethylenediaminetetraacetic acid-anticoagulated) plasma specimens were obtained after centrifugation, and stored at −80°C in aliquots for later assessment of HIV RNA viral-load and genotypic drug resistance testing. The ethical aspects of this study were approved by the local committee. For all participants, blood collection was performed after informed consent, under the supervision of local sanitary authorities.The CD3/CD4+ cells were enumerated by flowcytometry (BD FACSCount System) and the quantification of HIV-1 viral load (VL) was measured using NASBA, NucliSENS®EasyQ Assays (BioMerieux) at the central laboratory of “Hôpital General Peltier” in Djibouti.The plasma samples were shipped to the Laboratory of Microbiology of “Charles Nicolle” Hospital  for drug resistance genotyping tests.http://www.hivfrenchresistance.org/ANRS-procedures.pdf).For Drug Resistance genotyping, the viral RNA was isolated from plasma samples using DSP virus RNA kit (QIAGEN Inc.) and reverse transcribed to make complementary DNA (cDNA) copies using a cDNA synthesis kit the One Step RT-PCR (QIAGEN Inc). The reverse transcriptase (RT) and protease (PR) genes were amplified according to the ANRS consensus sets of primers: for the RT gene amplification, MJ3 and MJ4 primers were used for a first PCR (RT-PCR), and A(35) with NE1(35) as inner primers for nested PCR. For the protease gene amplification, 5′Prot1 and 3′Prot1 outer primers were used and the 5′Prot2 and 3′Prot2 as inner primers: . DNA bands were stained with RedSafe™ Nucleid Acid Staining Solution  . They were then visualized in UV light to assess the presence of fragments of the expected length compared with the standard marker SharpMass™50-DNA ladder . The amplification products were purified using QIAquick PCR Purification kit (QIAGEN Inc).PCR products (5 μL) were resolved by electrophoresis on a 1.5% agarose gel http://hivdb.stanford.edu/).The PCR products were finally sequenced on an ABI3130 Genetic Analyser (Applied Biosystems), and compared with HXB-2 sequence using SeqScape v2.6 software (Life technologies). All sequences were subjected to quality assessment and determination of DR mutation profile  using the Stanford Database  and 16 were first-line ART failures (45.8%). In ART naïve patients, the median CD4 count was 144 cells/μl and the average viral load was 5.58 log10/ml (388 643 copies/ml). For the patients with first-line ART failure, the median CD4 count was 178 cells/μl, viral loads mean was 5.62 log10/ml and the median time of therapy was 30 months (Table The ART naïve patients showed only NNRTI-associated mutations which were K103N (2 strains) and K101Q (1). No mutations of NRTI were detected. The minor PI mutation L10V was detected in one ART naïve patient .At the time of sampling, all the patients receiving ART were treated by first line. According to the Djiboutian National Program for fighting against AIDS (PNLS) recommendations , the firDrug susceptibility results were classified into three categories: susceptible, intermediate resistance and resistance. Of the 16 patients with first-line ART failure, 56.25% showed resistance to at least one antiretroviral molecule: two patients (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. We found that the resistance rates to Lamivudine and Nevirapine in the 16 patients with first-line ART failure were 37.5% and 43.75% respectively. Forty four percent (44%) of sequences from viral strains did not show any mutation associated to ARV drug resistance- 7out of 16 . TherefoMinimizing drug resistance to highly active antiretroviral therapy (HAART) is important. First, it maximizes the opportunity for successful second-line and subsequent therapies after viral rebound during first-line treatment; second, it limits the transmission of drug-resistant viruses. We report results of the HIV-1 genetic diversity and prevalence of mutations associated with antiretroviral drugs among ART naïve as well as patients who failed first line therapy. The HAART was introduced in Djibouti in 2004. Djibouti, surrounded by Eritrea, Ethiopia, and Somalia, is an important crossroad for trade and exchange.Our result revealed a majority of subtype C (~66%). In fact, Subtype C prevalence has been described in the Horn of Africa-14 and aFor the ART naïve patients, the low rate of CD4 and the high rate of VL can be explain by the fact that 80% of patients infected by HIV in Djibouti was already detected at an advanced stage of the epidemic according to WHO classification ,23. Amon. Mutations conferring resistance to NNRTIs were also seen, with one strain showing resistance only to NNRTIs. The NNRTI resistance-conferring mutations included K103N (25%), Y188L (12.5%), Y181C (6%), G190A (6%), H221Y (6%), M230L (6%). Our data suggest the non-epidemic circulation of resistant viruses, and the absence of multi-class drug resistant viral strains.Concerning first-line ART failures, 38% of 16 strains sequenced in this group had the M184V mutation. This mutation is selected by lamivudine (3TC), emtricitabine (FTC) or abacavir (ABC) use and confers high-level resistance to 3TC , toxicity and adverse events are all important considerations and will differ between patient populations. We noticed seven patients (~44%) did not show resistance-conferring mutations and the therapeutic failure may have been due to other factors -35. InfoThe escalating rates of transmission of drug resistant virus observed in the past few years, coupled with the poorer response to treatment in persons with drug resistant virus, are being considered as a basis for a recommendation that resistance tests should be performed routinely for persons with new HIV infection . This evThis study reveals primary mutations that confer resistance to NRTI, NNRTI in HIV-1 non-B and B subtype strains, isolated from ART naïve as well as patients who failed first line therapy in Djibouti. It also illustrates the importance of preliminary surveys of drug-resistance mutations in resource-poor countries. Therefore, we suggest the use of resistance testing to check the prevalence of the drug resistance mutation that arises following failure of the first line regimen. This will help in establishing guidelines for second line regimens in Djibouti. This allows the selection of adapted antiretroviral regimens in these regions. Our results provide a basis for repeated epidemiological studies to measure the population effects of HIV treatment programs over time.ART: Antiretroviral therapy; PR: Protease; RT: Reverse Transcriptase; ARV: Antiretroviral.The authors declare that they have no competing interests.AEA: wrote the manuscript, has made substantial contributions to acquisition of data, or analysis and interpretation of data. AEA and AJ: participated in data analysis and interpretation. HYD and MA BHK: revised the manuscript. AS: proposed the idea, revised the manuscript and save final approval of the version to be published. All authors read and approved the final manuscript.


Ensuring long-term adherence to therapy is essential for the success of HIV treatment. As access to viral load monitoring and genotyping is poor in resource-limited settings, a simple tool to monitor adherence is needed. We assessed the relationship between an indicator based on timeliness of clinic attendance and virological response and HIV drug resistance.Data from 7 virological cross-sectional studies were pooled. An adherence indicator was calculated as the number of appointments attended with delay divided by the number of months between antiretroviral treatment (ART) initiation and date of virological testing and multiplying this by 100. Delays of 1 or more to 5 or more days were considered in turn. Multivariate random-intercept logistic regression was fitted to examine the effect on outcomes, separately for adults and children.A total of 3580 adults and 253 children were included. Adults were followed for a median of 26.0 months (IQR 12.8-45.0) and attended a median of 24 visits (IQR 13–34). The 1-day delay adherence indicator was strongly associated with viral load suppression , virological failure  and HIV drug resistance  after adjusting for initial age and CD4 count, previous ART experience, type of regimen and Tuberculosis diagnosis at start of therapy. Similar results were observed in children.An adherence indicator based on timeliness of clinic attendance predicts strongly both virological response and drug resistance, and could help to timely identify non-adherent patients in settings where viral load monitoring is not available. Since the introduction of combined active antiretroviral therapy (ART), mortality and morbidity due to HIV have been drastically reduced in high-, middle- and low-income countries Viral load monitoring and genotyping are frequently used to identify non-adherent patients. However, access to these laboratory diagnostics is often poor in resource-limited settings. Simple alternative tools for rapidly detecting patients who do not regularly take their antiretroviral drugs as prescribed are therefore urgently needed No gold standard method exists for assessing treatment adherence. Many tools have been proposed, including pill-count, self-reported adherence, various biological markers, attendance to drug refill visits and/or clinic attendance, and quantitative determination of drugs in blood To monitor the emergence of drug resistance in patients receiving ART, the World Health Organization (WHO) recommends that HIV programmes use a number of indicators, called “early warning indicators” To assess whether a simple tool based on regularity of patients’ follow-up clinic attendance can be used as a proxy for treatment adherence, we pooled data from cross-sectional virological studies conducted in HIV programmes supported by Médecins Sans Frontières (MSF), linked them back to longitudinal monitoring data and examined its relationship with viral load suppression, virological failure and resistance to antiretroviral drugs.This is a cross-sectional secondary data analysis of cross-sectional virological evaluations conducted among patients receiving ART for 6 months or more in MSF-supported HIV programmes. Data were linked back to individual patient monitoring data collected in the HIV programs. Of 11 cross-sectional studies conducted between 2004 and 2009 in Africa and Asia, 4 were excluded because virological information was either unreliable or missing . Immunovirological testing was done in all studies and genotype testing in 5 studies . In two Routine clinical and laboratory data, such as appointment dates and the dates of clinic visits, were collected prospectively in each of the sites using the FUCHIA software . Viral load testing was not routinely performed in these programs and it was only used when treatment failure was suspected.http://hivdb.stanford.edu/) In the analyses, viral load suppression was defined as <400 copies/mL and virological failure as >5000 copies/mL  and paediatric (<15 years) patients. Patient characteristics, numbers of visits, and intervals between consecutive visits during the study follow-up were summarised using standard descriptive statistics. Comparison of visits across studies was assessed using Kruskal-Wallis tests.Five separate continuous adherence indicators were calculated for each patient, as explained before, using 1 or more, to 5 or more days of delay in clinic attendance in turn. Using univariate random-intercept logistic regression and Wald statistics, we selected the delay (in number of days) for which the continuous adherence indicator was most strongly associated with viral load suppression, virological failure and HIV drug resistance and used this in subsequent analyses.Univariate and multivariate random-intercept logistic regression models were fitted to assess the association between the continuous and categorical adherence indicators and viral load suppression, virological failure and HIV drug resistance after adjustment for other confounding factors at baseline: sex, age, CD4 cell count , WHO clinical stage , body mass index (BMI), tuberculosis diagnosis, history of ART use and type of regimen (nevirapine and efavirenz for non-nucleoside reverse transcriptase inhibitor (NNRTI), zidovudine and stavudine for nucleoside reverse transcriptase inhibitor (NRTI)) at ART initiation.Two sensitive analyses were performed. First, we did separate analyses for patients who received ART for <24 months and for ≥24 months. We also repeated the analyses using only data from the subgroup of patients with complete CD4 cell count, clinical stage and BMI information.Statistical analyses were performed with Stata 11.2 software .Before conducting the cross-sectional studies included in the analysis, ethics committee approval had been obtained. The ethics committees for the different studies were: the Uganda National Council for Science and Technology, the Ugandan AIDS Research Committee, the National Ethics Committee of Cambodia, KEMRI National Ethical Review Committee of Nairobi (Kenya), the National Health Science Research Committee of Malawi and the Consultative Committee for People involved in Biochemical Research (CPPRB) of Saint Germain-en-Laye, Paris, France. All participating patients had provided written informed consent A total of 3840 patients participated in the 7 cross-sectional studies included in our analysis. Seven patients (0.2%) were excluded because their date of birth was missing. Of the remaining 3833 patients, 3580 were adults and 253 children.2 (IQR 18.2–21.8) and 305 patients (8.5%) were diagnosed with tuberculosis. Among children, 51% were in clinical stage 3 and 17.4% in stage 4, median BMI was 14.9 kg/m2 (IQR 13.8–16.2) and 7 patients (2.8%) were diagnosed with tuberculosis. The most commonly prescribed initial ART regimens were a combination of stavudine, lamivudine and either nevirapine (83.7% for adults and 91.1% for children) or efavirenz .Patient characteristics at ART initiation are shown in Median follow-up time from ART initiation was 26.0 months (IQR 12.8–45.0) for adults and 22.6 months (IQR 11.1–28.1) for children. The median numbers of visits per patient were 24 (IQR 13–34) and 17 (IQR 11–24), respectively. Median intervals between consecutive visits were 0.95 months (IQR 0.82–1.84) and 1.02 months (IQR 0.89–1.84), respectively, and remained stable over time on ART .At the time of the cross-sectional evaluation, 3021 out of 3580 adult patients (84.5%) were virologically suppressed while 248 (6.9%) had virological failure. Genotype testing was performed for 2205 adult patients who had a viral load >1000 copies/mL. A total of 123 (5.6%) patients were classified as having HIV drug resistance to at least one antiretroviral drug. Similarly, 144 out of 253 pediatric patients (56.9%) failed to suppress viral load while 79 (31.2%) had virological failure. Genotype testing was performed for 33 children who had a viral load >1000 copies/mL. A total of 29 (11.5%) children had HIV drug resistance to at least one antiretroviral drug. The frequency of profiles of NRTI and NNRTI mutations encountered is shown in The use of the continuous adherence indicator based on a delay of 1 or more days after the scheduled appointment date showed the strongest association with viral load suppression, virological failure and HIV drug resistance in univariate analyses  and was therefore used in the subsequent risk factor analysis.P<0.001).The overall proportion of appointments attended with delay was 6.38% (95% CI: 6.22–6.55%) for adults and 12.08% (95% CI: 11.13–13.07%) for children, and decreased slightly following ART initiation. The median numbers of appointments attended with delay per patient were 1 (IQR 0–2) and 2 (IQR 0–3), respectively. These represent medians of 4.59% (IQR 0–11.11) and 9.09% (IQR 0–20.0%) of appointments attended with delay per patient, respectively. Median numbers and proportions of appointments attended with delay differed across studies  and ranged from 0% (IQR 0%–2.01%) to 7.93% (IQR 0%–9.91%) across studies. According to the study classification, 58.0% of patients showed good adherence, 35.6% moderate and 6.4% poor. The categorisation of patients according to adherence levels  showed higher proportions of individuals with poor and moderate adherence among those with virological failure and HIV drug resistance, and higher proportions of individuals with good adherence among those with viral load suppression .In univariate analysis using the continuous adherence indicator, patients with higher adherence indicator values (e.g. higher incidence of appointments attended with delay) were less likely to achieve viral load suppression (Odds Ratio (OR) 0.96, 95% CI 0.95–0.97 per unit increase), and more likely to experience virological failure  and HIV drug resistance .P<0.001) and variance due to studies represented less than 5% of the total variance, suggesting low heterogeneity between studies.In multivariate analyses , the catIn the full dataset, the median value of the continuous adherence indicator among children was 7.90% (IQR 0–16.65%). Overall, 43.0% were classified as showing good adherence, 42.7% as moderately adherent and 14.2% as poorly adherent. In multivariate analyses, the categorical adherence indicator remained strongly associated with viral load suppression  and with virological failure . A borderline statistical association was observed for drug resistance despite of the small sample size , and analyses restricted to patients with complete data showed consistent results (data not shown).In this large multicentric cross-sectional study including 3833 patients, we observed a strong association between an ART adherence indicator based on the regularity of attendance to scheduled clinic appointments and viral load suppression, virological failure and HIV drug resistance. Patients classified as having lower levels of adherence as defined by our indicator were less likely to have undetectable HIV viral load and were two to three times more likely to be in virological failure and have developed resistance mutations to antiretroviral drugs. These associations were seen in both adults and children and did not vary with duration of ART use.In our study population virological and drug resistance outcomes were more strongly associated with the adherence indicator based on a delay of one or more days after the scheduled clinical visit was used in the calculation. This delay seems reasonable in the context of HIV treatment programmes in resource-limited settings. In our study 6% of patient appointments were attended with a delay of one or more days. Although previous studies have suggested that adherence might decline with time on ART Overall, approximately 57% of patients showed good adherence when the indicator evaluated was used. This figure is lower than estimates reported in previous studies conducted in resource-limited settings, but those studies primarily used self-reported measures of adherence and a 95% cut-off to define good adherence The data used for this evaluation included patients from several African and one Asian HIV programme. At the time of the cross-sectional evaluations about 85% of study patients were virologically suppressed, 7% had virological failure and 6% had resistant mutations to antiretroviral drugs. Despite the relatively low rates of failure and drug resistance, a high and statistically significant increase in the probability of these two outcomes was observed in patients classified by the proposed indicator as moderate or low adherent, compared with patients classified as good adherent. This effect was independent of age, previous exposure to ART or the CD4 cell count level at ART initiation, and of the type of antiretroviral regimen taken. Although the absence of viral load monitoring in the study sites did not allow us to distinguish between episodes of long- versus short-term viremia in the analyses, our findings are strengthened by the consistent results of an association between the three virological outcomes studied and the adherence indicator . These outcomes are key markers of true adherence and treatment success.The association between the virological outcomes and the adherence indicator was also strong in children despite the smaller sample size of this group of patients, and it was of borderline significance for antiretroviral resistance, probably due to lack of power. This is an important finding that supports the use of this same indicator to identify non-adherent adult and children patients.We previously described strong associations between the adherence indicator evaluated, failure to first- and second-line therapy , and mortality among patients using first- and second-line therapy Our study has several limitations. First, patient follow-up on ART differed greatly according to site, from about 6 to 60 months, depending on the eligibility criteria used in each cross-sectional evaluation. As a result, the number of clinic visits per patient varied widely across sites, which could affect the reliability of the adherence indicator. To assess whether the duration of follow-up affects our findings, we performed a sensitivity analysis to identify possible differences in the degree of association between patients followed for less than 24 months and those followed for more than 24 months. No evidence of differences was detected for viral suppression, virological failure or HIV drug resistance. In addition, the length of delay in clinic attendance was not considered in the calculation of the indicator. Nevertheless, the median duration of clinic delay was 3 days and only 3.2% of visits had delays of more than 5 days.Our indicator might tend to underestimate patient adherence, since patients coming for more than one day after their scheduled visit are considered non-adherent for the entire period between the last visit and the actual visit, which is probably not the case (buffer drugs given to patients could prevent treatment interruptions). Because we calculated the adherence indicator over the whole period of ART use, we could not assess the temporal relationship between exposure and outcome and the effect of time itself was not really taken into account. Nevertheless, the homogeneous distribution of appointments attended with delay over time gives further confidence in the indicator. For the same reason, another important finding is that the interval between successive visits (on average about 1 month) was almost constant over time on ART.In conclusion, in this multicentric study conducted in resource-limited settings we observed a strong association between an indicator of adherence based on regularity of clinic attendance and both virological response and drug resistance. This indicator represents a simple tool that could be used in HIV treatment programmes for prompt identification of non-adherent patients who need strong, targeted support and counselling to prevent virological failure and drug resistance development.Table S1Number of adult patients with specific profiles of NRTI and NNRTI mutations stratified by amount of viral load.(DOC)Click here for additional data file.Table S2Number of paediatric patients with specific profiles of NRTI and NNRTI mutations stratified by amount of viral load.(DOC)Click here for additional data file.


To determine the contribution of transmission clusters on transmitted drug resistance (TDR) in newly diagnosed antiretroviral naive patients in Northern Greece, during 2000 -2007.Viral reverse transcriptase and protease genes from 369 individuals with newly diagnosed HIV-1 infection were sequenced at baseline. A maximum-likelihood phylogenetic analysis method was employed to examine for linkage between viral isolates. Clinical data were retrieved from the database and cross-referenced with the patients' medical files. Transmitted drug resistance was defined in accordance with the Surveillance Drug Resistance Mutation (SDRM) 2009 list.The study population characteristics were as follows: 82.8% male, 89.7% of Greek nationality, mean age 38, median CD4 cell count at diagnosis 295 cells/µl and mean HIV-1 RNA 4.94 log10 copies/ml. The most prevalent risk exposure category was men who have sex with men 59.1% (n=218) followed by heterosexual transmission 21.4% (n=79) and intravenous drug use 7.6% (n=28). Subtype B viruses were most prevalent (53.1%), followed by subtype A (32.5%). At least one drug resistance mutation was identified in 46/369 patients (12.4%). Twenty-eight patients (7.6%) harbored resistance mutations to nucleoside/nucleotide RT inhibitors, 20 patients (5.4%) to non-nucleoside RT inhibitors and 12 patients (3.3%) to protease inhibitors (PIs). Dual-class resistance mutations were identified in 14 patients (3.8%). The median CD4 cell count in patients with TDR was not significantly different compared to patients without (p=0.072). Phylogenetic analyses, supported by bootstrapping >90% and genetic distance <0.015, revealed three transmission clusters involving drug resistant strains, including one cluster of 11 patients infected with a strain carrying RT mutations Y181C and T215 variants conferring NRTI and NNRTI resistance.The overall prevalence of TDR in our study population was 12.4%. Phylogenetic analyses of viral sequences from these new diagnoses demonstrated the impact of transmission clusters on the primary drug resistance. The outbreak of dual-class TDR, coupled with late HIV diagnosis in this population may require improved public health interventions.


HIV-associated anemia is common and associated with poor prognosis. However, its response to antiretroviral treatment (ART) in rural Africa is poorly understood.HIV-infected adults (≥15 years) who enrolled in HIV care at Haydom Lutheran Hospital in northern Tanzania were included in the study. The effect of ART (zidovudine/stavudine + lamivudine + efavirenz/nevirapine) on HIV-associated anemia was studied in a subset of patients who were anemic at the time they started ART and had a follow-up hemoglobin measurement 12 months later. Pregnant women were excluded from the study, as were women who had given birth within the past 6 weeks. Anemia was defined as hemoglobin <12 g/dL in women and <13 g/dL in men. We applied paired sample T-tests to compare hemoglobin levels before and one year after ART initiation, and logistic regression models to identify predictors of persistent anemia.P < 0.001); however, 39 patients (38.2%) were still anemic after 12 months of ART. Independent predictors of persistent anemia were mean cell volume in the lower quartile  and a zidovudine-containing initial regimen .At enrollment, mean hemoglobin was 10.3 g/dL, and 649 of 838 patients (77.4%) were anemic. Of the anemic patients, 254 (39.1%) had microcytosis and hypochromia. Among 102 patients who were anemic at ART initiation and had a follow-up hemoglobin measurement after 12 months, the mean hemoglobin increased by 2.5 g/dL (Most patients had anemia at enrollment, of whom nearly 40% had microcytosis and hypochromia suggestive of iron deficiency. The mean hemoglobin increased significantly in patients who received ART, but one third were still anemic 12 months after ART initiation indicating that additional interventions to treat HIV-associated anemia in rural Africa might be warranted, particularly in patients with microcytosis and those treated with zidovudine. Anemia is a common feature of HIV infection, occurring in approximately 35% of patients who initiate antiretroviral treatment (ART) in Europe and North America . In addiIt is uncertain whether the association between anemia and mortality is causal or whether anemia acts as a surrogate marker of underlying disease. Previous studies have found that the incidence of anemia increases with progression of HIV infection -11, and Management of HIV-associated anemia in high-income countries includes erythropoietin treatment, which has been associated with recovery from anemia and improved survival, but high costs restrict its use in resource-limited settings . FurtherOur study was carried out in Tanzania, a low-income country in East Africa, with an estimated adult HIV prevalence of 5.7% . Haydom The HIV Care and Treatment Centre in Haydom has provided ART free of charge since October 2003. Clinical officers, under supervision of a physician, have been responsible for medical follow-up of patients. Most of the patients who enroll in the HIV program are diagnosed either through voluntary testing and counselling in the villages or testing of hospitalized patients with clinical suspicion of HIV/AIDS. Hence, many patients have advanced immunodeficiency at the time of enrollment.In the present study we describe the baseline hematological profile in a cohort of treatment-naïve individuals aged 15 years or older who enrolled in HIV care at Haydom Lutheran Hospital between October 2003 and January 2008. In a subset of patients who were anemic at the time they started ART and had a follow-up hemoglobin measurement 12 months later we studied the effect of ART on HIV-associated anemia. Women who were pregnant at baseline or became pregnant during the observation period were excluded from the study, as were women who had given birth within the past 6 weeks, since the physiological hemodilution in pregnancy and subsequent blood loss during delivery would have precluded the results. Ethical approval was obtained from the Medical Research Coordinating Committee of the National Institute for Medical Research in Tanzania and Regional Committee for Medical Research Ethics in Norway. Patients gave written consent to participate in the study.ART was initiated in accordance with guidelines from the World Health Organization (WHO) and the National AIDS Control Program -21: WHO First-line treatment comprised stavudine or zidovudine, combined with lamivudine, and either nevirapine or efavirenz. Regimen choice was subject to availability, with use of a generic fixed-dose combination of stavudine, lamivudine and nevirapine whenever possible. Efavirenz was used in patients with concomitant TB treatment. None of the patients switched to second-line ART during the observation period. Patients with CD4 ≤200 cells/μL or WHO stage 3 or 4 disease got co-trimoxazole prophylaxis 960 mg thrice weekly or 480 mg daily. Patients were seen by a clinical officer every 3 months, and CD4 cell count and full blood count (including hemoglobin) was scheduled every 3-6 months as part of routine care.HIV infection was established using two different rapid antibody tests, Determine HIV-1/2  and Capillus HIV-1/2 , in accordance with recommendations from the National AIDS Control Program . StandarAnemia was defined as a hemoglobin level <12 g/dL for women and <13 g/dL for men, in accordance with WHO guidelines . We clasIn general, we applied the first available laboratory result as the enrollment value. If two values were obtained within a month, the mean was employed. In the study on the effect of ART on HIV-associated anemia, only patients who were anemic at the time they started ART and had a follow-up hemoglobin measurement 12 months later were considered. The laboratory result obtained closest to the start of ART was used as baseline, and this value had to be no more than 3 months prior to ART initiation. Laboratory results obtained during the first year on ART were rounded off to the nearest 3 months.P < 0.2 in bivariable analyses were then entered in a multivariable model, using the forward stepwise  method. Multicollinearity was excluded using Spearman's correlation coefficient with a cutoff at 0.7. Data were analysed with SPSS version 16.0 for Windows . All tests were two-sided and level of significance was set at P < 0.05.To avoid loss of power we attempted to avoid dichotomization of continuous variables, such as BMI, MCV and MCH . Instead2 (SD 3.4). At enrollment, 399 patients (47.6%) had clinical AIDS (WHO stage 4), 245 (29.2%) had WHO stage 3, 97 (11.6%) had WHO stage 2, and 97 (11.6%) had WHO stage 1 disease. Ninety-eight patients (11.7%) were on TB treatment when they enrolled in care.A total of 838 HIV-infected adults who enrolled in the HIV program between October 3, 2003, and January 31, 2008, were included in this study , the mean MCV was 82.5 fL (SD 7.8), and the mean MCH was 26.6 pg (SD 3.2). Overall, 649 of 838 patients (77.4%) met the definition of anemia: 269 patients (32.1%) had mild anemia, 238 (28.4%) had moderate anemia, and 142 (16.9%) had severe anemia. Out of 649 anemic patients, 254 (39.1%) had microcytosis and hypochromia, suggestive of iron deficiency, whereas 52 (8.0%) had hypochromia with normocytosis, and 18 (2.8%) had microcytosis with normochromia.Table Out of 838 adults included in the study, 102 patients who were anemic at ART initiation and had a follow-up hemoglobin measurement 12 months later were selected for the sub-study on ART and anemia. Reasons for not being included in this sub-study are given in figure 2 (SD 3.3). Sixty-one patients (59.8%) had clinical AIDS at ART initiation. Fourteen patients (13.7%) were on TB treatment when they initiated ART and 8 more started TB treatment during the observation period of 12 months. Initial ART regimen was stavudine/lamivudine/nevirapine in 62 patients (60.8%), stavudine/lamivudine/efavirenz in 17 (16.7%), zidovudine/lamivudine/nevirapine in 16 (15.7%), and zidovudine/lamivudine/efavirenz in 7 (6.9%). Only 17 patients had a baseline CD4 measurement; the mean CD4 cell count was 140 cells/μL (SD 149).Among 102 patients in this sub-study, the mean age was 36 years (SD 10) and 75 patients (73.5%) were women. Mean BMI was 18.2 kg/mP = 0.470). After receiving ART for 12 months the mean hemoglobin increased to 12.4 g/dL (SD 1.9) (P < 0.001) . Eleven patients (10.8%) had severe anemia, 37 (36.3%) had moderate anemia and 54 52.9%) had mild anemia. Patients with severe anemia were less likely to start a zidovudine-containing initial regimen  than patients with moderate  or mild anemia , but the difference was not statistically significant (.9% had mP = 0.191). Adjusted for baseline hemoglobin, patients who did not start ART had a 6-fold increased risk of persistent anemia after 12 months compared to those who started ART .To assess whether the hemoglobin increase could be a result of other factors associated with enrollment in HIV care rather than ART itself, we studied the hemoglobin evolution in 18 non-pregnant adults who were anemic at enrollment and had a follow-up hemoglobin measurement 12 months later, but did not start ART. In this group there was no statistically significant hemoglobin increase: the mean hemoglobin was 10.1 g/dL (SD 1.5) at enrollment and 10.9 g/dL (SD 2.1) after 12 months (P < 0.001).Sixty-five patients who were alive and in care 12 months after ART initiation, but who did not have a follow-up hemoglobin measurement, were excluded from our analysis. These patients had a similar age (mean 38 years) and sex  distribution as in the main analysis. Forty-one of these patients had a hemoglobin measurement at 9 months, and they showed a similar improvement as patients in the main analysis, from a mean hemoglobin of 9.6 g/dL (SD 1.8) at baseline to 12.3 g/dL (SD 1.6) at 9 months (P < 0.001). We reviewed the records of these patients and found that anemia contributed to the death in 15 of 98 patients. Thirteen of 15 anemia-related deaths occurred in patients who received a stavudine-containing regimen, of whom 8 had severe anemia already at the time of ART initiation. Two patients died of presumed hematotoxic effects of zidovudine, 58 and 94 days after ART initiation, respectively.Ninety-eight patients who died during the first year on ART were also excluded from our analysis, 68 of whom died within the first three months. At the time of ART initiation the mean hemoglobin among patients who died was 8.8 g/dL (SD 1.7), significantly lower than among patients in the main analysis (P = 0.023) and a zidovudine-containing initial regimen  were significantly associated with persistent anemia.Among patients who were anemic at ART initiation, only MCV in the lower quartile (<76.0 fL) significantly predicted persistent anemia 12 months later in bivariable logistic regression analysis adjusted for baseline hemoglobin, whereas MCH in the lower quartile (<23.8 pg) and a zidovudine-containing initial regimen were borderline significant (Table P = 0.029) and a zidovudine-containing initial regimen  remained strong and significant predictors of persistent anemia.During the first year on ART, 3 patients switched from stavudine to zidovudine and 4 from zidovudine to stavudine, 2 of whom switched because of severe anemia. To assess whether drug substitutions might have confounded our results, we conducted a sensitivity analysis where these patients were excluded. In the resulting multivariable model, both MCV in the lower quartile  and TB, in line with previous studies -11,27,28The hemoglobin level increased significantly in patients who received ART; on average the hemoglobin increased 2.5 g/dL over the first 12 months among patients who were anemic at ART initiation. Previous studies have reported that ART is associated with resolution of HIV-associated anemia in Europe and North America ,14,15. MHowever, not all patients on ART achieved a normal hemoglobin level. A low MCV was a strong and independent predictor of persistent anemia in our study. Microcytosis was also associated with persistent anemia in the Women's Interagency HIV Study in USA , and witAn initial zidovudine-containing ART regimen was also associated with persistent anemia in our study, increasing the risk by nearly 3 times compared to regimens containing stavudine. The potential myelosuppressive effect of zidovudine has been known for more than two decades , but itsBefore the HIV era, recommendations for management of anemia in classical tropical medicine included screening for malaria, treatment for helminthic infections and iron/folic acid supplementation . In the There were some limitations of our study. Most importantly there may have been a selection bias towards patients with a more favorable prognosis in the sub-study on the effect of ART on anemia, since we only included patients with a follow-up hemoglobin measurement after 12 months. Patients who died during the first year on ART might have had a different hemoglobin evolution, but since the majority of deaths occurred within the first 3 months, few of these had a second laboratory measurement. We also excluded patients who were still receiving ART, but did not have a hemoglobin measurement after 12 months. If these patients were less likely to be anemic than the others, our results could be biased (selection by indication); however, patients who were excluded had a similar hemoglobin increase at 9 months as the patients in our main analysis. Furthermore, our study was limited by lack of adherence data, and it is possible that poor adherence contributed to persistent anemia in some patients. On the other hand, in a previous study from the same cohort, we found that 94.8% of patients had suppressed viraemia after 1 year of ART, indicating good adherence in the majority of subjects . FinallyIn conclusion, we found that the majority of patients who enrolled in HIV care at a rural hospital in Tanzania were anemic, and that nearly 40% of the anemic patients had microcytosis and hypochromia suggestive of iron deficiency. Patients who were anemic at the time of ART initiation had a strong and significant hemoglobin increase over the initial 12 months of ART. However, more than one third of patients did not reach normal hemoglobin levels while on ART, and a low MCV and a zidovudine-containing initial regimen were independent predictors of persistent anemia. Our study indicate that although ART effectively reverses HIV-associated anemia in the majority of individuals, additional interventions might be warranted in a subset of patients.The authors declare that they have no competing interests.AJ analyzed the data and drafted the manuscript. EN collected the data. SGG and JNB conceived the study and participated in its design and coordination. All authors critically revised the manuscript and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/190/prepub


Adherence to antiretroviral drugs in the treatment of paediatric HIV infection is complicated because of many factors including stigma and drug intake logistics. It is therefore important to identify children with non-adherence in order to intervene before they become at risk of developing treatment failure or drug resistance. The aim of this study was to determine the level of adherence to antiretroviral therapy (ART), measured by caretaker report, medication return and nevirapine plasma concentration. In addition, the association between level of adherence and patient’s immune status was compared across the three methods of measuring adherence.This was a descriptive cross-sectional study involving HIV infected children aged 2–14 years, on nevirapine- based antiretroviral treatment for at least six months, attending care and treatment clinic in three municipal hospitals in Dar- Es- Salaam City. Eligible patients and their accompanying caretakers were consecutively enrolled after obtaining written informed consent. Structured questionnaires were administered to caretakers to assess patient’s adherence by caretaker report and medication return whereas a single blood sample for CD4 cell count/percent and determination of nevirapine plasma concentration was taken from patients on the day of assessment.A total of 300 patients and accompanying caretakers were enrolled and the mean patient age (SD) was 8 (3) years. Caretakers’ report and medication return showed good adherence (98% and 97%) respectively. However, the level of adherence assessed by nevirapine plasma concentration (85%) was significantly lower than caretaker report and medication return (p < 0.001). The agreement between nevirapine plasma concentration and medication return and between nevirapine plasma concentration and caretaker report was weak (k = 0. 131) (k = 0. 09) respectively. Nevirapine plasma concentration below 3 μg/ml was associated with immunosuppression (p = 0. 021) whereas medication return (>5% of prescribed doses) and caretaker reported missing more than one dose within 72 hours prior to interview were not associated with immunosuppression (p = 0. 474), (p = 0. 569) respectively.Lower adherence level observed using nevirapine plasma concentration and its association with immunological response supports the validity of the method and indicates that adherence data obtained from caretaker report and medication return may overestimate the true adherence in paediatric antiretroviral therapy. Introduction of antiretroviral therapy has resulted in a decrease in mortality and morbidity in HIV/AIDS patients worldwide . AntiretAdherence to antiretroviral therapy in paediatric and adolescent patients has been reported to be problematic due to multiple factors. These include the pill burden, poor palatability, unpleasant flavour, side effects and long term toxicity . In addiIn low- and middle- income countries, lack of a simple and cost-effective gold standard for measuring adherence to ART is a major challenge for paediatric HIV/AIDS treatment. A number of strategies such as pill count, electronic monitors, diaries and self-report questionnaires have been employed. However, each of these methods has limitations and can provide different estimates of adherence. Importantly, these limitations have been reported to be more pronounced in paediatric patients ,10.Despite its limitations, antiretroviral drug plasma concentration may be considered the gold standard. However, it is often not feasible, particularly, in resource-poor countries. In adult patients, the antiretroviral plasma drug concentration has been reported to correlate with viral load response; therefore, giving a more reliable adherence estimate than self reporting . HoweverAlthough studies done to establish an association between adherence (measured by self report and medication return) and immunological response among paediatric HIV/AIDS patients have shown conflicting findings -14, adheThis was a descriptive cross-sectional study conducted from May to October 2011 at three municipal hospitals  in Dar-Es Salaam, Tanzania. HIV/AIDS patients aged 2-14 years on paediatrics nevirapine-based antiretroviral treatment regimen, for at least six months, and their accompanying caretakers were enrolled consecutively as they attended HIV care and treatment clinic. The study protocol was reviewed and approved by the Muhimbili University of Health and Allied Sciences (MUHAS) Ethical Review Committee and permission to conduct the study was obtained from the respective District Medical Officers. Only eligible patients whose accompanying caretaker gave a written informed consent were enrolled and patients not on nevirapine-based regimen were excluded. Also excluded were patients on medications that are known to induce/inhibit nevirapine metabolizing enzymes (CYP 2B6 and CYP3A4) such as rifampicin, fluconazole, and ketaconazole. An assent was also obtained from patients aged 12 years and above before inclusion into the study.2 per dose given twice a day [A structured questionnaire was used to assess adherence to antiretroviral therapy and information on the patient’s concurrent use of other drugs and co-morbidity was collected. In addition, patient’s medical history including history of illness in the previous visit, history of cough, fever, ear discharge, vomiting of medication, skin or oral lesion was taken. The duration of antiretroviral therapy, the time at which the early morning dose prior to assessment was taken, and the type of ART regimen in use were checked and recorded. Socio-demographic characteristics of the caretaker were also recorded. Screened patients in the study were on ART regimens in line with the Tanzania guideline for diagnosis and management of HIV infection. All patients were on Paediatric Fixed Dose Combination (FDC) tablets, and patients who were selected into the study, were either on: Triomune Baby, FDC 6®  or Triomune Junior, FDC 12®  or a fixed-dose combination containing 60 mg Zidovudine, 30 mg lamivudine, and 50 mg nevirapine. The nevirapine dose was 160 - 200 mg per mce a day . HoweverA single blood sample for determination of nevirapine plasma concentration and CD4 cell count/percentage was collected and divided into two aliquots. The aliquot for CD4 cell count/percentage was placed in Ethylenediaminetetraacetic Acid (EDTA) tubes and transported to the Muhimbili HIV Reference Laboratory for flow cytometry whereas the aliquot for nevirapine plasma concentration was placed in heparinised tubes and immediately centrifuged to obtain plasma. The plasma samples were transferred into cryovials, transported in a cool box to the MUHAS-Sida Bioanalytical Laboratory and stored at −80°C until assay. Determination of nevirapine plasma concentration was done using a published reversed phase High Performance Liquid Chromatography (HPLC) method . Before Adherence to antiretroviral therapy was assessed using caretaker report, medication return and nevirapine plasma concentration. The caretaker reported the total number of missed doses for the past three days. Adherence was concluded when no more than one dose was missed three days prior interview. A patient assessed by medication return was categorized as adherent when less than 5% of the medications were returned on the day for a refill and the level of adherence was calculated as percentage of prescribed dose not returned. Nevirapine trough plasma concentration of 3 μg/ml has been shown to be the cut off for efficacy . Therefo1) was assumed to be 50% and the proportion of immunosupressed children (π0) to be 30%. Taking 5% significance level, and an 80% power to detect a significant difference, the minimum sample size was calculated to be 200 and thus a total of 300 patients  was recruited into the study.The sample size was calculated based on the comparison of two independent proportions from groups of unequal sample size (good immune status versus immunosupressed). Since no similar previous study could be traced in the literature, the proportion of patients with good immune status ; age and sex of the patient; caretaker type; disclosure of child; and duration of ARTIn this study, immune status was defined and graded according to WHO immunological classification . Good imData were entered in the Epi info software version 3.5.1 and then exported to the Statistical Package for Social Science (SPSS) version 15 for analysis. Mean and standard deviations were calculated for continuous variables whereas percentages were calculated for categorical variables. All continuous variables were categorized and analyzed using the Chi square test or Fisher’s exact test and P value < 0.05 was considered significant. The association was measured by Odds ratio (OR). Factors with P-value 0.2 or less at bivariate analysis were selected for further multivariate analysis and entered into a logistic regression model. The model was used to assess the independence and strength of association of predictive factors of interest using CD4 count as the outcome variable, as well as to control for possible confounders of the main predictor (adherence) and outcome. A difference of at least 10% between adjusted odds ratio and the crude odds ratio was considered confounding. The Kappa statistic was used to assess agreement between adherence measures . The sensitivity, specificity, positive predictive value and negative predictive value were used to determine the validity and precision, using CD4 cell count/percentage, to compare the three methods of measuring adherence  to detect adherence and non adherence.A total of 300 participants met inclusion criteria and were recruited into the study. The majority of these patients were above five years (81.3%) of age. The mean age (SD) was 8 (3) years and almost half of the participants were female (50.7%).The majority  had advanced stage of HIV disease and almost half were malnourished. Of the 176 children assessed for disclosure only 23.9%, (n = 42) with age of 7–14 years knew their HIV status. Most of the children  were on ART for more than one year and 31.7% (n = 95) had infections. More than three quarters  of children were immunosuppressed at baseline while only 28% (n = 84) were immunosuppressed when current CD4 cell %/ count was checked..The mean age of the caretakers was 35.9 ± 10.7 years ranging from 18 to 70 years. Of the 300 caretakers, 88.3% (n = 265) were female and only 21% (n = 63) had beyond primary school education. Most of the caretakers had primary school education . The majority of caretakers  were mothers of patients. Among the 300 caretakers 36% (n = 138) were married and only 16.7% (n = 50) were employedAs many as 85% of the participants n = 254) were adherent when assessed by nevirapine concentration whereas the proportion of adherence assessed by medication returned and caretaker report were ,  respectively  whereas adherence by caretaker report as well as by medication report were not associated with immunosuppression  and  respectively  , patients on antiretroviral therapy for less than one year  and infections . These infections included pneumonia, Otitis media, oral lesion and skin lesion.The final multivariate analysis was performed by a backward elimination procedure taking into account the biological knowledge about independent variables and how they relate to immune status. After controlling for confounders, nevirapine plasma concentration <3 μg/ml was independently associated with immunosuppression . Duration of antiretroviral therapy (ART) less than a year was also independently associated with immunosuppression (OR = 6.79. CI = 2. 69–17.16). The potential confounders of nevirapine plasma concentration were patients with young age (2-5 years)  and infections   and one seldom used method (drug plasma concentration). Our data are similar to what has been reported in adult patients and suggest that care taker report and medication return may overestimate the true level of adherence. Adherence to Antiretroviral therapy in paediatrics is critical in order to maximize the benefit of medication. Inadequate adherence is associated with immunological, and virological failure; drug resistance, and treatment failure . In our In addition, there was a weak agreement in estimating adherence between nevirapine plasma concentration method and care taker report (kappa 0.09) as well as medication return (kappa 0.131). This means, non-adherent patients were classified as adherent by caretaker report and medication return methods whereas in fact, were non-adherent.Using medication returns and care taker report, our data show that only 3% of the patients were non adherent to antiretroviral medication. These findings are similar those reported from Uganda where the level of adherence assessed using medication return and caretaker reporting was relatively high . NevirapOur data show that nevirapine plasma concentration may be a better predictor of adherence and correlates well with the immunological response compared to medication return and caretaker report. However, lack of ability to detect missed dose through determination of drug concentration by HPLC, for drugs with a long half-life, such as nevirapine is one of the limitations of our study. Failure to detect missed doses would underestimate non adherence to ART in some patients. In addition, only a single blood sample was taken, and the time of last dosing was dependent on the authenticity of the caretaker’s reporting, which may not be guaranteed. It is possible that some of the caretakers or older children damped medication due to reasons previously reported  so as to. However, it is important to note that, blood drug concentration can be affected not only by the level of adherence but also drug bioavailability, individual’s metabolic capacity, timing of drug intake and the time the blood sample for determination of drug concentration is taken as well as having diarrhoea or vomiting immediately after drug administration. In our study, no patient was reported to have vomited or experienced diarrhoea after drug intake. It is unlikely that the low plasma concentrations observed in some patients have been due to poor nevirapine bioavailability since, in all patients, we used same paediatric fixed-dose combination tablets obtained from WHO prequalified manufacturers. In this study, all patients who were using other drugs, which could interact with nevirapine, such as ketoconazole and rifampicin were excluded precluding the possible influence of enzyme inhibition or induction on the observed plasma concentration. However, in this study, the patients were not genotyped with respect to CYP 3A and 2B6 to rule out the role of indivudual’s metabolic capacity that could influence the plasma concentrations of nevirapine. A recent Pharmacogenetics study conducted in Tanzanian adults indicated the frequencies of CYP2B6 genotypes to be 37%, 45% and 16% for homozygous wild type (fast metabolizers), heterozygous (intermediate) and homozygous mutated (poor metabolizers) repectively [In this study, non-adherence by nevirapine plasma concentration was found to be 15%, which is alarming when considering the management of HIV/AIDS in childrenectively . This imDespite the above limitations, our findings support the use of drug plasma concentration as an objective method for determining adherence to ART where feasible. This would complement the information obtained from care taker report and medication returns particularly in groups of patients showing poor treatment response. Determination of plasma drug concentration to estimate adherence is often not feasible but nevirapine plasma concentration may be useful and cost-effective in the setting of a pharmacovigilance program with adherence monitoring where this may be limited to patients at sentinel sites and at regular intervals. Apart from estimating adherence, nevirapine plasma concentration may be used in the analysis of non-response that may lead to detection of early signs of drug resistance particularly in patients with well characterised genotypes of important metabolizing enzymes.Lower level of adherence by nevirapine plasma concentration and its association with immunological response supports the validity of the method and indicates that the adherence estimate obtained by care takers’ reporting and medication returns may be an overestimate of true adherence level. This may have important clinical implications in antiretroviral therapy. Therefore, nevirapine plasma concentration may be a good predictor of adherence and should be considered where it is feasible and cost-effective.The authors declare that they have no competing interests.MFW participated in the design of the study, coordination, data collection, data analysis and writing the manuscript. MO participated in the design, interpretation of nevirapine plasma concentration results, read and advised in writing final manuscript. MA participated in the design, advised and read the final manuscript. SP participated in design, data interpretation read and advised in writing the final manuscript. We have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/13/95/prepub