The HIV wasting syndrome represented the face of HIV/AIDS before the advent of highly active antiretroviral therapy (HAART). Although the incidence of wasting has declined since the introduction of HAART, weight loss remains common in patients receiving HAART, especially in the setting of a failing HAART regimen. As we are not aware of any previous reports from Nigeria, we report a case of the classical wasting syndrome in a Nigerian female who had both virological and immunological HAART failure due to poor adherence. The influence of a failing HAART regimen, socioeconomic status, and other clinical variables in the wasting syndrome are discussed. HIV wasting syndrome has been defined by the Centre for Disease Control (CDC), USA, as involuntary weight loss greater than 10% of baseline weight associated with either chronic diarrhoea for at least 30 days or chronic weakness or documented fever for at least 30 days in the absence of a concurrent illness or condition other than HIV infection that could explain findings  , 3, weigIn Nigeria, weight loss and severe wasting often complicates HIV/AIDS –9, but wA 32-year-old HIV-1-infected widow was admitted to our tertiary hospital with a 6-month history of progressive lethargy, anorexia, recurrent fever, vomiting, watery non-bloody diarrhoea, and progressive weight loss. She went from 50 kg to 21 kg in 5 months, a 42% weight loss. She was unemployed and had no source of regular income to care for herself or her 3 children. Hence, her meals before and during her illness were irregular and consisted mainly of carbohydrate diet. 2, and the mid-upper arm circumference was 10 cm  and low serum albumin of 22 mmol/l, other renal and liver function tests were normal. Chest X-ray and abdominopelvic ultrasound were normal. Hepatitis B and C serology and blood film for malaria parasites were also negative. She was on HAART  for 16 months but adherence was poor (<70%). On HAART, her viral reduced from 421,000 copies/ml to 41,000 copies/ml, but CD4+ T cell count dropped from 168 cells/ul to 34 cells/ul. Full blood count revealed microcytic hypochromic anaemia (packed cell volume of 24%) and leukopenia of 1.2 × 10She was rehydrated, and diarrhoea was controlled with an antimotility agent (tablets loperamide) after empiric treatment for infectious diarrhoea using tablets albendazole 400 mg daily for 3 days, intravenous ciprofloxacin 500 mg twice daily for one week, and tablets tinidazole 2 g start. Oral fluconazole 200 mg daily for oral thrush, prohylactic septrin 960 mg daily, multivitamins, and haematinics were also given. She had intensive nutritional rehabilitation with locally available high protein and high energy diet as well as fruits and vegetables. Specifically, “kwashiokor pap” or “kwashi pap”, a high protein local diet made up of a mixture of ground guinea corn, ground soya bean, and ground crayfish, ground dried fish, roasted ground groundnuts, and boiled water was given orally three to five times per day, along with variety of other local foods. Additional adherence counselling was given, and ART was switched to Emtricitabine/Tenofovir and Nevirapine on account of anaemia. All symptoms gradually resolved and she gradually regained her physical strength with improved physical activity. At the end of 6 weeks, her weight increased by about 8 kg and she was thereafter discharged to the social welfare, nutritional, and adherence counselling unit. The pathophysiological mechanisms underlying the HIV wasting syndrome are related to three major factors including inadequate nutrient intake, nutrient malabsorption, and disturbances in metabolism (reviewed in references –12). The burden of HIV infection impoverishes ; in sub-Cytomegalovirus, Clostridium difficile, and Mycobacterium avium intracellular bacteria, among others) [Both micronutrient and macronutrient malabsorption have been observed in the wasting syndrome, and nutrient malabsorption may occur with or without diarrhoea , 16. Chr others) . In AIDS others) . AIDS en others) , 19. Con others) . By leading to malabsorption and/or changes in pharmacokinetics of the ART medications, chronic diarrhoea may also contribute to HAART treatment failure. In a clinical study by Brantley et al. , AIDS-reVarious metabolic abnormalities leading to weight loss have been described in HIV-infected patients, and these abnormalities have been attributed to several etiologies including the HIV virus itself, concomitant opportunistic infections, cytokine dysregulation, and hormonal imbalances accompanying HIV infection, as well as ART medications , 11. α, interleukins-1 (IL-1), IL-6, and interferon gamma, have been implicated in these metabolic perturbations [A failing HAART regimen leads to persistent viral replication and progressive immunosuppression as reflected in the CD4 cell count. Correlations have been established between high HIV viral load, low CD4 cell counts, and weight loss , 23. In rbations . α also lead to muscle weakness and muscle atrophy [By interfering with lipid metabolism in muscle, elevated levels of proinflammatory cytokines such as TNF- atrophy ; both arThe use of HAART has also been independently associated with an increase in resting energy expenditure, and this has been suggested as one of the factors perpetuating weight loss in the HAART era , 10. Alt Hypogonadism, represented by low levels androgen such as testosterone, may accompany HIV infection in men , and it Intensive nutritional rehabilitation to prevent or reverse weight loss remains the cornerstone of management of the wasting syndrome , 12. TheIn combination with adequate caloric intake, fitness training by progressive resistance exercises  increases muscle function and strength, as well as lean body mass and weight . ConversPharmacological treatments are usually reserved for patients who fail nutritional therapy. Appetite stimulants , recombinant human growth hormone (Serostim), and androgenic steroids  in men with hypogonadism have all been approved for the treatment of the wasting syndrome . CytokinHIV wasting syndrome is a disorder characterised by multiple pathophysiological mechanisms, most mediated by the HIV virus itself and driven by nutritional abnormalities. HAART treatment failure is an emerging global challenge, especially in developing countries where HIV infection is still endemic and the use of HAART is being scaled up . A failiTo prevent or reverse the resurgence of AIDS defining illness such as the wasting syndrome in this HAART era, ensuring effective uninterrupted HAART, socioeconomic empowerment of HIV-infected patients, and prevention of opportunistic infections are priorities for developing countries such as Nigeria.We declare that this work was done by the authors and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. The first author conceived the report; all authors were involved in patient management, manuscript preparation, and review, and they approved the final version for publication.No conflict of interests associated with this work.Consent was obtained from the patient for clinical photos.


Aprepitant is a NK1R antagonist approved by FDA as an antiemetic.Neurokinin-1 receptor (NK1R) antagonists have anti-HIV activity in monocyte-derived macrophages, decrease CCR5 expression and improve natural killer cell function 3 and plasma viral load ≥2,000 copies/ml. Subjects were stratified by viral load  and randomized within each stratum to receive aprepitant at 125 mg QD(Low), or 250 mg QD(High), or placebo(PL) for 14 days, and followed for 42 days.We conducted a phase IB randomized, placebo controlled, double masked study to evaluate the safety, antiviral activity, pharmacokinetics and immune-modulatory effects of aprepitant in HIV-infected adults not receiving antiretroviral therapy, with CD4+ cell count ≥350 cells/mmThirty subjects were randomized and 27 completed treatment , 250 (High), and PL groups). 63% were male; 37% white; mean (SD) age 43 (9.3) years. Geometric mean baseline viral load (copies/ml) for Low, High, and PL was 15,709, 33,013, and 19,450, respectively. Mean (95%CI) change in log10 viral load at day 14 for Low, High, and PL was −0.02, −0.05, and +0.04, respectively. The number of subjects with AEs was 4(44.4%), 5(62.5%), and 1(10%) for Low, High, and PL. No Grade 4 AEs occurred.Adverse events of aprepitant were more common in the treated groups. At the dose used in this two-week phase IB study, aprepitant showed biological activity, but no significant antiviral activity.NCT00428519ClinicalTrials.gov  The current paradigm of HIV treatment is the continuous use of multiple antiretroviral drugs that target several steps of the life cycle of the HIV virus. This strategy has been associated with substantial decreases in the morbidity and mortality associated with HIV infection A continued challenge in anti-HIV drug development is to make available antiretroviral agents with new target and mechanisms of action and activity against drug-resistant virus. Alternative paradigms for the treatment of HIV infection also need to be explored. Recent studies have demonstrated the key role that immune activation plays in the pathogenesis of HIV infection and disease progression The use of pharmacological agents and immunomodulators that target this residual inflammation acting at the cellular level, rather than at the HIV virus level might be useful in preventing inflammatory and neurocognitive events associated with HIV infection ex vivo studied.Substance P (SP), a member of the tachykinin family Preliminary data suggest that aprepitant, the only approved SP antagonist has anti-HIV activity similar to CP-96,345 in the same ex vivo system The purpose of this clinical trial was to determine the in vivo safety and antiviral activity of aprepitant by comparing the change in HIV RNA viral load after 2 weeks of aprepitant monotherapy in patients with HIV infection not receiving any other antiretroviral treatment. The study was designed as a two-week trial as mandated by the FDA in order to avoid emergence of resistance or changes in tropism if aprepitant had significant antiviral activity.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see 3 and plasma viral load ≥2,000 copies/ml. Thirty patients with HIV-1 infection, not receiving antiretroviral therapy were stratified by viral load  and randomized within each stratum to receive aprepitant 125 mg QD(Low), 250 mg QD(High), or placebo (PL) for 14 days, and followed for 42 days. The drug was masked by over encapsulation. The investigators were blinded to the study assignment of the patients.This was a phase IB randomized, placebo controlled, double masked study to evaluate the safety, antiviral activity, pharmacokinetics and immune modulatory effects of aprepitant in HIV infected adults not receiving antiretroviral therapy, with CD4+ cell count ≥350 cells/mmAt the screening visit, previous antiretroviral treatment (if any) was assessed, safety laboratory tests were conducted, and all patients underwent testing for HIV-1 co-receptor tropism with the use of a validated phenotypic tropism assay . Patients were also tested for plasma levels of HIV-1 RNA . Participants were then randomized and evaluated at day 0, 3, 7, 10, 14, while they were receiving aprepitant or placebo, and at day 42, 4 weeks after discontinuing study medication. Additionally, and 8 hour pharmacokinetic assessment was done after the first dose and at day 14.A modified commercially available antigen competition enzyme immunoassay (EIA) from Cayman Chemical Company  was used for the quantitation of SP, as previously described max, Tmax) and exposure metrics (AUC) were calculated using WinNonlin version 5.2 (Pharsight Corporation). PK data were summarized with descriptive statistics and graphical presentation was made using GraphPad Prizm version 4.A validated, liquid chromatography-tandem mass spectrometry method was utilized for the quantification of aprepitant in HIV-infected patients Viral tropism was assessed using the Trofile™ assay. Samples were shipped to Monogram Biosciences Inc., San Francisco, CA at day 0 and 14 of the study to evaluate any changes in the tropism of the HIV virus of the participants.primary objectives of the study were to assess the safety and tolerability of two different doses of aprepitant for 2 weeks in HIV infected individuals and to assess the response of plasma HIV-1 RNA. Our secondary objectives were to evaluate the dose-response and pharmacokinetic and pharmacodynamic relationship between viral RNA change and aprepitant plasma levels, the effects on CD4+ and CD8+ T-cell counts, circulating SP levels, , the effects of aprepitant on viral tropism and to provide preliminary description of any change mediated by aprepitant in sleep quality, anxious mood, depressed mood and neurocognitive measures , the Hamilton Anxiety Scale (HAM-A) and the Pittsburgh Sleep Quality Index Score.Our 3, HIV RNA viral load greater than 2,000 copies and an R5 tropic virus (Monogram). We excluded individuals with a history of cancer and other serious illness, pregnancy, chronic hepatitis B or C infection, individuals with significant laboratory abnormalities, or were using steroids or any other immunomodulators or chemotherapy. We also excluded individuals with allergy or hypersensitivity to aprepitant.We included HIV infected individuals, older than 18 years of age, not receiving antiretroviral therapy for at least 16 weeks, with CD4 cell count greater than 350 cells/mmThe study was conducted at AIDS Clinical Trials Unit and the Clinical and Translational Research Center (CTRC) of the Hospital of the University of Pennsylvania in Philadelphia, PA, USA. All patients signed a written informed consent. The study was sponsored by the National Institutes of Mental Health, approved by the IRB of the University of Pennsylvania, the US Food and Drug Administration , and registered in Clinical Trials.gov #NCT00428519.10), the mean and 95% confidence intervals of the intra-individual difference  was computed within each treatment group, whereas , for SP, the percent change from baseline was computed. For CD4+ cell count, the geometric mean at each visit was calculated for each of the three treatment groups. Linear mixed-effects models were fit to examine the potential change over time in viral load and CD4+ at days 0, 3, 7, 10 and 14 among the three treatment groups. The basic model has the viral load (or CD4+) as the outcome variable; and treatment group, visit, and treatment-by-visit as explanatory variables. Kruskal-Wallis nonparametric analysis of variance (ANOVA) was used to compare viral load at day 42, CD4+ at day 42, and SP at days 14 and 42 among the three treatment groups. Fisher's exact tests were used to compare the distribution of the grade 2–4 adverse events among the three treatment groups, and between the aprepitant-treated and the placebo groups. Wilcoxon signed-rank test was performed for the paired comparison of peak aprepitant concentration from 0 for each treatment group at day 1 and day 14. Wilcoxon signed-rank test was also performed for the paired comparison of peak aprepitant concentration from day 1 to 14 within each treatment group. Wilcoxon rank-sum test was used for the comparison of the difference in peak aprepitant concentration at day 1 or 14 between two aprepitant-treated groups. The P-values<0.05 were considered statistically significant. All data analyses were conducted using SAS 9.2.A sample size of 9 subjects within a dose group, gave us a 95% probability to observe one or more adverse events assuming the true underlying event rate within the dose group was at least 30%. We allowed the substitution of participants that did not complete the trial. The analyses of adverse events were primarily descriptive. The number and percent of subjects with an adverse event were calculated within each body system and overall for each of the three treatment groups. The geometric mean viral load (copies/ml) at baseline was calculated for each of the three treatment groups. For viral load , and 27 completed treatment , High (250 mg daily), and placebo (PL), respectively). 22 participants were naïve to antiretroviral therapy, eight had prior experience. Geometric mean viral load (copies/ml) at baseline for 125 mg (Low), 250 mg (High), and PL was 15,709, 33,013, and 19,450 copies/mL. There were no statistically or clinically significant changes in HIV RNA viral load in any of the three groups during the drug or placebo administration . The meaSixteen Grade 2 AEs and 1 Grade 3 AEs were reported (excluding pre-existing and ongoing events). The number of subjects with grade 2–4 adverse events (co-primary endpoint of the study) was 4(44.4%), 5(62.5%), and 1(10%) for Low, High, and PL, respectively (P = 0.056). Subjects in the aprepitant-treated groups experienced more grade 2–4 adverse events than that of the placebo group (P = 0.042). Neurological AEs were reported by 4 subjects in the High group  and 1 subject in the Low group (insomnia at 2 visits) .Mean values of SP concentration in plasma were relatively stable in the placebo group during the study  but theyMean (+standard deviation) aprepitant plasma concentration profiles for both dose groups are shown in Individual peak aprepitant plasma concentrations (Cmax) on days 1 and 14 are listed in There were no changes in the tropism of the HIV virus in study participants. All participants' viruses remained R5 tropic, as they were at the initiation of the study.Aprepitant (dosed at 125 and 250 mg daily for two weeks) was safe in patients with HIV infection not receiving antiretroviral therapy. Aprepitant showed biological activity, but not clinically  significant antiviral or immunologic improvement at this dose range and duration of therapy. Adverse events were more common in the aprepitant-treated groups, but the safety profile was similar to patterns and frequency observed that in HIV-uninfected patients treated for chemotherapy induced nausea and vomiting.We observed moderate declines of uncertain clinical or a statistical significance in SP in both treated groups. SP enhances inflammatory cytokine  production by immune cells such as macrophages through activation of NF-kB The observed reduction of plasma SP levels is of interest, since we have previously demonstrated increased levels of SP in men In another context, plasma SP levels have been studied in relationship to neuropsychiatric disorders. SP levels are elevated in the cerebrospinal fluid of patients with major depression A significant proportion of individuals successfully treated with antiretroviral therapy maintain elevated levels of immune activation and inflammation 2–6 years after the initiation of antiretroviral therapy We did not observe changes in sleep quality, anxious mood, depressed mood and neurocognitive measures. The absence of changes in these parameters most likely reflects the lack of significant pathologic characteristics at baseline in our study population rather than a lack of effect of this compound in mood and depression. Neurokinin-1 receptor antagonists originally showed promise as novel antidepressants Aprepitant pharmacokinetic results in HIV-infected patients are generally consistent with data previously published in healthy volunteers in vitro, however its in vivo effects seem to be limited:We propose several explanations of why aprepitant seems to have an antiviral effect The dose used in this study is probably too low to see an antiviral effect. The IC50 of aprepitant is approximately 5 µM in vitro (2.5 µg/ml) and the concentrations reached in this study were clearly subtherapeutic. There are a number of strategies that can be employed to boost the exposure of aprepitant to yield exposures in the therapeutic range including the co-administration of inhibitors of the p450 system such as ritonavir or cobicistat, as is done with many antiretroviral drugs. The coadministation with these boosters is expected to result in increased plasma levels of the drug due to inhibition of the induction phenomenon that has been reported in the past with extended aprepitant administration The duration of the administration of aprepitant was too short. Because anti-inflammatory compounds do not target viral proteins and act through an indirect mechanism it is not surprising that they do not show antiviral activity after the short term administration that characterize antiretroviral trials. We have recently completed an in vivo study in Rhesus macaques evaluating the antiviral activity of aprepitant in the setting of SIV infection. The study was conducted in the Non Human Primate center of Tulane University. Two groups of Rhesus macaques (n = 4 in each group) were infected with SIVmac251. One group was untreated and served as control and the other group received aprepitant (125 mg q.d.). There was a difference in viral load set point of approximately 1 log that became apparent only after 90 days of treatment with aprepitant .In conclusion, at the dose used in this exploratory phase IB study, aprepitant was safe and showed biological activity but did not show significant antiviral activity. Pharmacokinetic studies showed concentrations could reach as high as 5.5 µg/ml without enzymatic induction of the aprepitant metabolism although this must be revisited over longer drug administration. Further studies evaluating larger doses and/or co-administration of aprepitant with ritonavir or cobecistat are planned. Hence, pharmacokinetic strategies are possible to achieve higher exposure targets and overcome any presumed liability due to nonlinearities in drug clearance. A study of 375 mg of aprepitant daily in HIV infected individuals is currently ongoing . Future studies evaluating the therapeutic window of aprepitant in Neuro AIDS patients at doses and study durations targeted to show the multimodal effects demonstrated in vitro and in preclinical models are necessary before proof-of-concept can be established. The therapeutic goals for this class of compounds will need to be defined in terms of their ability to improve both virologic and neurocognitive outcomes.Checklist S1CONSORT Checklist.(TIF)Click here for additional data file.Protocol S1Trial Protocol.(PDF)Click here for additional data file.


HIV diagnosis, prognostic and treatment requires T CD4 lymphocytes’ number from flow cytometry, an expensive technique often not available to people in developing countries. The aim of this work is to apply a previous developed methodology that predicts T CD4 lymphocytes’ value based on total white blood cell (WBC) count and lymphocytes count applying sets theory, from information taken from the Complete Blood Count (CBC).3, lymphocytes/mm3, and CD4/μL3 subpopulation per flow cytometry of 800 HIV diagnosed patients. Union between sets A and C, and B and D were assessed, and intersection between both unions was described in order to establish the belonging percentage to these sets. Results were classified into eight ranges taken by 1000 leucocytes/mm3, calculating the belonging percentage of each range with respect to the whole sample.Sets theory was used to classify into groups named A, B, C and D the number of leucocytes/mmIntersection (A ∪ C) ∩ (B ∪ D) showed an effectiveness in the prediction of 81.44% for the range between 4000 and 4999 leukocytes, 91.89% for the range between 3000 and 3999, and 100% for the range below 3000.Usefulness and clinical applicability of a methodology based on sets theory were confirmed to predict the T CD4 lymphocytes’ value, beginning with WBC and lymphocytes’ count from CBC. This methodology is new, objective, and has lower costs than the flow cytometry which is currently considered as Gold Standard. HIV infection has affected around 60 million people to date . In 20093[HIV is a retrovirus that mainly affects T cells and those cells that express CD4, such as macrophages, follicular dendritic cells and lymph nodes [3. Its det3,7. Howev3-9.3; however, when this prediction was compared to the deduction based on total lymphocytes, it was found that in male patients sensitivity increased with no changes in specificity, and in female patients sensitivity did not change and specificity decreased [Given the large impact that HIV/AIDS represent for global public health, it has been sought to make flow cytometry more accessible by implementing simplified flow cytometers that are chargeable by battery or solar panels . On the ecreased . A crossecreased . Howeverecreased . Anotherecreased . This moecreased . In the ecreased .3 each, for its evaluation. The conclusion was that ranges below 5000–4000 leukocytes/ml3 predict CD4 counts lower than 570 CD4/μL3 with effectiveness percentages between 90-100% [In a previous study, Rodríguez et al.  develope 90-100% . This shThe aim of this work is to validate the clinical application of the methodology developed based on sets theory, applying it to a larger sample of HIV-positive cases.Determined Sets for the study of leukocytes/mm3, lymphocytes/mm3and CD4/μL3populations[:pulations:A. {/x > =6.800 ^ y > =1.800}B. {/x > =6.800 ^ z > =300}C. {/x < 6.800 ^ y < =2.600}D. {/x < 6.800 ^ z < =570}Where  is a triplet of values, being “x” the number of WBC, “y” the number of lymphocytes and “z” the T CD4 lymphocytes’ count.It is a study in which a physical–mathematical previously developed methodology based on sets theory is applied in order to predict T-CD4 lymphocytes’ count. It is based on the mathematical analysis of the total WBC and lymphocytes’ count in HIV-positive patients.Printed CBCs of 800 HIV diagnosed patients were used, without distinction of gender, age, population kind, or clinical variables such as infection stage, hemoglobin value or medications used. The CBCs were taken from storage tests in a physical data-base of the infectologist who participated in the study.3, lymphocytes/mm3 and CD4/μL3 subpopulation counts measured by flow cytometry were taken. Then, they were organized in descending order according to the WBC number, establishing ranges of 1000 leukocytes/mm3. Values higher than 10.000/mm3 were assigned to a single range as well as values lower than 3.000/mm3, so a total of 9 ranges were established in order to observe mathematical relationships between populations, independent of time or patient’s evolution.First, records of leukocytes/mmAccording to the previously developed methodology, records were evaluated by establishing if they belonged or not to sets A∪C and B∪D, as well as to set (A∪C) ∩ (B∪D), using a software that was previously developed based on sets algebra . This so3, with a lymphocyte value equal to or higher than 1800/mm3 and with a CD4 cell value equal to or higher than 300/mm3 or it may have a leukocyte value lesser than 6800/mm3, with a lymphocyte value equal to or lesser than 2600/mm3 and with a CD4 cell value equal to or lesser than 570/mm3.Results for the 9 leukocytes ranges were assessed, determining the elements number that belong to each set in each range and the percentage of success to which it corresponds to, according to the total number found for each range. In addition, the same values were established for the whole sample. In this work, the belonging percentage of each range to each one of sets is equivalent to the effectiveness percentage of prediction for such range. When a triplet of values belongs to all sets, this fact means that this triplet met with the condition of have a leukocyte value equal to or higher than 6800/mmSome performance measures were established for each range through a binary classification performance measurement, where True positive (TP) is the number of cases with a correct prediction in the range with respect to real values, False negative (FN) is the number of wrong predictions in the range with respect to real values, and finally True negative (TN) is the total number of correct predictions in the other ranges. The performance measures calculated for each range were Sensitivity (SENS), and Negative Predictive Value (NPV); the first one which was calculated with the next equation:Otherwise, Negative Predictive Value (NPV) and was calculated by means of the next equation:This study follows the laws established on articles 11 and 13 of the 008430 Colombia’s Health Ministry resolution of 1993 given that physical calculations were made based on results of medically prescribed tests of the clinical practice, from an anonymous database retrospectively evaluated, with no risks to patients, protecting the integrity and anonymity of participants and with no need of informed consents. The approval of an ethics committee of a specific institution is not needed because it was accessed only numerical values of the database , collected specifically for research purposes by one of the authors.Belonging of leukocytes, lymphocytes and CD4 cells values to each set in 27 specific samples is shown at the Table Table TP values ranged between 17 and 136, TN values were between 450 and 569, and FN between 0 and 59. Values for SENS ranged between 0.56 and 1, and values for NPV were between 0.89 and 1. The highest SENS values were for the ranges of 10000 leukocytes or more, between 9999–9000, 3999–3000, and for the range of 2999 leukocytes or less; the first three had values of 0.99 and the last one of 1.The NPVs showed values equal to or greater than 0.98 in 5 out of the 9 assessed ranges (See Table This is the first work in which a new predictive methodology of T CD4 lymphocytes’ count is applied to a sample of 800 HIV-positive patients. This methodology was developed beginning with the analysis of WBC and lymphocytes’ count from CBC, and it is based on sets theory. Its predictive percentages are equal or above 73.91% for 6 out of 9 measured ranges, confirming its predictive capacity and clinical applicability independently of epidemiological and clinical variables. Sensitivity values over 0.80 were founding 5 of the 9 measured ranges; specificity was not calculated, given that there are no False Positives.3, this predictive methodology showed an effectiveness percentage of prediction of 100% when leukocyte values were less than 3000.This means that a value of CD4 less than 570/mm3 is predicted for all these cases.Taking into account that the starting of anti-retroviral treatment is suggested at 300 CD4 cells/cmThe belonging percentage to set A ∪ C is greater than the percentage to set B ∪ D, showing the specificity of T CD4 lymphocytes’ values and evidencing the difficulty to find results that allow their prediction. In contradistinction to the previous work , one morThe mathematical theory through which predictions are obtained does not allow the establishment of False Positives in the statistical analysis, given that each set, as well as the intersections that constitute the prediction, exclude the possibility of finding triplets that allow obtaining a False Positive prediction. This is the reason why it is not possible to establish a positive predictive value, showing that this mathematical inductive way of thinking can’t be taken directly from traditional statistical parameters; instead of that, the sets algebra way of thinking achieves deductive predictions of clinical importance.Works performed with the aim to simplify and reduce costs of HIV patients follow-up are mostly epidemiological, with limitations in the prediction of immunological biomarkers -13, giveBased on neural networks and machine learning, some methodologies propose viral load measurement as marker of treatment response in HIV-infected patients. The limitation of these experimental methodologies is that they don’t take into account the immune response of the patient, but genotypic virus characteristics -19, whicIn the context of dynamic systems, a descriptive but not predictive model of the immune response to HIV dynamics was developed by plotting how T CD4, CD8, B lymphocytes and antibodies act, and how the viral load progresses . The preLikewise in this work, where predictions are achieved from mathematical theories, in different areas of medicine other phenomena have also characterized and predicted from several mathematical and physical theories, evidencing the mathematical self-organizations of these phenomena -30.This study confirms the predictive capacity of the developed methodology based on sets theory to determine the number of T CD4 cells based on WBC and lymphocytes’ count, achieving a 91.89% effectiveness for the range between 3000 and 3999 leukocytes, and 100% for the range below 3000 leukocytes. This methodology can be useful to determine the number of CD4 in places where there is no easy access to flow cytometry, reducing costs in determining the state of patients with HIV/AIDS.The authors declare that they have no competing interests.JOR developed the original idea and coordinated the study, SEP and CC made the data analysis and contributed in drafting and revising the manuscript; CEP revised the manuscript and made data recollection and its systematization; JTM and JB revised the manuscript and participated in data recollection and its systematization; YS and LA contributed in drafting and revising the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1756-6649/13/3/prepub


It was an observational cohort of pregnant HIV positive women ignorant of antiretroviral therapy with CD4 cell count of>350/mm3. Exclusion criteria: highly active antiretroviral therapy prior to pregnancy.Routine CD4 cell count assessment in HIV positive pregnant women completed by non exclusive measurement of the viral load by PCR /ARN in those with CD4 cell count>350/mm3, median: 528 (IQR: 421–625). 145 underwent measurement of viral load by PCR/RNA at a median gestational of 23 weeks of pregnancy (IQR: 19–28). Median viral load 4.4log10/ml, IQR (3.5–4.9).19/145(13%) had an undetectable viral load of=1.8log10/ml. 89/145(61%) had a viral load of=4 log10/ml and were eligible for maternal triple ARV prophylaxis.Between January and December 2010, CD4 cell count was systematically performed in all pregnant women diagnosed as HIV-infected (n=266) in a referral center of 25 antenatal clinics. 63% (N=170) had CD4 cell count>350/mm3 may require triple antiretroviral for prophylaxis of MTCT. Regardless of cost, such results are conclusive and may be considered in HIV high burden countries for universal access to triple antiretroviral prophylaxis in order to move towards virtual elimination of HIV MTCT.More than 6 in 10 pregnant HIV positive women with CD4 cell count of>350/mm Sub-Saharan Africa continues to record the highest burden of the HIV-1 epidemic, contributing more than 90% in the new cases of perinatally acquired HIV pediatric infections . Mother-3. Maternal baseline serum samples were quantified for HIV-1 RNA load using real Time reverse transcriptase PCR . All the samples were analyzed with the Laboratoire Centre Pasteur in Yaoundé acting as service provider at a unit cost of 30US$. ZDV was started for all the others non eligible for triple antiretroviral treatment at 14 weeks of pregnancy, while waiting for the result of the viral load.The study population was an observational cohort of pregnant HIV positive women. Pregnant women with<36 weeks of amenorrhea were enrolled in 25 antenatal clinics in the Djoungolo Health District. After opt-out counseling, serial HIV-1/2 algorithm antibody tests were done using Determine HIV-1/2 assay and SD bioline HIV 1/3 3.0  rapid kits on mothers' serum samples. For all HIV positive women, CD4 cell counts were measured in the referral laboratory. The method used a flow cytometer for measuring the percentage of CD4 T cells; an automated blood cell counter helped to measure the total number of lymphocytes. Results were produced in absolute numbers and in percentages. CD4 cell count screening was followed by non exclusive measurement of viral load when CD4 cell counts were>350/mm10 was considered as undetectable viral load. For this descriptive analysis we considered . A confidence interval of 95% was considered as the margin for percentage accuracy.The following qualitative variables were considered: maternal age, WHO clinical stages. Quantitative variables included: CD4 cell count and HIV Viral load. Viral load values were log10 transformed and stored in an Excel sheet form. A threshold of≤1.8 logAll interventions within this project were implemented under the supervision of the District Office of the Ministry of Public Health. Data collection and monitoring of the intervention was part of the routine activities of the Health District. Free measurement of HIV1 RNA viral load was offered to all the eligible mothers at the referral center where all activities were coordinated by the Center for HAART treatment and the local board in charge of fighting against HIV and AIDS.Mothers′ mean age (SD) was 27.0 years IQR 22.5–30 and 15/172(8.7%) were adolescents. All the mothers were classified in WHO clinical stage 1 or 2.3, IQR (442–646) and 68/170 had CD4 cell counts of between 350 and 500/mm3.Median CD4 cell count was 534 cell/mmMaternal viral load and transmission10. The median log10 viral load was 4.4(IQR 3.5–4.9). 13%, 95% CI (7.5–18) had an undetectable viral load. A viral load of>3log10 was recorded for 107/145(74%); 89/145(61%) had a viral load of≥4log10 and for 29/145  a very high level of viral load as usually observed during primary acute infection, was noted (≥5log10).3, making them eligible for maternal triple ARV prophylaxis as required elsewhere. Taking into account that an average of 4 in 10 HIV positive pregnant women are yet to be eligible for HAART because of CD4 cell count of<350/mm3, it may be deduced that among the remaining 6 in 10, at least 4 may require HAART for prophylaxis according to their HIV viral load. Altogether 8 in 10 women at least may be eligible for HAART during pregnancy based upon biological criteria. Such results vindicate the opinion of those who claim that universal access to maternal triple ARV prophylaxis during pregnancy regardless of CD4 cell count is a must and are consistent with the latest WHO recommendations for women not yet in need of treatment for themselves, namely option B [To our knowledge, this is the first study in Cameroon where viral load is systematically determined in pregnant women in order to assess the eligibility to more effective antiretroviral regimen especially at high levels of CD4 cell count. Our key finding is that the level of HIV viral load is≥4log 10/ml in more than 60% of HIV pregnant women, ignorant of HAART, with CD4 cell count>350/mmIn Sub-Saharan Africa, some countries have opted for triple antiretroviral therapy for all pregnant women according to the level of care . In the 10 in more than 60% newly diagnosed pregnant women in Cameroon with high levels of CD4 cell count. Such results suggest the promotion of the routine measurement of viral load during pregnancy in A option country or the switch to B option for all, in order to ensure larger access to maternal antiretroviral triple prophylaxis. This approach is likely to be more effective in lowering maternal viral load and reducing mother-to-child transmission of HIV.We concluded that antenatal serum HIV-1 RNA viral load, stands above 4log


At the end of 2009, a total of 501 AIDS patients were receiving antiretroviral therapy (ART) in Fujian Province in China, yet there were no assessments to determine treatment efficacy and HIV-1 preventive potency under the current health care delivery system.+ T-lymphocyte (CD4) count, plasma viral load (VL), and patient characteristics were analysed. The results were compared between 4 groups divided by the baseline CD4 values at the 25, 50 (median), and 75 percentiles.During the period of 2005–2009, we assessed the outcomes of initial ART by following up 381 patients for 12 months in Fujian Province. CD4P < 0.001), but VL sharply declined regardless of the baseline CD4 count (P = 0.136). Although this VL decline indicates the potency of ART as an HIV-1 prevention tool, the time between positive diagnosis and ART initiation suggests serious delay in both diagnosis and treatment; the medians of periods for the lowest and highest baseline CD4 quartiles were 1.2 and 9.6 months, respectively.Over three-quarters of the subjects reported heterosexual contact as the probable route of transmission. After 12 months of ART, CD4 recovery varied between the 4 groups , and there is no link between ART and the STI care delivery system. This may interfere with the HIV-1 preventive potency of ART. We highly recommend establishing a collaborating mechanism with STI care, strengthening the VL determination system, and promoting HIV tests and early ART initiation. At the end of 2011, the number of people living with HIV (PLHIV) in China was 780,000, accounting for 0.058% of the total population. With this low national prevalence rate, China has lower-level HIV endemics compared to many other countries, but the number of PLHIV and AIDS patients is continuously increasing. The major modes of transmission are sexual intercourse and injecting drug use. The proportions of PLHIV who contracted HIV-1 through sexual contact and injecting drug use are 75.2% and 17.3%, respectively . The number of cases that met this criterion varied significantly among the baseline CD4 quartiles (P = 0.001). Some HIV clinical indicators, categorised as stage 3 according to WHO [In summary, the mean age of the subjects was 39.0 years, 67% were married, and 76% of the self-reported probable route of transmission was heterosexual intercourse. After 12 months of ART, both the CD4 count and VL improved. While the CD4 increment depended on the baseline CD4 counts  after 12 months of ART. Regardless of the baseline CD4 count, the virological responses were almost of the same level Click here for additional data file.Table S1Immunological failure and virological failure based on WHO criteria (N=112).(DOCX)Click here for additional data file.


Within a prospective observational study, we measured adjustment of methadone doses and responses to treatment after once-daily raltegravir (RGV)-based highly active antiretroviral therapy (HAART) was initiated in injection drug users (IDUs).We evaluated HIV-infected IDUs attending an inner city clinic in Vancouver who were receiving RGV-based HAART and methadone within a directly observed therapy program. Follow-up was according to clinical standards, with changes in methadone dose being made as required to achieve clinical stabilization within the first month of HAART. The change in methadone dosing associated with the initiation of HAART was calculated as the difference between the post- and pre-HAART methadone doses. The most recent on treatment CD4 cell count and HIV plasma viral load were used to evaluate HAART efficacy after initiation of therapy.3 and 243 copies/mL, respectively. At month 3, the mean methadone dose was 97.9 mg/day with the observed mean methadone dose change from baseline being 0.4 mg/day (p=NS). In these patients, 7 (21%) required increases, 8 (24%) required decreases, while 19 (56%) required no change in daily methadone dose from baseline. At most recent follow-up, the mean CD4 cell count was 355 cells/mm3 while virologic suppression (HIV RNA <50 and <400 copies/mL) was achieved in 23 (68%) and 31 (91%) of patients receiving RGV-based therapy.The study included 34 subjects  with a median follow-up period of 16.5 months. All patients were treatment experienced and co-infected with hepatitis C virus. Most patients received RGV-based HAART along with emtricitabine and tenofovir (n=16) or lamivudine and abacavir (n=9). At baseline, the mean methadone dose, mean CD4 cell count and median plasma viral load were 97.4 mg/day, 286 cells/mmLack of drug interactions with methadone and improved immunologic and virologic responses support the use of once-daily RGV-based HAART in this vulnerable population.


Both population and ultra-deep sequencing (UDS) of the HIV-1 V3 loop are useful in selecting candidates for maraviroc (MVC) therapy. We used mathematical modeling to determine that patients whose non-R5 HIV comprises <2% of the viral population by UDS are likely to respond to a MVC-containing regimen. However, the predictive value of absolute amount of non-R5 HIV is unknown.To determine whether non-R5 viral load contributes to predicting response to a MVC-containing regimen.Patients enrolled in the MERIT study (MVC or efavirenz plus zidovudine/lamivudine in treatment-naïve patients) with R5 virus at screening  and randomized to the twice-daily MVC arm were included. UDS was performed with a 454/Roche GS-FLX instrument. Tropism was predicted using the "geno2pheno" co-receptor algorithm (g2p). A sample was considered R5 if <2% of variants had a score below 3.5 FPR. MVC responses at Week 48 were predicted by descriptive statistics and mathematical modeling.Samples for 343 patients  were available. Baseline median CD4 and mean viral load (VL) were 247 and 232 cells/µL and 4.9 and 4.6 log10 c/mL in patients with R5 and non-R5 virus. No CXCR4-using viruses were detected in 249/343 (73%) patients. Among the 94 patients with detectable CXCR4-use, median  percent and absolute levels of CXCR4-using viruses were 0.8% (0.4-8.1) and 2.9 (2.3-3.5) log10 c/mL, respectively. Week 48 virologic responses are shown in Table In univariate models, baseline CD4 and percent of CXCR4-using virus were not significant predictors of week 48 response ; VL and absolute amount of CXCR4-using virus were significant  and were included in the multivariate model .In MVC-treated patients in the MERIT study, baseline VL and absolute amount of CXCR4-using virus were predictive of Week 48 response. It is possible that total burden of CXCR4-using virus in drug-naive individuals may play a greater role than the percentage of such virus in predicting response to regimens containing a CCR5 antagonist.


Prior studies have suggested that HAART initiation may vary by race/ethnicity. Utilizing the U.S. military healthcare system, which minimizes confounding from healthcare access, we analyzed whether timing of HAART initiation and the appropriate initiation of primary prophylaxis among those at high risk for pneumocystis pneumonia (PCP) varies by race/ethnicity.3. Multiple logistic regression was used to compare those who started vs. delayed therapy; comparisons were expressed as odds ratios (OR).Participants in the U.S. Military HIV Natural History Study from 1998-2009 who had not initiated HAART before 1998 and who, based on DHHS guidelines, had a definite indication for HAART , an indication to consider HAART  or electively started HAART  were analyzed for factors associated with HAART initiation. In a secondary analysis, participants were also evaluated for factors associated with starting primary PCP prophylaxis within four months of a CD4 count <200 cells/mm1262 participants were evaluated in the analysis of HAART initiation . Race/ethnicity was not associated with HAART initiation in Groups A or B. In Group C, African American race/ethnicity was associated with lower odds of initiating HAART . Race and ethnicity were also not associated with the initiation of primary PCP prophylaxis among the 408 participants who were at risk.No disparities in the initiation of HAART or primary PCP prophylaxis according to race/ethnicity were seen among those with an indication for therapy. Among those electively initiating HAART at the highest CD4 cell counts, African American race/ethnicity was associated with decreased odds of starting. This suggests that free healthcare can potentially overcome some of the observed disparities in HIV care, but that unmeasured factors may contribute to differences in elective care decisions. HIV has become a treatable illness in the era of highly active antiretroviral therapy (HAART), and HAART is associated with a reduction in morbidity and mortality among those with severe immunosuppression . There iDespite the known benefits of HAART, certain groups appear to be more vulnerable to treatment disparities, and these disparities have persisted over time. In a U.S. study of HIV-infected persons, delays of three or more months between diagnosis and an initial specialty encounter occurred more frequently among patients who lacked access to care at diagnosis and among those of African American or Latino race/ethnicity . Delays The etiology of these disparities is complex and likely multifactorial. Factors that have been hypothesized from previous studies include lack of health insurance or other barriers impeding healthcare access, variations in patient education or health literacy, and aspects of the physician-provider relationship ,13. UndeIn this study, we evaluated factors associated with HAART initiation within a defined time window in a United States military cohort of persons with HIV. As a secondary analysis, we also analyzed factors associated with the initiation of primary pneumocystis pneumonia (PCP) prophylaxis among persons at highest risk for PCP. Our population consists of an ethnically diverse group of early-diagnosed HIV patients with free healthcare and medications . FurtherThe U.S. Military HIV Natural History Study (NHS), a cohort study of military beneficiaries with HIV infection including active duty personnel, retirees and dependents, enrolled 5261 participants from 1986 to 2009. Active duty personnel must be HIV negative prior to entry into U.S. military service, and subsequently undergo HIV screening every one to five years. Service members and beneficiaries identified with HIV are referred to military treatment facilities (MTFs) across the U.S. for evaluation and treatment. Those participating in the study provide written informed consent prior to assuming a schedule of evaluations by an HIV specialist at least every six months at one of five participating MTFs. Data including demographic characteristics, medical history, and laboratory measurements are collected at each study visit as previously described . In comp3, an AIDS-defining condition, or any severe symptoms  [Participants who were enrolled in the NHS from 1998-2009 and who had not initiated HAART before 1998 were included in this substudy. Participants were excluded from these analyses if they had died (N = 1195), were lost-to-follow-up (N = 969), or had initiated HAART prior to January 1, 1998 (N = 1514). The analysis also excluded those participants who, prior to the three-month window preceding the first study date, i.e. September 1, 1997, had a CD4 cell count below 200 cells/mmParticipants were followed from the later of January 1, 1998 or their date of initial diagnosis with HIV until meeting criteria for assignment in one of three groups based on concurrent DHHS criteria. The details of this classification as well as changes in DHHS criteria during the study period of 1998-2009 are summarized in Table • Group A – those participants with a definite indication for HAART. Participants were classified as “initiators” if they started HAART within four months of classification or “delayers” if they did not. Those with less than four months of follow-up between the date of classification and last study visit were excluded from analysis (N = 18).• Group B – those participants with an indication to consider HAART. Initiators started HAART within six months of classification and delayers did not start HAART within this timeframe. Those with less than six months of follow-up between the date of classification and last study visit were excluded from analysis (N = 77).• Group C – those participants who may have electively started HAART; initiators started HAART prior to being categorized into either Groups A or B; delayers were HAART-naive as of their last study visit and had not yet been categorized into Groups A or B.The different initiator/delayer classification timeframes for Groups A and B were selected based on practice patterns unique to the military, specifically that some participants do not live near an MTF offering specialty HIV care and receive care on an approximately four to six month cycle. Participants could be evaluated in more than one group if they later met criteria for inclusion in another group, up until the time when they initiated HAART, at which point they could no longer be analyzed in any other group.3 . This analysis included those without a history of PCP who met CD4 criteria for primary PCP prophylaxis and had not yet started this treatment. Initiators started PCP prophylaxis within four months and delayers did not.To further assess for evidence of disparity, the analysis was repeated evaluating the association between study factors and starting primary pneumocystis pneumonia (PCP) prophylaxis within four months of the first verified CD4 count <200 cells/mmFactors studied included demographic characteristics , HIV-specific factors , comorbid medical conditions  and history of a mental health disorder .3, AIDS-defining condition, or severe symptoms). The Group C analyses additionally adjusted for HIV RNA and CD4 cell count. To account for changes in recommendations, models for the analyses were stratified by HIV treatment guideline era. Odds ratios are given with 95% confidence intervals (CI). All p-values are two-sided. Analyses were conducted using SAS .Descriptive statistics are presented as means with standard deviations (SD) or medians with interquartile ranges (IQR). For Groups A and B, factors were compared as of the date the participant was categorized into the group. For Group C, comparisons used the date of HAART initiation for initiators and date of last visit for delayers. Within each group, study variables among participants who initiated vs. delayed HAART were compared using logistic regression. Analyses were repeated among participants who initiated vs. delayed primary PCP prophylaxis when indicated. All multivariate models adjusted for gender, age, race/ethnicity, marital status, rank, duty status, military site, branch of service, duration of HIV disease, prior ART use, prior comorbid illnesses, and history of mental health diagnosis. The Group A analyses additionally adjusted for HIV RNA and reason for classification  with a mean age of 33.8 years (SD 8.7). Forty percent of participants were Caucasian, 46% African American, 10% Hispanic and 5% other ethnic groups. Sixty-two participants were evaluated in both Group A and Group B.3, 99 (48%) due to severe symptoms, and 10 (5%) for an AIDS-defining condition. Of these, 121 (58%) initiated HAART within 4 months of indication, with a median of 11 days (IQR 3-36) from indication to HAART initiation. The remaining 87 (42%) were defined as delayers; reasons for delay were not captured in the study database. The median CD4 cell count was 176 (IQR 131 – 207) among initiators and 417 (IQR 267-554) among delayers.Of the 1262 participants, 208 were categorized as having a definite indication to start HAART (Group A), including 99 (48%) due to a CD4 count below 200 cells/mm10 increase). Participants classified into Group A due to severe symptoms were less likely to initiate HAART when compared to those classified because their CD4 cell count fell below 200 cells/mm3 . There was no significant difference between participants classified into Group A for an AIDS-defining condition when compared with those classified for a CD4 cell count <200 cells/mm3  (Table In both the univariate (UV) and multivariate (MV) models, race/ethnicity and other demographic variables were not significantly associated with the timing of HAART in Group A. Participants with higher HIV RNA levels were more likely to initiate HAART  initiators and 389 (61%) delayers. Of these, 476 (75%) were classified into this group because of CD4 count, 82 (13%) due to HIV RNA level, and 79 (12%) because of meeting a combination of these factors. As in Group A, race/ethnicity was not significantly associated with the timing of HAART in Group B in either model. Factors associated with initiating HAART in both the UV and MV models were duration of HIV infection  and prior non-HAART antiretroviral therapy (ART) use . Additional factors that were significant only in univariate analyses were age, military status, and comorbid illnesses  participants initiated HAART. In UV models, African Americans were half as likely to initiate HAART in this group when compared to Caucasians . In the MV model, African American race remained significantly associated with delayed HAART initiation   as were those with CD4 cell counts closer to 200 cells/mm3   were lack of regular follow-up, active substance use, and the patient’s desire to not use HAART . There aAfrican Americans in this study were less likely than Caucasians to electively start HAART at higher CD4 counts. And while there were no statistically significant ethnic differences among those with an indication for HAART, there remained a trend towards lower odds of HAART initiation at lower CD4 count thresholds among African American participants when compared with Caucasian participants. A number of previous studies observed disparities in HAART use among certain race/ethnic groups and women although these did not specifically evaluate the elective initiation of HAART at high CD4 counts ,21,23-26The implication of the ethnic difference in elective HAART initiation in this study is important because some studies have demonstrated a differential response to HAART between Caucasian and non-Caucasian groups ,21,31,323) was a prominent factor in multivariate models [Depression was not associated with the timing of HAART in this study among those with an indication to start, although it has been shown to impact the decision to start HAART in some previous studies ,34 but ne models .There are some study limitations. It is a retrospective analysis covering a broad timeframe and involves five DoD research sites among which practice patterns may vary. Statistical models were adjusted to account for site of visit and service affiliation. While the cohort has a balance of male African American and Caucasian participants, other ethnic groups and women are represented in much smaller proportions, making it difficult to draw conclusions about the timing of HAART among these groups. Overall, the cohort is relatively small, and while we observed many trends that did not reach statistical significance, it is possible that these trends would represent clinically important differences in a larger cohort. Further, it is likely that there are additional confounding factors that we were not able to fully account for in the analysis. The criteria for determining eligibility for HAART initiation in this study are limited to the DHHS guidelines, even though other societies in the U.S. and worldwide have published guidelines that may have influenced practice patterns. And importantly, there are limited data on substance use in this cohort, although it is estimated to be quite low, with no IVDU reported at last survey .In a cohort with free access to HIV care and medications, race/ethnicity, age, and gender did not impact HAART timing among those with an indication for therapy, but African American race/ethnicity was associated with lower odds of electively initiating antiretrovirals. This study reinforces that free healthcare can potentially overcome some of the observed disparities in HIV care, particularly those related to access. However additional unmeasured factors may result in persistent differences in elective care decisions.The authors declare that they have no competing interests.ENJ, MPR, and BKA designed and conducted the study, interpreted the data, and prepared and edited the manuscript. MLL, NFC, ACW, AG, JFO, and GEM assisted with study design, data interpretation, and editing the manuscript. All authors read and approved the final manuscript.


To introduce the possible role of hydroxyurea (HU), a well-characterized antineoplastic drug with established antiviral effects, in the treatment of chronic hepatitis B.Four antiretroviral-naïve patients with chronic hepatitis B were enrolled in this limited pilot trial, and given 1000 mg/day of hydroxyurea for 4 weeks; then, the administration of the drug was suspended for 4 weeks. A clinical study and laboratory safety assessments and measurements of viral load were made at baseline, after drug therapy, and after one month suspension of the treatmentAll 4 patients showed a significant decrease in viral load after 4 weeks of hydroxyurea therapy and the viral load of 2 patients increased again after a 4-week suspension of hydroxyureaOur data demonstrate that the old low-cost antineoplastic drug, hydroxyurea, efficiently blocks hepatitis B virus (HBV) replication. We suggest that HU will play an important role in the treatment of chronic hepatitis in the fore- seeable future. Further studies including those that evaluate optimal dosing in long-term use will continue to define the role of HU in the treatment of HBV infection alone or in combination with other antiviral drugs Hepatitis B virus (HBV) has affected more than 2 billion people worldwide (about one third of the world's population) with more than 350 million people chronically infected. Chronic hepatitis B is a dangerous disease that can lead to liver cirrhosis and subsequently to hepatocellular carcinoma (HCC).HBV is a double-stranded DNA virus belonging to the hepadnavirus family replicating an intermediate RNA, the pregenomic RNA (pgRNA); this very much resembles the mechanism of HIV virus replication2]..2].Hydroxyurea (HU) is a drug with a history of more than 40 years use in the treatment of hematological malignancies such as chronic myeloid leukemia (CML) and diseases like sickle cell anemia, polycythemia vera, and psoriasis. HU is a free radical quencher, and inhibits the cellular enzyme ribonuclotide reductase, the rate limiting enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides, which is essential for DNA synthesis (3). This amazing biological effect has stimulated interest in HU for the treatment of diseases which are mediated by DNA viruses such as human immunodeficiency virus (HIV), herpes simplex virus (HSV) and polyomavirus6]..6].This astonishing characteristic of HU has marked the last decade for its initiation of the application of HU to AIDS therapy, for the first time in history.Since there is abundant evidence for the positive effects of HU in decreasing HIV and HSV replication, and because of the similarity of the HIV and HBV replication processes, we have postulated HU's possible effect in decreasing HBV replication.To explore whether HU could be used to decrease HBV viral load, we conducted a limited trial on patients with chronic hepatitis B.The four patients whose chronic hepatitis B positivity was definite were male with an age range of 29-58. They had not received any kind of treatment before they participated in our research. None of them had any sign of being previously or presently affected by hepatitis A, C, D, and HIV. Liver biopsy also indicated their active chronic hepatitis with HBV. Other routine tests including complete blood count (CBC), albumin and prothrombin time showed completely normal results.All patients were fully informed of the effects of the drug and instructed in its use. Afterwards, they gave their informed consent so as to be allowed to participate in the study. For four weeks on a daily basis, the patients received 1000 mg HU medication. During this four-week therapy period, the patients were examined once a week for any possible side effects and underwent a CBC test. We evaluated the HBV replication rate by estimating HBV viral load before and after the first month of treatment with HU, by means of the polymerase chain reaction (PCR), similar to the method followed in previous studies . In ordeThe levels of viral load prior to the study were more than 106 copies/mL. Each patient was given this test three times: prior to the treatment, at the end of drug therapy, and one month after the end of therapy.Finally, after two months, none of the patients showed any side effects; on the contrary, wonderful effects of the drug were revealed. Complete viral load results during the study appear inFig 1As mentioned before, the most important goal in the treatment of chronic hepatitis B is to reduce HBV load; and HU was shown to be capable of putting this into practice. Moreover, a recurring increase in HBV in half of the patients (two out of four) after a month of drug suspension strengthens our conclusions. Finally, given the HBV load of zero in one of the patients, the hypothesis of the HU effect in hepatitis B treatment is further confirmed.The findings of the present study are to be considered as the primary basis upon which further comprehensive and more detailed studies are recommended for the evaluation of the impact of HU as a possible drug effective in the treatment of hepatitis B. HU is simply absorbed orally and any probable or rare side effects, in case of occurrence, can be prevented by discontinuation of the drug 7]..7].It is worth mentioning that any claim that a certain drug is capable of fully hindering HBV replication should be accompanied by its practical effect on the reduction of initial viral load by more than 2 logs ; this haAltogether, there are reasons to support the claim that HU is a very excellent drug for the treatment of hepatitis B:1.HU has been a well-recognized drug throughout its years of use.2.It is simply absorbed orally and is easily absorbed into all bodily fluids.3.Toxicity of this drug is very low and in case of any side effects mere discontinuation can end complications4.The most important reason for drug resistance is mutation. HU, by causing a reduction in the production and replication of HBV in fact lowers the chance of mutations, thereby reducing resistance.In any case, it seems that further and more comprehensive studies of HU in isolation or in combination with other drugs can be very helpful in shedding light on all relevant aspects.


Data on combination antiretroviral therapy (cART) in remote rural African regions is increasing.We assessed prospectively initial cART in HIV-infected adults treated from 2005 to 2008 at St. Francis Designated District Hospital, Ifakara, Tanzania. Adherence was assisted by personal adherence supporters. We estimated risk factors of death or loss to follow-up by Cox regression during the first 12 months of cART.Overall, 1,463 individuals initiated cART, which was nevirapine-based in 84.6%. The median age was 40 years (IQR 34-47), 35.4% were males, 7.6% had proven tuberculosis. Median CD4 cell count was 131 cells/μl and 24.8% had WHO stage 4. Median CD4 cell count increased by 61 and 130 cells/μl after 6 and 12 months, respectively. 215 (14.7%) patients modified their treatment, mostly due to toxicity (56%), in particular polyneuropathy and anemia. Overall, 129 patients died (8.8%) and 189 (12.9%) were lost to follow-up. In a multivariate analysis, low CD4 cells at starting cART were associated with poorer survival and loss to follow-up . Higher weight was strongly associated with better survival .cART initiation at higher CD4 cell counts and better general health condition reduces HIV related mortality in a rural African setting. Efforts must be made to promote earlier HIV diagnosis to start cART timely. More research is needed to evaluate effective strategies to follow cART at a peripheral level with limited technical possibilities. WHO launched the ambitious "3 by 5" strategy in 2003 following the dramatically improved prognosis of HIV-infected patients receiving combination antiretroviral therapies (cART) in industrialized countries ,2. This In 2007, the HIV-prevalence in Tanzania was estimated to be 5.7% with about 1,867,918 HIV infected Tanzanian children and adults . The HIVWe aimed to assess the clinical and immunological response to cART in a rural treatment centre in Tanzania emphasizing immunological recovery and risk factors of death or loss to follow-up during the first year after starting cART.st January 2005 and 20th December 2008 at the SFDDH were included in this study.We analyzed data of a local prospective cohort study of HIV-infected individuals at the Care and Treatment Centre of St. Francis Designated District Hospital (SFDDH) in Ifakara, Tanzania. All HIV-infected adults initiating cART between 1The SFDDH is the most important health care facility in the rural Kilombero and Ulanga District of the Morogoro Region in Southern Tanzania, providing treatment and care for a population of about 600,000 inhabitants and an estimated 30,000 patients living with HIV/AIDS . EstabliIn the survival analysis, we included all HIV-infected patients who died from all causes within the first year after starting cART. We also considered all patients lost to follow-up as failures, since a meta-analysis of Brinkhof et al.  showed tAll patients were treated according to NACP guidelines  which arPatients were followed up every month until resolution of clinical symptoms of an AIDS defining illness and to the point of achieving two consecutively increasing CD4 cell counts under cART. Thereafter, appointments were scheduled on a three-monthly basis. At each visit, signs of tuberculosis were actively investigated. Laboratory controls included a blood cell count and chemistry two weeks after initiation of cART and a CD4 cell count after 3 months and every 6 months thereafter. Measurement of HIV-RNA was not available for the present study. cART switches due to toxicity or other reasons were performed in line with the NACP . The samEach patient receiving cART designated a personal adherence supporter - mostly a family member or any other trustworthy person living in the vicinity of the patient. The supporters received counseling about cART and side effects, provided assistance in taking cART throughout the study period and accompanied the patient at cART initiation and from then on request at follow-up visit.Patients with cART who missed visits were contacted or traced by home based care volunteers where possible.Data was collected prospectively using standardized record forms completed at each follow-up visit including information on the clinical course, occurrence of tuberculosis, WHO stage, adherence, laboratory results and treatment history. Data was entered into the NACP and the Kilombero and Ulanga Antiretroviral Cohort (KIULARCO) databases. Quality control was performed by double data entering and cross-checking procedures.Staging of HIV disease followed the case and stage definition according to WHO guidelines . TubercucART defined as a combination of at least 3 antiretroviral drugs was prescribed following the recommendations of the NACP . The firLoss to follow-up was defined as not showing up on two consecutive visits, i.e. for a period of 6 months.Basic socio-demographic characteristics, co-infection with tuberculosis, HIV clinical stage, CD4 cell count, and antiretroviral treatment were compared using the Chi-square test or Fisher's exact test for categorical variables, and the Mann-Whitney test for continuous variables. Cox proportional hazard models were used to investigate factors associated with survival and loss to follow-up after starting cART. All patients were censored at 12 months after starting cART if no death or loss to follow-up had occurred. Patients who were lost to follow-up were considered as failures and censored at the last visit date. The proportional hazards assumption was checked using the Schoenfeld test. Logistic regression was used to explore risk factors of being lost to follow-up during the first year of cART and as well as predictors of treatment modification. Multivariate models were built using a forward stepwise approach, adding each factor significant at the level of 0.1 and other factors defined in the literature as prognostic in the model one by one. The final model was examined for interaction using the likelihood ratio test.All analyses were performed using STATA™ software version 9.2 for Windows .The research and ethical clearance was obtained from the Medical Research Coordination Board of the National Institute for Medical Research, Tanzania, through the Tanzania Commission for Science & Technology and from the Ethical Review Board of the Cantons Baselstadt and Baselland, Switzerland. Written informed consent was obtained from all participants enrolled in the Kilombero and Ulanga Antiretroviral Cohort (KIULARCO).st January 2005 and 20th December 2008. The median age was 40 years (interquartile range [IQR] 34-47), 35.4% were males, and 7.6% had confirmed tuberculosis. 2.6% of the female individuals were pregnant. The median CD4 cell count was 131 cells/μl (IQR 54-234) and 24.3% had a WHO clinical stage 4. Patients co-infected with tuberculosis tended to start cART at higher CD4 cell counts than patients without tuberculosis . Antiretroviral therapy consisted of a nevirapine-based regimen in 83.1% of individuals. 75% of all patients were started on nevirapine/lamivudine/stavudine, 8.1% on nevirapine/lamivudine/zidovudine, 13.3% on efavirenz/lamivudine/zidovudine, 1.8% on efavirenz/lamivudine/stavudine, and 1.8% on other drug combinations.Overall, 1,463 treatment-naïve, HIV-infected adults initiated cART between 1General characteristics of the study population according to the outcome at 12 months are listed in Table The median CD4 cell count increase was 61 cells/μl (IQR 10-167) and 130 cells/μl (IQR 43-222) after 6 and 12 months, respectively. In particular, at 6 months and 12 months 49.9% and 70.1% of patients with available data had CD4 cell counts > 200 cells/μl, respectively, compared with only 23.6% at starting cART. During the follow-up, the median increase in body weight was 3 kg (IQR 0-8), and 115 out of 181 patients (63.5%) with available data experienced a change of activity from either bedridden or ambulant to working. Overall, 129 patients (8.8%) died and 189 (12.9%) were lost to follow-up during the first year of cART.In a multivariate model adjusted for age, gender, body weight, CD4 cell count, the presence of tuberculosis and antiretroviral regimen, risk factors for poor survival were lower CD4 cell counts at starting cART   changed their antiretroviral treatment after a median time of 133 days (IQR 58-371). The most frequent reason for modifying cART was toxicity to cART (56.2%), i.e. peripheral polyneuropathy in 26.8%, concern of potential drug-drug interactions related to antituberculous therapy in 23.2%, anemia in 13.9% of patients, and treatment failure in 6.7%. Among patients with treatment change due to toxicity, the use of stavudine was clearly associated with an increased risk of polyneuropathy , whereas all 26 patients who changed their treatment due to anemia were initiated on a zidovudine-containing regimen. Only one woman modified her initial, efavirenz-containing antiretroviral therapy due to pregnancy.The immunological recovery at 12 months did not differ between patients who changed their treatment and those who did not . However, patients who changed cART showed a higher median increase in body weight than those who remained on their first regimen . No difference in the mortality rate was noted according to whether patients had changed their treatment .In a multivariate model adjusted for age, sex, CD4 cell count, body weight, presence of tuberculosis, and cART, an antiretroviral regimen containing efavirenz and zidovudine combined with lamivudine (odds ratio (OR) 1.80, 95% CI 1.11-2.90, p = 0.016, compared to a nevirapine based regimen) was the only risk factor of treatment modification within the first year of cART.During the study period, 189 (12.9%) patients were lost to follow-up. In a multivariate model adjusted for age, sex, marital status, distance between living place and the clinic, confirmed tuberculosis and CD4 cell count, independent risk factors of loss to follow-up were a CD4 cell count below 100 cells/μl at starting cART  and living more than 100 km away from the clinic .pneumocystis jirovecii pneumonia in 14.3%, waisting syndrome in 10.7%, Kaposi sarcoma in 10.7%, cerebral toxoplasmosis in 7.1%, cervical/anal cancer in 3.6% and candida osophagitis in 3.6%). 18 individuals died of other causes . The cause of death remained unknown in 55 patients.Overall, 129 patients (8.8%) died during the first year of antiretroviral therapy. 56 (75.7%) of all patients with a known cause of death died of an AIDS-defining event . However, as we considered that these latter patients experienced treatment failure this may have accounted for an underestimation of cART effectiveness. Thirdly, viral load could not be measured which has important implications for the definition of treatment failure. Moreover, the exact cause of death was not known in 43% of all patients. This may have lead to a survival bias as tuberculosis may have accounted for a substantial proportion of deaths. Adherence could not be included in our analysis due to inconsistency of data. Due to the large proportion of missing data for certain variables, we could not include all investigated factors in the multivariate analysis. WHO's 2010 guidelines  promote The strengths of our study are manifold. The care and treatment facility at St. Francis Designated District Hospital is among the largest centres offering cART in a remote rural setting of Eastern Africa. Data was collected prospectively allowing more precise assessment of treatment efficacy. Data was derived from one centre therefore avoiding any leveling effects and high heterogeneity. Also, our site strictly follows the Tanzanian guidelines of the NACP and was uniquely supplied by the NACP which directly reflects a reach-out antiretroviral treatment programme with a public health approach. All individuals in this study were treatment-naïve at the start of cART. Therefore, results are not affected by previous exposure to antiretrovirals. Finally, our cohort is one of the largest rural HIV-cohorts in sub-Saharan Africa ,23-27 shIn conclusion, cART initiation at higher CD4 cell counts and better general health condition can reduce HIV related mortality in a rural African setting. The principle of starting cART only with a treatment assistant may help to overcome the constraints of a rural setting and allows comparable treatment outcomes as in urban cohorts. Efforts must be made to promote earlier HIV diagnosis in order to start cART timely and improve survival in rural African regions with moderate to high HIV prevalence. This necessitates wider voluntary HIV testing and counselling.The authors declare that they have no competing interests.The authors designed and executed the study, had full access to the raw data, performed all analyses, wrote the article and had full responsibility for the decision to submit for publication. All authors contributed to the design of the present analysis and to the final draft of the article. The authors EM, MS, EGM and EC were members of the Chronic Disease Clinic of St. Francis Designated District Hospital at Ifakara, Kilombero District, United Republic of Tanzania and were actively involved in the management of patients and the collection of data. EM completed the first draft of the article. LE performed the statistical analyses.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/98/prepub


Modeling of short-term viral dynamics of hepatitis B with traditional biphasic model might be insufficient to explain long-term viral dynamics. The aim was to develop a novel method of mathematical modeling to shed light on the dissociation between early and long-term dynamics in previous studies.We investigated the viral decay pattern in 50 patients from the phase III clinical trial of 24-week clevudine therapy, who showed virological response and HBsAg decline. Immune effectors were added as a new compartment in the model equations. We determined some parameter values in the model using the non-linear least square minimization method.10copies/mL, and on-treatment viral load decline was 5.683 Log10copies/mL. The median half-life of free virus was 24.89 hours. The median half-life of infected hepatocytes was 7.39 days. The viral decay patterns were visualized as triphasic curves with decreasing slopes over time: fastest decay in the first phase; slowest in the third phase; the second phase in between.Median baseline viral load was 8.526 LogIn the present study, mathematical modeling of hepatitis B in patients with virological response and HBsAg decline during 24-week antiviral therapy showed triphasic viral dynamics with direct introduction of immune effectors as a new compartment, which was thought to reflect the reduction of clearance rate of infected cells over time. This modeling method seems more appropriate to describe long-term viral dynamics compared to the biphasic model, and needs further validation. Chronic hepatitis B virus (HBV) infection is a serious global health problem, affecting approximately 350 million people worldwide. Previous studies on HBV viral dynamics during antiviral treatment have provided valuable insights into the complex interaction between the host and the virus in patients with chronic hepatitis B (CHB).We therefore aimed to describe HBV dynamics for longer period than previous studies by directly introducing the immune activity of cytolytic and noncytolytic HBV clearance in the mathematical modeling.E” to characterize a non-zero steady state for immune effectors, “E”. We analyzed HBV viral dynamics using a modified model of viral infection originally suggested by Neumann et al. The model equations have several parameters which must be assigned for numerical simulation. We used literature values for some of the parameters, and they are listed in The MATLAB®-ODE solver  was used to numerically solve our model equations. We performed the individual non-linear least-square minimization method, using the Levenberg-Marguardt algorithm in order to estimate the parameters which were not predetermined.The applicability of modeling was evaluated in a total of 182 hepatitis B e antigen (HBeAg)-positive CHB patients who were enrolled in the treatment group in a randomized, phase III clinical study of clevudine. Inclusion and exclusion criteria were previously described in detail. 10 copies/mL during treatment were excluded due to concerns about the accuracy of the mathematical processing. Hence, a total of 50 patients from this group were included in our final analysis. Patients were evaluated at baseline and monitored on days 8, 15, and 29 and every 4 weeks thereafter, during a total period of 24 weeks. The analysis was conducted using the quantified data of serum HBV DNA, HBsAg and ALT from the blood samples obtained at each time point.Among a total of 182 enrolled patients, 110 patients who achieved an arbitrarily defined “response” at the end of the 24-week treatment were assessed for the applicability of modeling; “response” was defined as decline of serum HBV DNA level and ALT to below 10,000 copies/mL and below ULN, respectively. These patients were distinctively divided into two groups according to the patterns of quantified HBsAg kinetics: one group showed decline of HBsAg titer during the 6-month treatment period (n = 62); the other group did not (n = 48). The latter group was discarded from analysis in the present study. Among the first group of 62 patients, 12 patients with fluctuating HBV DNA levels more than 1 LogThe present study was approved by the institutional review board of Seoul National University Hospital. Written informed consents were obtained from all subjects. We conducted the study in accordance with the principles of the Declaration of Helsinki.Serum HBV DNA and ALT levels were obtained at each time of sampling used in the previous study. Serum HBsAg titer was measured for the present study in serial serum samples from each patient which were stored at –70°C until the time of analysis. HBsAg was quantified using the Architect HBsAg assay  after a 1∶100 dilution. Samples with HBsAg levels >250.0 IU/mL at 1∶100 dilution were retested at a final dilution of 1∶1000. Samples with HBsAg levels <0.005 IU/mL at 1∶100 dilution were retested undiluted.P value <0.05 was considered as statistically significant. All statistical tests were two-sided. Statistical analyses were performed using SPSS 17.0K .Continuous variables were presented as either mean with standard deviation or median with interquartile range (IQR). Correlation between variables of baseline or follow-up and estimated parameters was calculated using Pearson’s correlation coefficients. Simple and multiple linear regression analyses were performed to identify independent determinants of parameters among variables of interest. A The baseline characteristics of 50 enrolled patients were as follows: median age was 37 years; 32 patients were male (64%); median baseline ALT level was 124.5 IU/L; median baseline levels of HBV DNA and HBsAg were 8.526 Log10 copies/mL and 4.044 Log10 IU/mL, respectively. The HBV DNA data measured from baseline to the end of therapy were used to fit the model equations in the 50 selected patients with HBeAg-positive CHB. Estimated parameters are shown in 10 copies/mL. In concordance with the original data, visualization of the calculated viral decay curves followed a triphasic pattern in these patients . Among the calculated parameters in the model equations, parameter “α” (cytolytic activity-induced clearance rate of infected cells) was correlated with both antiviral efficacy (r = 0.337, P = 0.017; P = 0.001; In the present study, we analyzed the viral dynamics during 24 weeks of clevudine therapy in 50 patients with HBeAg-positive CHB who also showed the decline of serum HBsAg titers during therapy. Triphasic dynamics were demonstrated by solving our mathematical models with direct incorporation of immune effectors with cytolytic and non-cytolytic activities in the equations in these patients. To the best of our knowledge, this study was the first attempt to mathematically solve the model equations including immune effectors in chronic hepatitis B to show triphasic dynamics.Simulation of viral dynamics throughout long-term treatment requires additional assumptions for both the hepatocyte proliferation and the immune system activity. A median of 7.39 days as an estimated half-life of the infected cells was comparable to estimates from previous studies . This fiWhether the HBV infection is cleared or persistent is determined by a complex interaction between the virus and the host immune system. In There are some limitations in this study. First, given that viral dynamics vary according to various genotypes of the HCV, the effect of genotypes on the HBV dynamics needs to be elucidated, In conclusion, the present study demonstrated that the mathematical modeling of the HBV dynamics during 24 weeks of antiviral therapy was successful with adding immune effector as a new compartment in the model equations. Some of the estimated parameters demonstrated significant correlation with observed biologic phenomena, e.g., on-treatment viral decline and negative feedback of immune activity. The resultant triphasic dynamics seems more appropriate to explain long-term viral dynamics compared to the traditional biphasic model. Our methodology of modeling needs to be validated in clinical studies using other NUCs available for patients with CHB, to examine the possibility of offering insights for appropriate treatment endpoint or optimal treatment duration.


ABCB1 gene encodes P-glycoprotein, an ATP-dependent drug efflux pump, which is responsible for drug transport across extra- and intra-cellular membranes. The variability in the expression of ABCB1 may contribute to variable plasma efavirenz concentration which results in variability in the levels of suppression of the human immunodeficiency syndrome virus (HIV). The aim of the study was to evaluate the role of polymorphisms in ABCB1 gene on plasma efavirenz levels and treatment response in the form of change in viral load and CD-4 cell count in HIV/AIDS patients receiving efavirenz-containing highly active antiretroviral treatment regimens. Two hundred and eighty-two HIV-infected patients were recruited from Themba Lethu Clinic in Johannesburg and plasma efavirenz drug concentration levels were measured using LC-MS/MS. SNaPshot was used to genotype five known ABCB1 single nucleotide polymorphisms (SNPs). Genotype-phenotype correlations were computed. The ABCB1 4036A/G and 4036G/G genotypes were significantly associated with low plasma efavirenz concentrations (P = 0.0236), while the ABCB1 1236C/T and 1236T/T genotypes were associated with high efavirenz concentrations (P = 0.0282). A haplotype ABCB1 T-G-T-A is reported that is associated with significantly increased plasma efavirenz levels. This is the first report on 61A>G, 2677G>T/A, and 4036A>G SNPs in the South African population. ABCB1 plays a role in determining the plasma concentrations of efavirenz and should be taken into account in future design of assays for genotype-based dosing of efavirenz-containing regimens.The  Efavirenz provides the backbone to first-line highly active antiretroviral treatment (HAART) in South Africa. HAART effectively suppresses human immunodeficiency syndrome virus (HIV) replication in the majority of patients , 3435C>T (rs1045642), and 2677G>T/A (rs2032582), where multiple alleles have been reported. The 1236C>T SNP occurring in exon 13, does not result in an amino acid change, but may affect ABCB1 expression through codon usage , Asians (Chinese), Caucasians, and Yoruba individuals, respectively , coded by the a et al.  showed tn = 227) were female. Of the patients, 7 and 10% smoked and consumed alcohol. The clinical characteristic of the patients included viral load and CD-4 cell count  genotype. All ABCB1 SNPs were in Hardy–Weinberg Equilibrium (HWE). The ABCB1 61A/G (rs9282564), 3435T/T (rs1045642), 4036G/G (rs3842), 1236T/T (rs1128503), 2677T/A, and 2677G/A (rs2032582) genotypes were present at frequencies of 0.006, 0.024, 0.036, 0.015, 0.004, and 0.004, respectively, among the South Africans. No individuals with an ABCB1 3435A allele was observed in the South African cohort (Table http://hapmap.ncbi.nlm.nih.gov/).The genotypes for all the SNPs were observed in the HIV/AIDS patients, except the 61G/G rs982564 genrt Table . The allABCB1 4036A/G and 4036G/G genotypes were associated with significantly decreased efavirenz levels (P = 0.0236), compared to the 4036A/A genotype . No significant differences were observed in efavirenz concentrations between the ABCB1 2677G>T/A and ABCB1 3435C>T genotypes  compared to 0.49 and 0.65 μg/mL among patients with the T-G-C-A or T-G-T-G haplotypes, respectively  and were still significant after Bonferroni’s correction for multiple testing (cut-off significant P < 0.01).Haplotype and efavirenz plasma levels for each patient are presented in supplementary Table 10 HIV-RNA levels, ABCB1 haplotypes, ABCB1 1236C>T, 4036A>G, 3435C>T, and 2677G>T/A genotypes on plasma efavirenz concentrations , ABCB1 4036A>G , and ABCB1 2677G>T/A . The adjusted coefficient of determination (R2) for the regression was 0.16, indicating that 16% of the total variance in efavirenz concentrations was explained by the model. ABCB1 1236C>T, 4036A>G, and 2677G>T/A genotypes accounted for 29, 23, and 17% (respectively) of the total variance in plasma efavirenz concentrations. When repeating the multivariate analysis among female patients only, the adjusted coefficient of determination (R2) for the regression was 0.18, indicating that 18% of the total variance in efavirenz concentrations was explained by the model compared to the 16% explained when males and females were combined. However, the majority of HIV/AIDS patients in our study were female. Further statistical analysis comparing only the genetic- and non-genetic factors in the multivariate analysis, showed that the genetic factors alone explained 11% of variance in efavirenz levels, while the non-genetic factors only explained 3% compared to the 16% explained by the combined multivariate analysis.Univariate regression analysis was performed to determine the effect of age, gender, tobacco smoking, alcohol use, BMI at baseline, CD-4 cell count, logABCB1 1236C>T and 4036A>G were compared at baseline and also 6 months post-initiation of ARV therapy. The ABCB1 1236C/T and 4036G/G genotypes were associated with the biggest decreases in viral load at 6 months . The differences in allele frequencies between the African and Caucasian individuals show that therapeutic drugs, including efavirenz, may not have similar effectiveness in different populations when given at standard dosages. Similarly, fine scale genetic structure exists within the African population which, therefore, should not be treated as one population.To our knowledge, the present study is the first to report on the allele and genotype frequencies of the a et al. . This daABCB1 4036A/G and 4036G/G genotypes and this could perhaps be as a result of the disruption of a miRNA binding site in the 3′UTR of ABCB1. Five poorly conserved miRNAs namely; miR-129, miR-491, miR-4795, miR-561, and miR-4717 have been predicted to target the 3′UTR region surrounding the ABCB1 4036A>G SNP using the TargetScanHuman 6.1 miRNA target prediction software. Disruption of these sites could potentially cause reduced transport of efavirenz by ABCB1 resulting in lower plasma efavirenz levels. In a different study among Ugandans, the ABCB1 4036A/G and 4036G/G genotypes were associated with higher efavirenz bioavailability  increase in CD-4 cell count (cells/μL) and a 94% decrease in viral load (copies/mL) when baseline levels where compared to levels at 6 months post-initiation of treatment. Failure of reduction in viral load and emergence of opportunistic infections after 6 months led to ARV switching. Sustained viral load after 6 months and the presence of opportunistic infections are indications of possible treatment failure or non-adherence. On the other hand, other studies have shown that high plasma efavirenz concentrations are associated with development of adverse drug events leading to drug discontinuation  South African HIV/AIDS patients receiving efavirenz-based treatment for at least 6 months, were recruited to participate in this study. All subjects were of Bantu origin and comprised of Sotho/Tswana from Gauteng and Xhosa subjects from the Western Cape Province, South Africa. All subjects gave information on their ethnicity, age, health status (including self-reported adherence to treatment or pill counts), dietary, and smoking habits.All participants provided written informed consent and study approval was obtained from the University of Cape Town Health Science Faculty Research Ethics Committee, Cape Town, South Africa and the University of Witwatersrand Human Research Ethics Committee, Gauteng, South Africa. The research was performed in accordance with the guidelines of the Helsinki Declaration of 2008. Two hundred and eighty-two ; 40 pmol of the forward and reverse primers ; 1 U of GoTaq Flexi DNA Polymerase . The PCR reactions were carried out using a “MyCycler Thermal cycler” from Bio-Rad. PCR conditions were as follows: 3 min at 94°C; 40 cycles of 94°C for 30 s, the annealing temperature specific to each SNP for 30 s, 72°C for 50 s; and 10 min at 72°C for final extension.Each PCR reaction contained, 50 ng of genomic DNA, 1X Green GoTaq Flexi Reaction Buffer , 0.2 mM of each of the deoxynucleotide triphosphates ; 1.5 mM MgClExoI  in a total reaction volume of 20 μL. The shrimp alkaline phosphatase and ExoI reaction was incubated at 37°C for 1 h and the enzymes were inactivated at 75°C for 15 min. SNaPshot single base extension of the genetic polymorphisms was performed on the “GeneAmp® PCR System 9700 version 3.08′′  using the SNaPshot cycling programme as 96°C for 10 s, and then repeated for 25 cycles at 50°C for 5 s and 60°C for 30 s. The SNaPshot reaction (10 μL) contained 1 μL ABI Prism® SNaPshot™ Multiplex Kit  and the pooled internal SNaPshot primers . Following the SNaPshot reaction, the clean-up reaction was repeated using 1 U shrimp alkaline phosphatase using cycling conditions as mentioned before, and capillary electrophoresis was performed using a ABI 3130xl Genetic Analyzer . SNaPshot results were analyzed on the GeneMapper © Software version 4.1 .Five microliters of each PCR product was pooled and 10 μL of the combined PCR products were cleaned using 1.5 U shrimp alkaline phosphatase  and 2 U ABCB1 haplotypes were inferred using Phase v2.1 . Univariate regression analysis was applied to log10 efavirenz concentrations as dependant variable and the percentage change in efavirenz levels, with the 95% CI, was calculated as 100 × regression coefficient. Multivariate regression analysis was performed by including covariates from the univariate analysis, followed by stepwise backward removal. TargetScanHuman 6.1 miRNA target prediction software were used to predict miRNA binding to the ABCB1 3′UTR. The following equation was used to calculate the sample size required to achieve a 99% confidence-interval: N = [DEFF * Np(1−p)]/[(d2/Z21−α/2 * (N−1) + p * (1−p)]. DEFF is defined as the design effect, Z is the value for 99% confidence, d is an α-value = 0.05, while p is the frequency of the variant allele. A DEFF-value of 1 was used for random sampling, a Z-value of 1.96 and allele frequency of 0.1 was used to calculate the sample size of N = 239 samples. All statistical tests were performed two tailed, and statistical significance was defined as P < 0.05.Statistical analysis was performed using the Graphpad Prism  and STATA  statistical programs. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.