The Chinese government has provided health services to those infected by the human immunodeficiency virus (HIV) under the acquired immunodeficiency syndrome (AIDS) care policy since 2003. Detailed research on the actual expenditures and costs for providing care to patients with AIDS is needed for future financial planning of AIDS health care services and possible reform of HIV/AIDS-related policy. The purpose of the current study was to determine the actual expenditures and factors influencing costs for untreated AIDS patients in a rural area of China after initiating highly active antiretroviral therapy (HAART) under the national Free Care Program (China CARES).A retrospective cohort study was conducted in Yunnan and Shanxi Provinces, where HAART and all medical care are provided free to HIV-positive patients. Health expenditures and costs in the first treatment year were collected from medical records and prescriptions at local hospitals between January and June 2007. Multivariate linear regression was used to determine the factors associated with the actual expenditures in the first antiretroviral (ARV) treatment year.P = 0.02; and ≥200 cells/μL versus <100 cells/μL, P < 0.004), residence in Mangshi County (P < 0.0001), ADEs (P = 0.04) and OIs (P = 0.02) were significantly associated with total expenditures in the first ARV treatment year.Five ARV regimens are commonly used in China CARES: zidovudine (AZT) + lamivudine (3TC) + nevirapine (NVP), stavudine (D4T) + 3TC + efavirenz (EFV), D4T + 3TC + NVP, didanosine (DDI) + 3TC + NVP and combivir + EFV. The mean annual expenditure per person for ARV medications was US$2,242 (US$1 = 7 Chinese Yuan (CNY)) among 276 participants. The total costs for treating all adverse drug events (ADEs) and opportunistic infections (OIs) were US$29,703 and US$23,031, respectively. The expenses for treatment of peripheral neuritis and cytomegalovirus (CMV) infections were the highest among those patients with ADEs and OIs, respectively. On the basis of multivariate linear regression, CD4 cell counts (100-199 cells/μL versus <100 cells/μL, This is the first study to determine the actual costs of HIV treatment in rural areas of China. Costs for ARV drugs represented the major portion of HIV medical expenditures. Initiating HAART in patients with higher CD4 cell count levels is likely to reduce treatment expenses for ADEs and OIs in patients with AIDS. According to the most recent estimates by the Ministry of Health and the Joint United Nations Program on HIV/AIDS (UNAIDS), China had 560,000-920,000 people living with human immunodeficiency virus (HIV) and 97,000-112,000 people living with acquired immunodeficiency syndrome (AIDS) as of the end of 2009. A total of 320,000 HIV/AIDS cases have been reported from the national infectious disease surveillance system from 1985 to 2009 ). For ARV medications, expenditures were calculated on the basis of negotiated prices between provincial health bureaus and participating pharmaceutical companies. The average daily expense of ARV regimens was calculated using the total amount of the individual drug expenditure in 1 year divided by the total number of treatment days during the same time period, adjusted for standard dosages. The expenditure data over different calendar years were adjusted with a 3% annual inflation rate, using 2004 as the comparison year.2 tests were used to compare the proportions of costs among the three strata of CD4 cell counts. The potential independent factors which may affect the total expenditure included age, gender, treatment site, marital status, ethnic group, level of education, occupation, residential status, drug use, CD4 cell count and incidence of ADEs and OIs. A multivariate linear regression model was fitted to estimate the independent factors associated with total expenditures by eliminating nonsignificant variables in a stepwise manner. Only the factors significant at a level of 0.05 (two-tailed) were reported. R/S plus software version 2.8.1  was used for the analyses.We analyzed the expenditures in the first year after starting ARV therapy. We calculated the means and standard deviations (SD) of the total costs, as well as itemized costs for ARV medications, laboratory testing and treatment of ADEs and OIs. We further calculated the costs stratified by study site, demographic variables and baseline CD4 cell count. The CD4 cell count was categorized into three strata as follows: <100 cells/μL, 100-200 cells/μL and ≥200 cells/μL. The Kruskal-Wallis test was used to evaluate the expenditures associated with different CD4 cell count categories. Cochran-Mantel-Haenszel χTwo hundred seventy-six patients were included in the analysis. One hundred seventy-six patients (63.8%) were from Dehong Prefecture of Yunnan Province, including 31 patients (11.2%) from Mangshi County, 32 patients (11.6%) from Ruili County and 113 patients (41.0%) from Longchuan County. One hundred patients (36.2%) were from Wenxi County of Shanxi Province. The average age of the patients at entry into ARV treatment was 39 years . The patients had the following characteristics: 62% (170/276) were male, 71% (197/276) were married, 61% (167/276) were majority Han ethnicity, 21% (57/276) were Jingpo ethnicity, 14% (40/276) were Dai ethnicity, 4% (12/276) were other minority ethnicities, 12% (34/276) were illiterate, 45% (124/276) had an elementary school education, 84% (232/276) were farmers and 88% (242/276) were living with their families. Sixty-four patients (23.6%) had a history of drug use. The means of CD4 cell count to the time ARV treatment initiated was 181 ± 93 cells/μL (Table Eighty-five percent of patients (235/276) complied with ARV treatment for 1 year, and 85% of patients (235/276) remained in treatment or obtained prescriptions. Five ARV regimens were used by the patients, as follows: combivir + EFV (36% [100/276]), D4T + 3TC + NVP (23% [65/276]), AZT + 3TC + NVP (21% [57/276]), DDI + NVP + 3TC (13% [35/276]) and D4T + 3TC + EFV (7% [19/276]).The mean expenditure per patient during the first year after initiating ARV therapy was US$2,633 . The total expenditure for all 276 patients was US$726,847; 85.1% of the total expenditure was spent on ARV medications, 2.5% on medications for treating ADEs, 1.8% on medications for treating OIs, 7.6% on ARVs laboratory monitoring, 0.4% on laboratory tests for diagnosing ADEs, 0.4% for diagnosing OIs, 1.1% on other medical services related to ADEs and 1.0% on other medical services related to OIs (Table P < 0.01). On average, patients in Mangshi had higher expenditures than patients at the other three sites (P < 0.01). Non-drug users had greater expenditures than drug users (P < 0.01). The total expenditure was also associated with the level of education, but not associated with age, gender and ethnicity . The total expenditure on CD4 cell count monitoring, as a part of regular laboratory tests, was US$39,429 for all patients  had at least one documented ADE. The most common ADEs were hepatotoxicity 41.3%), allergic dermatitis (23.9%), gastrointestinal disorders (21.7%) and anemia . Huganpian and Gansukeli , diammonium glycyrrhizinate (capsules or injection) and glucurolactone tablets are commonly used in China to treat hepatotoxicity, with average expenses of US$4.21 per patient per month. The average expenditure of diagnosing ADEs ranged from US$0-US$17, and other inpatient and outpatient medical service charges ranged US$3-206, including medical care for peripheral neuritis (Table P = 0.32). Patients with lower CD4 cell counts had significantly higher expenditures for medications (P = 0.04), tests for diagnosing ADEs (P = 0.04), inpatient and outpatient medical services (P = 0.01) and total medical services (P = 0.04) compared to patients with higher CD4 cell counts  had at least one diagnosed OI in the first treatment year. The most frequent OIs were respiratory infections (23.6%), diarrhea (7.3%), fungal infections (6.2%), herpes simplex virus and zoster infections (4.7%), tuberculosis  and cytomegalovirus (CMV) infections ; tuberculosis, US$245 ; fungal infections, US$158 ; herpes simplex virus and zoster infections, US$107 ; bacterial respiratory infections, US$85 ; and diarrhea, US$58 (Table P < 0.01). The OI-related expenditures among patients with lower CD4 cell counts were significantly higher for medications (P < 0.01), diagnosing OIs (P < 0.01), inpatient and outpatient medical services (P < 0.01) and total medical expenditures (P < 0.01) compared to patients with higher CD4 cell counts , residence in Mangshi (P < 0.0001), ADE incidence (P = 0.04) and OI incidence . The average expenditures for the treatment of ADEs and OIs increased significantly among patients with low CD4 cell counts. Therefore, initiating HAART to patients with higher CD4 cell counts appears to be effective in controlling AIDS treatment-related expenses for ADEs and OIs.This is the first study to determine the expenditure data for AIDS treatment in rural areas of China. During the first year, the average annual expenditure for AIDS treatment per person was US$2,633. Expenditures were significantly higher for patients with low CD4 cell counts compared to those with high CD4 cell counts , tenofovir, emreicitabine and abacavir are used in cases of drug resistance. Some of these ARVs are now available as cost-free treatment. Merito et al.  showed t [et al.  showed t [et al.  showed t [et al. , the dir [et al.  reportedet al. [A higher level of education was associated with a higher cost of HAART-related expenses in our study, which was consistent with the results of Bozzette et al. . In ruraADEs contribute significantly to HIV/AIDS treatment costs, which can be mitigated through early symptom assessment and management. The ADEs associated with ARVs have been reported in many studies -20. BesiHAART has been shown to be effective in increasing the CD4 cell count and reducing the incidence of OIs -27. In tPneumocystis carinii pneumonia (PCP) was very low and may be attributed to regular prophylaxis using sulfamethoxazole. Because the sample size was limited in our study, the correlation between the use of sulfamethoxazole for prophylaxis and the incidence of PCP should be the subject of study in China.In this study, we also showed that the incidence of When to begin HAART is an important issue which has been much discussed in recent years. Studies in high-income countries have verified that initiating HAART in patients with higher CD4 cell counts can improve the efficacy of treatment . HoweverIn our study, we pooled the data from two distinct programs, the only difference in program design of which was the enrollment criteria for CD4 cell counts. However, the CD4 cell count was similar in the two programs after considering the WHO's clinical stages. Pooling data can increase the sample size and yield more information about HAART expenditures. Moreover, the price of medications and medical services was similar in the different sites of the program, although the different frequencies of follow-up may produce heterogeneity in expenses .The main limitation of the study was that the results cannot be extended to all of China because of the differences in the cost of living across China . Second,Despite the above limitations, this study is the first to provide important information on the cost of rural AIDS patients in China. The ARVs expense was the largest part of HAART cost, averaging US$2,242 per patient in the first ARV treatment year. These costs appear to increase over time in China, as consequences of the increasing number of possible occurrences of drug resistance in the continuing period of HAART and more second-line regimens are used. Over the long term, initiating HAART in patients with a higher CD4 cell count is likely to effectively control AIDS treatment expenses on ADEs and OIs, but some factors should be generally balanced when initiating HAART.Pneumocystis carinii pneumonia; TB: tuberculosis; WHO: World Health Organization; 3TC: lamivudine.ADEs: adverse drug events; AIDS: acquired immune deficiency syndrome; ARV: antiretroviral; ARVs: antiretroviral medications; AZT: zidovudine; China CARES: China Comprehensive AIDS Response; CMV: cytomegalovirus; CNY: Chinese yuan; D4T: stavudine; DDI: didanosine; EFV: efavirenz; HAART: highly active antiretroviral therapy; IDUs: injection drug use; IDV: indinavir; Kaletra: lopinavir/ritonavir; NRTI: nucleoside reverse transcriptase inhibitors; NNRTI: non-nucleoside reverse transcriptase inhibitors; NVP: nevirapine; OIs: opportunistic infections; PCP: The authors declare that they have no competing interests.FZ designed the project, collected data and drafted the first version of the manuscript. GFK revised the manuscript. HZQ oversaw the data analysis and revised the manuscript. SD collected laboratory data. JSW participated in data analysis. ZWG collected and analyzed data. XPZ reviewed medical records and collected data at the Wenxi site. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/6/prepub


Background. High rates of second-line antiretroviral treatment (ART) failure are reported. The association with resistance and nonadherence on switching to second-line ART requires clarification.Methods. Using prospectively collected data from patients in South Africa, we constructed a cohort of patients switched to second-line ART (1 January 2003 through 31 December 2008). Genotyping and drug concentrations  were measured on stored samples preswitch. Their association with viral load (VL) <400 copies/mL by 15 months was assessed using modified Poisson regression.Results. One hundred twenty-two of 417 patients  had genotyping (n = 115) and/or drug concentrations (n = 80) measured. Median CD4 count and VL at switch were 177 cells/µL  and 4.3 log10 copies/mL , respectively. Fifty-five percent (n = 44/80) had subtherapeutic drug concentrations preswitch. More patients with therapeutic vs subtherapeutic ART had resistance (n = 73): no major mutations (3% vs 51%), nonnucleoside reverse transcriptase inhibitor (94% vs 44%), M184V/I (94% vs 26%), and ≥1 thymidine analogue mutations (47% vs 18%), all P = .01; and nucleoside reverse transcriptase inhibitor (NRTI) cross-resistance mutations . Following switch, 68% (n = 83/122) achieved VL <400 copies/mL. Absence of NRTI mutations and subtherapeutic ART preswitch were associated with failure to achieve VL <400 copies/mL.Conclusions. Nonadherence, suggested by subtherapeutic ART with/without major resistance mutations, significantly contributed to failure when switching regimen. Unresolved nonadherence, not NRTI resistance, drives early second-line failure. The management of first-line, nonnucleoside reverse transcriptase inhibitor (NNRTI)–based antiretroviral treatment (ART) failure is challenging in resource-limited settings. In settings with viral load (VL) monitoring but without resistance testing, guidelines recommend excluding drug interactions and toxicity, intensifying adherence support, and switching to second-line, boosted protease inhibitor (PI)–based ART if a second VL remains elevated –3. This Nevertheless, under programmatic conditions, high rates of second-line virological failure are reported –8. DelayNonadherence on second-line ART is increasingly considered the main driver of early second-line failure –7. AlthoIn a South African treatment program, this study describes the contribution that resistance and nonadherence, as determined by subtherapeutic drug concentrations and the absence of major DRMs, make to first-line virological failure on switching regimens, and investigates the impact of NRTI resistance and nonadherence on response to second-line ART.This retrospective cohort analysis used prospectively collected clinic data and stored plasma from patients enrolled in a multisite workplace and community ART program managed by the Aurum Institute, South Africa , 27. PatInclusion criteria were age ≥15 years, switched from first- to second-line ART between 1 January 2003 and 31 December 2008, VL >400 copies/mL at switch with available stored plasma (6 months before to 1 week after switch), and potential for at least 15 months of follow-up (data included up to 31 March 2010). Stored samples, from patients with VL >400 copies/mL 12 months  following switch, were also analyzed.http://hivdb.stanford.edu/) with mixtures reported as mutant genotypes. HIV type 1 (HIV-1) subtype classifications were performed using Rega version 2.0. Plasma drug concentrations were measured using a protein precipitation procedure to extract the drugs, and analyzed using a validated liquid chromatography–tandem mass spectrometry assay. The accuracy and coefficient of variation statistics for 3TC, EFV, NVP, and LPV during sample analysis were 90.7%–108.2% and 1.1%–7.0%, at high, medium, and low quality control concentrations. Limits of quantification were 20 ng/mL for 3TC, 39.1 ng/mL for EFV, and 19.5 ng/mL for NVP and LPV.Human immunodeficiency virus (HIV) RNA was assayed using polymerase chain reaction  and genotyping performed on stored plasma using a modified validated in-house assay . MutatioViral suppression on second-line ART was defined as a VL <400 copies/mL 2 weeks to 15 months following switch. Patients without VL results were excluded if they transferred to another program; otherwise they were considered to have failed treatment. Alive in care was defined as no record of leaving the program or loss to follow-up  by 15 months.NRTI resistance at switch, one of the key exposures of interest, was categorized as (1) none, (2) M184V/I, (3) 1–2 thymidine analogue mutations (TAMs) ± M184V/I, and (4) NRTI cross-resistance mutations (≥3 TAMs and/or K65R and/or Q151M and/or T69ins) ± M184V/I.Major DRMs were defined using the 2011 update of the International Antiviral Society–USA drug mutations list . Cumulatnonadherence at switch, as determined by subtherapeutic first-line drug concentrations with or without major DRMs. Therapeutic drug monitoring (TDM) guidelines recommend measuring trough drug concentrations (Ctrough) to evaluate efficacy of NNRTIs and PIs, with recommended thresholds for NVP, EFV, and LPV of ≥3 mg/L, ≥1 mg/L, and ≥1 mg/L, respectively ) or NVP (36.1% [n = 44]) with the NRTI backbone d4T/3TC , ZDV/3TC , TDF/3TC , or d4T/ddI . Six patients on ZDV and 2 on TDF had prior exposure to d4T.On switching, the median age of patients was 36 years , with a median duration on first-line ART of 545 days ; duration of viremia was 306 days , CD4 count was 177 cells/µL , and VL was 4.3 logNinety-seven percent (n = 112/115) of patients genotyped were subtype C, 2 were subtype B, and 1 was subtype A. No major DRMs were detected in 23% (n = 26), ≥1 NNRTI mutations were found in 74% (n = 85), and ≥1 NRTI mutations were found in 70% (n = 80), of which M184V/I predominated . At least 1 TAM, K65R, or Q151M mutation was detected in 34% (n = 39), 3% (n = 3), and 3% (n = 3) of patients, respectively. Sixteen percent (n = 19) of patients harbored NRTI cross-resistance mutations.trough: 8% (n = 4) 0.01–2.9 mg/L and 30% (n = 10) BLQ. Thirty-eight percent (n = 18/47) of patients on EFV had concentrations greater than Ctrough: 25% (n = 12) <1 mg/L and 36% (n = 17) BLQ  of patients on 3TC had drug concentrations above the assay's limit of quantification . Fifty-eight percent (n = 19/33) of patients on NVP had drug concentrations greater than CFifty-five percent (n = 44/80) of patients had subtherapeutic drug concentrations to at least one first-line drug: NNRTI and 3TC were BLQ in 24, NNRTI alone in 8  and 3TC alone in 11 . One patient whose NVP concentration was BLQ was not on 3TC-containing ART at time of sampling. Data on patients’ self-reported adherence were available for one-third of patients at time of sampling (n = 27/80); 12 of 23 patients reporting adherence had subtherapeutic drug concentrations.P < .01). Major DRMs were more likely to be detected in patients on therapeutic vs subtherapeutic first-line ART: NNRTI mutations in 94% vs 44%, M184V/I in 94% vs 26%, ≥1 TAMs in 47% vs 18% , and NRTI cross-resistance mutations in 26% vs 13% (P = .23), respectively.Seventy-three patients had drug concentrations and genotyping performed Table . No majoP = .67) and those with therapeutic first-line ART preswitch with or without major DRM vs subtherapeutic ART plus major DRM vs subtherapeutic ART with no major DRM .Eighty-three percent (n = 101/122) of patients were alive in care 15 months following switch to second-line ART . There was no difference in retention for those with ≥1 NRTI mutations vs no NRTI mutations . Patients on subtherapeutic first-line ART with no major DRMs were less likely to achieve viral suppression than those with major DRMs and those on therapeutic first-line ART .Overall, 68% (n = 83/122) of patients achieved viral suppression on second-line ART. Five patients had no VL results: 4 died or were lost to follow-up and were considered to have failed treatment, whereas 1 patient transferred to another program and was excluded from subsequent analyses. Patients with ≥1 NRTI mutations preswitch were more likely than those without to achieve a VL <400 copies/mL  vs subtherapeutic ART plus major DRM  vs therapeutic ART with or without major DRM (reference); n = 70; trough.Of the original cohort of 417 patients, 287 had a VL 12 months following switch to second-line ART, of whom 112 (39%) were >400 copies/mL. Fifteen of 16 samples located were successfully genotyped: 6 patients had no major DRMs, 8 had NNRTI mutations, and 5 had ≥1 NRTI mutation . No major PI mutations were detected. Importantly, only 1 of 13 patients in whom drug concentrations were measured had LPV concentrations greater than CThis is the first study in a resource-limited setting to determine first-line ART drug concentrations among patients switching to second-line ART. The finding that 55% of patients had subtherapeutic drug concentrations on first-line ART, and that these patients were less likely to achieve viral suppression on second-line ART, suggests that efforts to intensify adherence support when switching regimens were often unsuccessful. Drug concentrations also influenced resistance patterns at switch; NRTI mutations were detected in 97% of patients on therapeutic first-line ART, compared to 31% on a subtherapeutic regimen. However, rather than being associated with virological failure, the presence of NRTI mutations preswitch was associated with achieving viral suppression.trough; however, this is the first study in a resource-limited setting to measure drug concentrations at time of switch [Previous cross-sectional studies of clinic cohorts, which included patients with virological suppression, found that 4%–16% of patients had drug concentrations greater than <Cf switch –39. The f switch . This suf switch . Certainf switch , 40. Murf switch . AlthougOverall, the pattern of DRMs observed in this study was consistent with others’ findings: no major DRMs in 12% (23% in this study), NNRTIs in 86%–96% (74%), NRTIs in 81%–85% (70%), M184V/I in 68%–74% (67%), and NRTI cross-resistance mutations in 16% (16%) , 16. HowAlthough the majority of patients achieved early viral suppression on second-line ART , failurOur study has several limitations. First, we have assumed that subtherapeutic drug concentrations are a marker of nonadherence; however, malabsorption or drug interactions may also contribute . One of In conclusion, this study highlights that ongoing nonadherence is a major contributor to first-line virological failure when switching regimens and leads to suboptimal second-line outcomes. Patients need intensified adherence support when switching regimens if successful outcomes are to be realized; however, the optimal strategy for achieving this is unclear. In particular, HCWs need better guidance on how to manage patients with first-line virological failure who continue to be nonadherent, despite intensified support. At an individual level, switching nonadherent patients to a boosted PI–based regimen that is less susceptible to development of resistance may be appropriate and is the recommended strategy in many high-income settings. However, the cost-effectiveness of this approach for a public health program, where second- and third-line regimens are costly and treatment options limited, needs to be explored.Supplementary materials are available at The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author


Antiretroviral therapy inhibits HIV replication, maintains health, and preserves life. However, both antiretroviral therapy and HIV infection have been reported to have short- and long-term effects on bone metabolism. A cross-sectional study was performed to compare serum bone profiles in HIV positive patients on highly active antiretroviral therapy and compare them to therapy-naïve patients. Serum levels of calcium, magnesium, phosphate, and albumin were measured in 40 female participants on highly active antiretroviral therapy, recruited sequentially from Parirenyatwa Opportunistic Infections Clinic, Harare, Zimbabwe. The 40 women were matched for age with 40 antiretroviral therapy-naïve women. Magnesium, phosphate, and albumin levels were significantly higher in the therapy-naïve than in therapy-experienced patients. There was no statistically significant difference in calcium levels of the two groups of women. Evidence from this study suggests that highly active antiretroviral therapy lowers levels of magnesium, phosphate, and albumin but has no effect on levels of serum calcium. Zimbabwe is in sub-Saharan Africa which is at the epicenter of the human immune deficiency virus (HIV) epidemic. According to UNAIDS the prevalence of HIV in Zimbabwe has decreased to about 1 in 10 adults 2012) from a high one of almost 1 in 4 in 2002 [012 from The high disease burden of HIV has necessitated a rapid increase in the use of highly active antiretroviral therapy (HAART). As of 2012, over 476 thousand HIV-infected Zimbabweans were on HAART compared to only 8000 in 2003 [HAART has reduced both the morbidity and mortality of HIV-infected people due to AIDS. However HAART is reported to have adverse side effects, one of which is bone mineralization. Changes in serum levels of biochemical markers of bone metabolism have been shown elsewhere in experimental and clinical studies –7. A numOsteopenia and osteoporosis are conditions involving the weakening of bones which differ in the degree to which bones weaken . OsteopeA European study showed that Tenofovir increases bone turnover and decreases bone mineral density in treated HIV-infected adults . ResultsTenofovir, a drug recently introduced to Zimbabwe's First line treatment, has been associated with osteomalacia, low serum levels of phosphate, and elevated alkaline phosphate in a Swiss study . A NigerThere is no published evidence for the bone effect of prescribed first and second lines HAART regimens on Zimbabweans. The objective of this study was to provide evidence of changes in serum bone profiles, if any, in a Zimbabwean population of women using currently prescribed HAART regimens and compare them to a similar group of HIV-infected women who are not yet on HAART . The broParirenyatwa Group of Hospitals is a group of public and teaching hospitals found in the capital city of Zimbabwe. The group of hospitals are referral hospitals servicing casualties from Harare City Centre and its environs, together with referred cases from a number of clinics around Harare and other Zimbabwean provinces and districts. The clientele attending the Parirenyatwa Opportunistic Infections (OI) Clinic, the site of our study, is diverse in terms of geographical location, but many are from low-income groups. Those participants who were on HAART were taking a combination of tenofovir, lamivudine and nevirapine for the first line treatment and zidovudine, didanosine, and lopinavir/ritonavir for second line treatment. We sequentially recruited 40 HIV-infected women, aged 18 to 40 years who had been on first line HAART for more than 6 months and consented to be part of our study. 40 HIV-infected women who were HAART-naïve were matched for age with the 40 consenting HAART-experienced women. All the women had no evidence of critical illness at the time of the study. Permission to work with the Parirenyatwa OI Clinic was sought from the Clinical Director of Parirenyatwa Group of Hospitals, whilst ethical clearance was given by the Joint Research Ethics Committee of the Parirenyatwa Group of Hospitals and University of Zimbabwe (JREC) after they were satisfied that the research protocol was ethically sound. Participants gave written consent after being informed fully about their role, risks, and benefits in the study and being satisfied that their identities would be protected using anonymous numerical identifiers on all documents and specimen containers. n = minimum sample size, z = test statistic (at 5%), p1 = proportion with desired characteristic (0.05), Zα/2 = 1.96, Z1 − β, p1 = 0.63, p2 = 0.28,–n = ((1.96+0.84)2/(0.63−0.84)2)(0.63(1 − 0.63) + 0.28(1 − 0.28)) = 27.14 = 28,–n = 28 + Refusal Rate ,Final –n = 28 + 12.17 = 40.17.ConsiderTherefore overall minimum sample size = 80 (40 on HAART and 40 HAART-naïve).Demographic data was obtained from the patients and clinic records . Blood wSerum samples were thawed once at room temperature, and analysis was carried out on a BS120 Mindray Chemistry analyzer. The chemistry analyzer was calibrated, and then controls and samples were assayed for total calcium, magnesium, phosphate, and albumin. Corrected calcium was calculated for participants with low albumin levels.The mean assayed values for the female patients on HAART  were: total calcium 6.92 mg/dL (reference range 8.80–9.50 mg/dL), Magnesium 1.78 mg/dL (Reference Range 1.46–2.68 mg/dL), Phosphate 5.10 mg/dL (reference range 1.80–5.10 mg/dL) and Albumin 2.87 mg/dL (reference 2.80–5.30 mg/dL). The means of the HAART-naïve women were; Total calcium 8.40 mg/dL, Magnesium 2.55 mg/dL, Phosphate 5.10 mg/dL and Albumin 3.25 mg/dL. There was no significant difference between total calcium levels in patients on HAART and HAART-naïve patients. However, the values of calcium for the two groups in our study fall below the normal range. Our results suggest that use of both HIV and HAART may lead to hypocalcaemia. This contradicts with studies done in America where elevation in serum calcium levels in women on antiretroviral therapy including tenofovir was linked to an increase in bone loss . Our resThere were other interesting differences in serum bone profiles of the women in our study. Magnesium and albumin levels were within normal ranges for both groups of women, but the HAART-experienced had lower magnesium and albumin levels than the HAART-naïve patients. Our findings for magnesium do not however agree with two studies: a Brazilian study that showed hypomagnesaemia occurring in 29% of patients on antiretroviral therapy  and a stPhosphate levels were elevated in HAART-naïve patients, and their levels were above the normal range whilst the HAART-experienced women have on average phosphate levels that fall within normal ranges. Our study contradicts with studies which showed a link between use of tenofovir and hypophosphatemia  and studThe results of our study show both expected and unexpected results. It is important to improve this study by increasing the number of study sites, increasing the power by enlarging the sample size, and also measuring the levels of vitamin D . The results of this and future studies will be used to advocate for change in practice in treatment of HIV-infected women on HAART. Clinicians and decision makers may have to consider giving patients on HAART vitamin D supplements to avoid weakening of bones in Zimbabweans on HAART. Another study involving both men and women is also necessary in order to determine what serum bone profiles changes may be happening in men due to HIV infection and/or HAART. Is serum bone profiling a potentially noninvasive, inexpensive method that can be used routinely to detect changes in bone metabolism in the HIV infected patients in our resource-limited setting? This study shows evidence of changes in serum bone profiles. A larger study that also aims to determine association between serum bone profiles and measurement of the WHO gold standard  may help answer that question.


To compare the efficacy and safety of an individualized treatment-simplification strategy consisting of switching from a highly-active anti-retroviral treatment (HAART) with a ritonavir-boosted protease inhibitor (PI/r) and 2 nucleoside reverse-transcriptase inhibitors (NRTIs) to lopinavir/ritonavir (LPV/r) monotherapy, with intensification by 2 NRTIs if necessary, to that of continuing their HAART.This is a one-year, randomized, open-label, multi-center study in virologically-suppressed HIV-1-infected adults on their first PI/r-containing treatment, randomized to either LPV/r-monotherapy or continue their current treatment. Treatment efficacy was determined by plasma HIV-1 RNA viral load (VL), time-to-virologic rebound, patient-reported outcomes (PROs) and CD4+T-cell-count changes. Safety was assessed with the incidence of treatment-emergent adverse events (AE).Forty-one patients were randomized to LPV/r and 39 to continue their HAART. No statistically-significant differences between the two study groups in demographics and baseline characteristics were observed. At day-360, 71 patients completed treatment, while 9 discontinued. In a Last Observation Carried Forward Intent-to-Treat analysis, 40(98%) patients on LPV/r and 37(95%) on HAART had VL<200copies/mL (P = 0.61). Time-to-virologic rebound, changes in PROs, CD4+ T-cell-count and VL from baseline, also exhibited no statistically-significant between-group differences. Most frequent AEs were diarrhea (19%), headache (18%) and influenza (16%). Four (10%) patients on LPV/r were intensified with 2 NRTIs, all regaining virologic control. Eight serious AEs were reported by 5 patients.At day-360, virologic efficacy and safety of LPV/r appears comparable to that of a PI+2NRTIs HAART. These results suggest that our individualized, simplified maintenance strategy with LPV/r-monotherapy and protocol-mandated NRTI re-introduction upon viral rebound, in virologically-suppressed patients merits further prospective long-term evaluation.NCT00159224ClinicalTrials.gov  The standard treatment approach in HIV-1 infection involves using a combination of at least three antiretroviral (ARV) drugs, designated highly active antiretroviral therapy (HAART) to fully suppress plasma HIV-1 RNA viral load (VL), in a sustainable fashion. Currently recommended first line antiretroviral regimens consist of two nucleoside (NRTI) or nucleotide (NtRTI) analog reverse transcriptase inhibitors and either a non-nucleoside reverse transcriptase inhibitor (NNRTI), an integrase strand transfer inhibitor (INSTI) or a ritonavir-boosted protease inhibitor (PI/r) in vitro activity against HIV A number of regimen simplification treatment approaches have been explored to improve adherence, reduce the risk of virologic failure and long-term toxicities, and enhance the patient's quality of life The objective of this pilot study was to assess the efficacy and safety of a simplified strategy aimed to optimize the use of LPV/r monotherapy maintenance, whereby intensification with two NRTIs was allowed if VL in plasma became detectable, among patients stably suppressed on PI/r triple-combination therapy.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.3. Patients were excluded if they were HBsAg+, had active tuberculosis or an opportunistic infection, active malignancy (except Kaposi's Sarcoma), elevated hepatic transaminases , or an uncontrolled substance abuse or psychiatric illness that could preclude compliance with the protocol. Patients were also excluded if they were pregnant or lactating, had received an investigational drug within 30 days prior to study initiation, or had modified their ART within three months of study entry or were intending to do so during the course of the study.Eligible patients were HIV-1 infected adults who: i) were on their first ART regimen, composed of any two NRTIs plus LPV/r or a PI/r combination; and ii) had been virologically suppressed with a HIV-1 RNA viral load of <50 copies/ml for at least 6 months prior to study entry and a CD4+ T-cell count ≥100 cells/mmThis was a one-year pilot, prospective, open-label, randomized, comparative, multi-center study. The study was conducted according to the tenets of the Declaration of Helsinki and approved by an independent ethics review board . All participating patients provided written informed consent prior to study entry. Patients were recruited between January 2005 and July 2007 across 9 sites in Canada, Argentina, and Mexico, and were randomized to receive in a 1∶1 ratio either monotherapy with LPV/r (IM) or the standard HAART regimen (ST). Randomization was centrally coordinated by a third-party data management center and was stratified by center. A sealed envelope containing the randomized allocation was sent by the data management center to the physician who was blinded for the randomization schedule, and was opened by the patient. The allocation document was subsequently signed by the physician and mailed back to the data management center. Patients randomized to the IM group were provided with co-formulated LPV/r 133.3/33.3 mg soft gel capsules and were instructed to take 3 capsules BID orally with food. Clinical assessments took place at Screening/Baseline (Day -1) and Days 15, 30, 60, 90, 120, 150, 180, 240, 300, and 360. Efficacy measures included plasma HIV-1 RNA levels and CD4+ T-cell counts. Safety was assessed with the incidence of treatment emergent adverse events (AE), vital signs, clinical laboratory data, including venous lactic acid and serum lipid levels.Patients with HIV-1 RNA >50 copies/ml in one visit were retested between 7 and 30 days later. If the second viral load was <50 copies/ml the patients continued on their randomized therapy, while if it was >50 and <200 copies/mL the patients were followed on protocol and were retested until either <50 or >200 copies/ml was confirmed. If the second viral load was >200 copies/ml, patients in the ST arm were considered to have met the endpoint of virologic failure, and treatment was to be modified at the discretion of the investigator/treating physician. In the monotherapy arm, if the second viral load was >200 copies/ml, intensification with two NRTIs was allowed (either the same NRTIs as before randomization or different ones) and the patient was maintained on the randomized treatment starting the visit schedule from the beginning. Intensified patients who developed a viral load of >50 copies/ml and a subsequent viral load of >200 copies/ml were considered to have reached the study endpoint of virologic failure, and therapy was to be modified at the discretion of the investigator or the treating physician.The primary efficacy endpoint was the percentage of patients with plasma HIV-1 RNA level <200 copies/ml at Day 360. Secondary efficacy measures were the percentage of patients with plasma HIV-1 RNA <50 copies/mL at Day 360   = 50 copies/mL), the time to confirmed virologic rebound (≥200 copies/ml and ≥50 copies/ml) or meeting the criteria for virologic failure as described above through Day 360, as well as the mean change in Viral Load and CD4+ T-cell count from baseline to final assessment.The impact on patient-reported outcomes (PROs) was assessed using the Symptoms Distress Module (SDM) which was administered at each visit. This questionnaire was developed by the NIAID AIDS Clinical Trials group and consists of 20 questions evaluating the impact of specific symptoms, possibly related to the treatment, on the patient's life. The total score is calculated as the sum of the five point response to the 20 questions where 0 =  symptom not reported, 1 =  I have this symptom and it doesn't bother me, 2 =  I have this symptom and it bothers me a little, 3 =  I have this symptom and it bothers me, and 4 =  I have this symptom and it bothers me a lot. The SDM score ranges from 0 to 80 and higher values indicate worse PROs.Safety was determined by the incidence of treatment emergent adverse events (AE), changes in vital signs and clinical laboratory data, as well as the occurrence of metabolic toxicity as indicated by the venous lactic acid and fasting serum lipid levels. AE relationship to the study medication was based on the judgment of the treating physician.Sample size calculations for the current study were based on the expected difference between the two treatment groups in the proportion of patients with virologic control defined as <200 copies/mL at Day 360. Previous studies have shown that at 48 weeks, approximately 90% of virologically suppressed patients treated with LPV/r remain virologically controlled Between-group differences in the rates of virologic control  were assessed with the Chi-Square test. The odds ratio (OR) with 95% confidence intervals was used as the measure of treatment effect. In this analysis the Last Observation Carried Forward (LOCF) approach was used for patients that discontinued the study prior to the 360 day follow up. The time to first confirmed virologic rebound was estimated using the Kaplan Meier Survival function, and the maximum likelihood test was used to compare the two groups with respect to the rate of virologic rebound. The Student's t-test for independent samples was used to assess between group differences with respect to the change in CD4+ T-cell count, VL and SDM score from baseline to final assessment. Repeated Measures Analysis of variance with Mixed Effects to account for unequal follow up were used to assess the treatment effect on CD4+ T-cell count, VL and SDM over time. Paired Student's t-test was used to descriptively assess the change in CD4+ T-cell count, VL and SDM within the two treatment groups, while simple linear regression models were used to assess these changes over time within the two treatment groups. Safety was assessed by the incidence of adverse events. All analyses were performed using the intent-to-treat population, defined as all patients enrolled who had taken at least one dose of the study medications and had completed at least one follow up visit.A total of 80 patients were enrolled in the study, met the intent-to-treat (ITT) criteria and were randomly assigned to treatment, of which, 71 (89%) completed the study and 9 (11%) prematurely discontinued. Among these 9 discontinued patients, 7 belonged to the ST group and 2 to the IM group. Reasons for discontinuation and patient disposition are described in 10 HIV-1 RNA were 383 (195) cells/mm3 and 1.68 (0.08) log10copies/ml, respectively. The most common ARV medications used prior to randomization, were: lamivudine 44 (55%), LPV/r 44 (55%), low dose ritonavir 34 (42%), zidovudine/lamivudine 31 (39%), and zidovudine 26 (33%). There were 23 patients on a LPV/r combination and 18 patients on a non-LPV/r regimen in the IM group, prior to randomization. Similarly, 23 patients in the ST group were on a LPV/r combination while 16 patients were on a non-LPV/r combination.As summarized in The primary outcome measure of the study was the proportion of patients with plasma HIV-1 RNA <200 copies/ml at 360 days. In an ITT analysis using the LOCF principle, 37 of the 39 patients (95%) in the ST group and 40 of the 41 patients (98%) in the IM group had plasma HIV-1 RNA <200 copies/ml . With respect to the proportion of patients with plasma HIV-1 RNA <50 copies/ml at 360 days, applying again the LOCF principle, there were 36 patients (92%) for the ST and 39 (95%) for the IM group . Four (10%) patients on LPV/r were intensified with 2 NRTIs and all of them regained virologic control, as demonstrated by achieving a plasma HIV-1 RNA <50 copies/mL following the intensification.The Kaplan Meier estimates of the proportion of patients with sustained virologic response are shown in The results in Using the SDM to assess the PROs, it was determined that the patients in the IM group experienced a decline in the SDM from 31.7 at baseline to 26.2 at 360 days (P = 0.003), indicating a statistically significant improvement in the PROs. On the contrary, patients in the ST group experienced a statistically non-significant decline from 31.8 at baseline to 29.6 at 360 days (P = 0.094). Nevertheless, the difference in the change in SDM from baseline to 360 days of treatment between the two treatment groups was not statistically significant (P = 0.131). Similarly, linear regression analysis showed that the change in SDM over time was statistically significant for the IM group (P = 0.001), but not for the ST group (P = 0.949) . HoweverA total of 658 AEs were reported for 66 (83%) patients. Of these, 269 AEs were reported by 32 (82%) patients in the ST group while 389 AEs were reported by 34 (83%) patients in the IM group. Both the incidence and the profile of adverse events were comparable between the two groups, showing no apparent differences. The most frequently reported adverse events were diarrhea (19%), headache (18%), influenza (16%), nasopharyngitis (13%), back pain (10%), hypertriglyceremia (8%) and insomnia (8%). Adverse events were predominantly mild in severity and judged unrelated to the study drug. There were three SAEs reported by two patients in the IM group  and five SAEs reported by three patients in the ST group, of which seven were considered severe and one in the IM group was moderate. All SAEs were considered unrelated to the study drug.The goal of this pilot, randomized clinical trial was to compare an individualized, simplified maintenance LPV/r-based strategy with reintroduction of two NRTIs upon viral rebound in plasma, to the standard continued triple-drug therapy with respect to sustained virologic response over 360 days, among virologically suppressed HIV-1-infected patients on their first PI/r-based HAART regimen. Our results demonstrate comparable safety, efficacy and tolerability for the induction/maintenance strategy and the continued standard HAART treatment. Overall, virologic success rates of over 90% and 95% were documented when using the 50 and 200 copies/mL plasma viral load thresholds, respectively. Importantly, intensification by NRTIs was required in only 10% of the patients randomized to LPV/r which, in all instances, resulted in regaining virologic control as defined by a sustained plasma HIV-1 RNA level of <50 copies/mL. Additional immunologic and virologic parameters including the change in the CD4-T-cell count and the viral load from baseline to final assessment, the rate of change in these two parameters over the 360 days, and time to virologic rebound defined as >200 HIV-1 RNA copies/ml, were also not statistically different between the two groups. With regards to the time to VL>50 HIV-1 RNA copies/ml, a trend towards the favor of the ST was observed, which was however not statistically significant. Previous studies have shown that ritonavir-boosted PI monotherapy is associated with low-level viremia (50–200 copies/mL), the clinical relevance of which is still not clear The results of the current study are in agreement with those from the OK04 study Use of class-sparing regimens, such as the one described in this study, offers the advantage of saving alternative ARV classes as a “back-up” option for new ARV combinations, in the case of ART failure. Furthermore, such regimens could help avoid the side effects associated with nucleoside analogue-containing regimens including renal or bone toxicity and high cardiovascular risk associated with tenofovir disoproxil fumarate and abacavir, respectively One of the possible limitations of the current study is the small sample size and the fact that the calculated sample size was not achieved due to low recruitment rate. However, the differences between the two groups with respect to virologic suppression and immunological changes were clinically non-important in addition to not being statistically significant. This observed similarity between the treatment groups provides evidence for the comparability of their effectiveness. Nevertheless, larger studies with longer follow up would be helpful in confirming these conclusions. The open label design of the study represents a methodological limitation. However, this design is in line with real-life practice while the objective and blinded ascertainment of virologic and immunologic parameters precludes the possibility of differential ascertainment bias.In conclusion, our study reports encouraging preliminary safety and efficacy outcomes using an individualized, simplification maintenance strategy of LPV/r monotherapy with NRTI re-introduction upon viral rebound in plasma, among virologically suppressed patients on their first PI/r-based HAART. Based on these results, our strategy of simplified maintenance with LPV/r monotherapy merits further prospective long term evaluation of its safety and effectiveness in larger cohorts. Evaluation of the simplified strategy proposed here is particularly important as it provides a potentially simple and more affordable strategy for long term ART, that may be particularly relevant to the current global effort to expand access to HAART to millions in need.Protocol S1(PDF)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Early institution of therapy has been advocated worldwide. However, until recently, NACO guidelines recommended therapy in HIV positive patients with CD4 count <250/µL. In this study we observed the clinical and immunological disease progression for a year in treatment naïve patients with CD4 count between 250-500/µL.3 in WHO clinical stage 1 or 2 were included in the study and enrolled into 2 groups at 2:1 ratio and followed for 1 year. Group A with CD4 count between 250-350/mm3 & Group B with CD4 count between 350-500/mm3. At the end of study each group was evaluated for number of opportunistic infection, ART conversion, conversion to stage III and IV, lost to follow up (LFU) and death. Statistical analysis was done by SPSS 16.0 version. Data were expressed as mean (±SD) for continuous variable and percentage for categorical variable. An independent sample t-test was used to detect differences in clinical and laboratory results.150 treatment naïve adults PLHIV with CD4 count between 250 -500/mmAs compared to Group B, Group A had significantly higher number of opportunistic infection , ART conversion , death  and LFU (20% vs 6% p=0.037).This study reinforces the need of early initiation of treatment in patients with CD4 <350/ µL.


Whether tenofovir nephrotoxicity is reversible after its withdrawal is unknown. Furthermore, there are no data on the viral efficacy of raltegravir (RAL) plus ritonavir-boosted Darunavir (DRV/r) in patients with suppressed viral load.This multicenter, randomized trial compared renal function and viral efficacy in patients with suppressed viral load treated with RAL+DRV/r and ritonavir-boosted lopinavir (LPV/r) plus tenofovir/emtricitabine (TVD), who had been previously on LPV/r+TVD. The primary endpoint was the proportion of patients with >10% improvement in estimated glomerular filtration rate (eGFR) at 48 weeks calculated with Cockcroft-Gault equation.58 randomized and treatment-exposed patients were analyzed (28 on RAL+DRV/r and 30 on LPV/r+TVD). Greater than 10% improvement in eGFR was noted in 6 (25%) out of 24 with RAL+DRV/r and 3 (11%) of 28 with LPV/r+TVD, and the difference was not statistically significant . Sensitivity analyses using three other equations for eGFR showed the same results. Urinary β2 microglobulin, a sensitive marker of tenofovir tubulopathy, significantly improved with RAL+DRV/r than with LPV/r+TVD . Per protocol analysis showed that the HIV-RNA was <50 copies/mL at week 48 in all patients of both arms (24 in RAL+DRV and 29 in LPV/r+TVD).Switching LPV/r+TVD to RAL+DRV/r did not significantly increase the proportion of patients who showed >10% improvement in renal function among those with relatively preserved eGFR. However, the switch improved urinary β2 microglobulin, suggesting that discontinuation of TDF might be beneficial in the long-term. RAL+DRV/r showed favorable viral efficacy in patients with suppressed viral load.ClinicalTrials.gov NCT01294761 http://clinicaltrials.gov/ct2/show/NCT01294761?term=SPARE&rank=2, Umin Clinical Trials Registry UMIN000005116 http://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000006083&language=J) Tenofovir disoproxil fumarate (TDF) is one of the most widely used nucleotide reverse transcriptase inhibitors (NRTI) for patients with HIV infection, with proven efficacy and safety -6. HowevClinical manifestations such as lipoatrophy and neuropathy caused by NRTI-induced mitochondria toxicity are difficult to reverse ,23, but Recently, antiretroviral therapy (ART) not containing NRTIs (NRTI sparing regimens) has gained a wide attention, since these combinations can avoid NRTI toxicity. Despite high expectations, the results of studies on the efficacy and safety of NRTI sparing regimens for treatment-naïve patients showed dismal results. A small single arm study of CCR5 inhibitor maraviroc plus ritonavir-boosted Darunavir (DRV/r) showed a high rate of virologic failure, especially in patients with high baseline viral load of >100,000 copies/mL . RaltegrAt this stage, it is important to elucidate the effectiveness of NRTI sparing regimen for patients with suppressed HIV-1 viral load, because longer exposure with NRTIs tends to result in clinically overt NRTI-associated mitochondrial toxicity ,32, and Based on the above background, this multicenter randomized trial was conducted to elucidate 1) the reversibility of tenofovir nephrotoxicity, and 2) efficacy and safety of RAL+DRV/r for patients with suppressed viral load.This clinical trial was designed and reported according to the recommendations of the Consolidated Standard of Reporting Trials (CONSORT) statement . The proDeclarationofHelsinki.The Research Ethics Committees of Hokkaido University Hospital, Higashisaitama National Hospital, Niigata University Medical and Dental Hospital, the Institute of Medical Science, the University of Tokyo, Juntendo University School of Medicine, Shirakaba Clinic, Saku Central Hospital, Hiroshima University Hospital, Ehime University Hospital, National Hospital Organization Kyushu Medical Center, Kumamoto University Graduate School of Medical Sciences and National Center for Global Health and Medicine approved the study protocol. All patients enrolled in this study provided a written informed consent. The study was conducted according to the principles expressed in the The SPARE trial is an on-going phase 3B, multi-center, randomized, open-label, parallel group study conducted in Japan to compare renal function and viral efficacy of NRTI-sparing regimen of RAL+DRV/r and a standard regimen of PI/r + 2NRTIs [(lopinavir/ritonavir (LPV/r) plus fixed dose of tenofovir/emtricitabine (TVD)] for 96 weeks, randomly allocated to patients on LPV/r+TVD with suppressed viral load. With one to one ratio, patients with suppressed viral load on LPV/r (800 mg/200 mg) plus fixed dose of TDF (300 mg)/emtricitabine (200 mg) were randomly assigned to either RAL (800 mg) plus DRV/r (800 mg/100 mg) or to continue LPV/r+TVD. Patient enrollment remained open between February 21, 2011 and December 2011, and the follow-up period is scheduled to end in December 2013. This report summarizes the findings after 48 weeks of treatment, including the primary endpoint. Randomization was stratified based on baseline body weight of 60 kg because low body weight, especially body weight of <60 kg, is an important risk for tenofovir nephrotoxicity ,18,34. RThe study population included Japanese patients with HIV-1 infection, aged ≥20 years, who were on LPV/r plus TVD and with suppressed HIV-1 RNA viral load of <50 copies/ml over a period of more than 15 weeks. Patients were screened and excluded if found positive for hepatitis B surface antigen, or had history of virologic failure with regimens including protease inhibitor or integrase inhibitor, or if they were considered inappropriate for the study by the attending physicians. Candidates were also excluded if the level of alanine aminotransferase was 2.5 times the upper limit of normal, estimated glomerular filtration rate (eGFR) calculated by Cockcroft-Gault equation (CG equation) was <60 ml/min, {[(140 - age) × weight (kg)] / (serum creatinine × 72)] } , or on taspartateaminotransferase, alanine aminotransferase, lactatedehydrogenase, alkaline phosphatase, creatine kinase, blood urea nitrogen, serum creatinine, sodium, potassium, calcium, phosphate, triglyceride, total cholesterol, low-density lipoprotein cholesterol, high density lipoprotein cholesterol, glucose), and urine examination , and albumin). The values of urinary β2 microglobulin, NAG, and albumin were expressed relative to urinary creatinine of 1 g/L (/g Cr). Percent tubular resorption of phosphate was calculated by the following formula: {1 – [(urine phosphate × serum creatinine) / (urine creatinine × serum phosphate)]} × 100 [2) the Modification of Diet in Renal Disease (MDRD) equation adjusted with coefficient for the Japanese [0.808 × 175 × (serum creatinine)-1.154 × (age)-0.203× ] [α× max  -1.209 × (0.993)age× ]  [38]. Furthermore, the percent improvement in eGFR from baseline to week 48, calculated with all four equations described above, was compared between the two arms by the Student’s t-test. Because the percent improvement in eGFR may depend on the baseline value, a correlation between the percent improvement in eGFR and the baseline value was tested, and the results showed very weak correlation (0.001<r<0.2) for all four equations for eGFR. Accordingly, the comparison of the percent improvement was conducted by the t-test as described above.The primary endpoint was the proportion of patients with >10% improvement in eGFR at 48 weeks from the baseline calculated with the CG equation 35]. The baseline eGFR was estimated from the average of serum creatinine measured at baseline and at screening for enrollment. eGFR at week 48 was estimated from the average of serum creatinine at weeks 36 and 48. The proportion of such patients was compared between the two arms by the Fisher exact test. The following three equations for eGFR were also used for sensitivity analysis: 1) A 3-variable equation for the Japanese set by the Japanese Society of Nephrology (JSN equation): [194 × (serum creatinine)atients)], 2) the tients)] , and 3) 5. The ba2 test or Fisher’s exact test for categorical variables. Statistical significance was defined at two-sided p values <0.05. All statistical analyses were performed with The Statistical Package for Social Sciences ver. 21.0 .The secondary renal endpoint was changes in per protocol renal tubular markers from the baseline to week 48, and the results were compared by the Mann-Whitney test. The secondary efficacy endpoint was the proportions of patients with HIV-1 RNA <50 copies/mL at weeks 24 and 48. Data of both per protocol population and the intent-to-treat (ITT) population, comprising all randomized treatment-exposed subjects were used for the assessment of efficacy. With regard to analysis on the viral efficacy in this study, per protocol analyses were more important than ITT analyses, because some patients enrolled in the RAL+DRV/r arm were expected to develop adverse events due to switching to the new medications and subsequent discontinuation of the allocated regimen, whereas new adverse events were not likely in patients of the LPV/r+TVD arm solely by continuing the same regimen as before. Baseline parameters were compared between the two arms by the Student’s t-test for continuous variables and by either the χBetween February and December of 2011, 59 patients from 11 centers were enrolled in the study and randomized. Of these, 29 and 30 patients were allocated to the RAL+DRV/r and the LPV/r+TVD arm, respectively . One patAt week 48, six patients (25%) out of 24 in the RAL+DRV/r arm and 3 patients (11%) out of 28 in the LPV/r+TVD arm, experienced >10% improvement in eGFR from baseline, and the difference was not statistically significant . Sensitivity analysis with three other equations for eGFR  showed the same results; no difference in the proportion of patients with improvement of >10% in eGFR was noted between the two arms    .Additional analysis showed that the percent improvement in eGFR from the baseline to week 48 calculated using all four equations was not significantly different between the two arms     . Thus, tAmong the four renal tubular markers used in this study, the improvement in urinary β2 microglobulin from baseline to week 48 was significantly larger in the RAL+DRV/r arm (n=23) than in the LPV/r+TVD arm (n=28)  . HoweverAmong the per protocol population, the proportion of patients with HIV RNA <50 copies/mL was 96.2% for the RAL+DRV/r arm and 96.7% for the LPV/r+TVD arm at week 24, with a difference of -0.5% , and 100% for the both arms at week 48, with a difference of 0% (95% CI -0.1 to 0.1) . ITT anaOne patient from each arm was lost to follow-up. Three patients of the RAL+DRV/r arm discontinued the allocated regimen by week 48 . The other patient withdrew consent at week 24, because it was easier for him to maintain a good medication adherence with once-daily LPV/r+TVD (the regimen the patient used before enrollment). None of the patients of the LPV/r+TVD arm discontinued the allocated regimen by week 48. Thus, at week 48, 24 patients (86%) out of 28 in the RAL+DRV/r arm and 29 (97%) of 30 in the LPV/r+TVD arm, were on the allocated regimens.The following grade 3 or 4 laboratory data or abnormal symptoms that were at least one grade higher than the baseline were encountered in this study: RAL+DRV/r arm: a rise in ALT , and elevated LDL-cholesterol (n=3), LPV/r+TVD arm: elevated LDL-cholesterol (n=1), and hypophosphatemia (n=3). The above side effects did not lead to discontinuation of the study drugs.2 , with only one patient with CKD stage 3 due to persistent +1 proteinuria, and no patients with stage 4 or more. Although the number of patients is relatively small, a previous pilot study of 21 patients reported improvement of eGFR (by CG equation) in most patients after switching from PI/r+TVD to PI/r+RAL in patients with proteinuria and suppressed HIV viral load [39]. Thus, improvement of eGFR after discontinuation of TDF might be more significant in patients with severe to moderately impaired renal function. Larger studies are needed to investigate this issue thoroughly. 2) Study patients had been on TDF for a long period of time at enrollment , although shorter duration of TDF therapy is likely to be associated with greater eGFR improvement after discontinuation [26]. Furthermore, because TDF-induced renal dysfunction is mainly observed during the first 6 months after commencement of such therapy , it is possible that patients who developed severe renal dysfunction soon after starting TDF might have already discontinued TDF and therefore not included in the study.This randomized trial elucidated the recovery of TDF-associated nephropathy after discontinuation of TDF. The results demonstrated no significant increase in the proportion of patients who showed >10% improvement in eGFR after switching to NRTI sparing regimen of RAL+DRV/r, compared to continuation of LPV/r+TVD. This finding could be due to any of the following reasons; 1) Relatively preserved baseline renal function of the enrolled patients, with a median eGFR of 86 ml/min/1.73 mAlthough the present study did not show an increase in eGFR after discontinuation of TDF, it is noteworthy that the value of urinary β2 microglobulin, a sensitive marker for TDF-induced tubulopathy , improved significantly in the RAL+DRV/r arm compared to LPV/r+TVD, even in patients with relatively preserved eGFR. It is of importance considering that proximal tubulopathy is associated with bone mineral density abnormality and possible long-term nephrotoxic effect . Further large and long-term studies are needed to elucidate the long-term impact of TDF-induced tubulopathy on GFR.http://aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf). Because the number of enrolled patients is relatively small and this study does not have sufficient power to elucidate viral efficacy, further studies are needed to confirm the viral efficacy of RAL+DRV/r in patients with suppressed viral load. If the NRTI sparing regimen of RAL+DRV/r is proved to be efficacious in maintaining viral suppression in treatment-experienced patients, switching to this combination for patients with suppressed viral load should become an attractive treatment option for patients who cannot tolerate NRTI toxicity or to prevent further NRTI toxicity.With regard to the viral efficacy and safety of RAL+DRV/r, all patients in that arm who continued the allocated regimen accomplished viral suppression of <50 copies/ml at week 48 (n=24). Only one (3.6%) patient discontinued RAL+DRV/r due to a side effect possibly related to RAL+DRV/r (weakness of the lower extremities), confirming the safety of this combination. To our knowledge, this is the first study to examine the viral efficacy of RAL+DRV/r in patients with suppressed viral load. The KITE study, an industry-sponsored pilot study, examined the viral efficacy of RAL+LPV/r in patients with suppressed viral load [46]. However, LPV/r is placed as an alternative PI in the American Department of Health and Human Services Guidelines, mainly because of the higher rates of gastrointestinal side effects and hyperlipidemia compared with other PIs . Further studies are needed to determine whether the findings of this study are also applicable to females, patients with different routes of transmission, and patients of different racial background.In conclusion, this trial showed that discontinuation of LPV/r+TVD and switching to NRTI-sparing regimen of RAL+DRV/r did not result in improvement of renal function among patients with relatively preserved eGFR and suppressed HIV viral load. However, urinary β2 microglobulin, a sensitive marker of TDF-induced tubulopathy, improved after discontinuation of TDF plus ritonavir-boosted PI, suggesting switching TDF to NRTI sparing regimen might be beneficial in the long-term. RAL+DRV/r showed favorable viral efficacy and safety in patients with suppressed viral load, but further larger studies are needed to confirm the viral efficacy of this combination. Protocol S1Trial protocol.(DOCX)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.


Tenofovir (TDF) is part of the WHO recommended first-line antiretroviral therapy (ART); however, there are limited data comparing TDF to other nucleoside reverse transcriptase inhibitors in resource-limited-settings. Using a routine workplace and community-based ART cohort in South Africa, we assessed single drug substitution, HIV RNA suppression, CD4 count increase, loss-from-care, and mortality between TDF, stavudine (d4T) 30 mg dose, and zidovudine (AZT).In a prospective cohort study we included ART naïve patients aged ≥17 years-old who initiated ART containing TDF, d4T, or AZT between 2007 and 2009. For analysis of single drug substitutions we used a competing-risks time-to-event analysis; for loss-from-care, mixed-effect Poisson modeling; for HIV RNA suppression, competing-risks logistic regression; for CD4 count slope, mixed-effects linear regression; and for mortality, proportional hazards modeling.Of 6,196 patients, the initial drug was TDF for 665 (11%), d4T for 4,179 (68%), and AZT for 1,352 (22%). During the first 6 months of ART, the adjusted hazard ratio for a single drug substitution was 2.3 for d4T  and 5.2 for AZT , compared to TDF; whereas, after 6 months, it was 10  and 4.4  for d4T and AZT, respectively. Virologic suppression was similar by agent; however, CD4 count rise was lowest for AZT. The adjusted hazard ratio for loss-from-care, when compared to TDF, was 1.5  for d4T and 1.2  for AZT. The adjusted hazard ratio for mortality, when compared to TDF, was 2.7  and 1.4  and for d4T and AZT, respectively.In routine care, TDF appeared to perform better than either d4T or AZT, most notably with less drug substitution and mortality than for either other agent. Decisions on first-line combination antiretroviral therapy (ART) regimens for use in resource-limited-settings are made based on trade-offs regarding cost, need for laboratory monitoring, severity and frequency of side effects, and effectiveness. Stavudine (d4T) was initially recommended by the World Health Organization (WHO) largely due to less need for laboratory monitoring and a lower price than zidovudine (AZT) Despite these changes in WHO recommendations, d4T and AZT remain widely used due to the higher cost of a month supply of TDF compared to either d4T or AZT. An additional limiting factor for TDF uptake is concern related to monitoring patients receiving TDF for renal toxicity, a potential severe side effect of TDF. However, in addition to the direct cost of the medication, other factors need to be considered when selecting an agent for long-term use, such as the costs of monitoring, need for managing common toxicities, frequency of the need to change regimens, and the impact, from a public health standpoint, of loss of patients from care related to medication side effects. Thus outcome assessments comparing agents during routine patient care can be valuable for informing policy regarding ART agent selection.Two studies set in resource-limited-settings and using routine program data have compared TDF, d4T (30 mg), and AZT-based regimens. One was a study from Zambia that compared single drug substitutions and a composite mortality and loss-from-care endpoint All data were analyzed anonymously and all aspects of this study were conducted according to the principles expressed in the Declaration of Helsinki. Human subject research approval for this study was received from the University of KwaZulu-Natal Biomedical Research Ethics Committee and the Johns Hopkins Institutional Review Board.3 or a CD4 count less than 350 cells/mm3 with WHO clinical stage III or any CD4 and WHO clinical stage IV disease; whereas the community program required a CD4 count less than 250 cells/mm3 or any CD4 with WHO clinical stage IV disease. Other patient monitoring and management was similar between the two programs with similar use of guidelines and program monitoring and evaluation activities.The study population consisted of HIV-infected patients enrolled in community or workplace HIV care programs applying standardized treatment and monitoring approaches and overseen by a single HIV management organization For this study, we included patients who were ART naïve at study entry, ≥17 years old, initiated ART between June 1, 2007 and June 30, 2009, and were initiated on a regimen of TDF, d4T (30 mg twice daily), or AZT with 3TC or FTC and either EFV or NVP. Patients were excluded if they received 40 mg of d4T or if the dose was not recorded. Entry into observation was defined as the date of ART initiation. Exit was defined as the earliest date amongst patient death, time of change of any ART agent, or 24 months elapsed on ART. The study period closed on June 30, 2011, six months prior to the cohort closure, to allow for a potential 24 months of follow-up with an additional six months for assignment of appropriate loss to follow-up status for all participants.Patient deaths were identified through clinical records and linkage with the South African National vital status registry for patients with recorded national identification numbers. We used inverse probability weighting to adjust for under-ascertainment of death among patients without recorded national identification numbers who were lost from care, up weighting individuals lost from care who had identification numbers and down weighting those lost from care who did not have identification numbers Loss-from-care was defined as any patient whose last clinic or laboratory record was before closure of the database for the specific site, in the absence of a recorded treatment stop reason or death.We compared patient characteristics at ART initiation by initial NRTI  using chi-squared or Kruskal-Wallis testing. We performed time to event analysis for each of single-agent substitution, loss-from-care and mortality. We used single agent substitution as a proxy for severe side effects, assessing risk of substitution using a competing-risks time-to-event analysis, with death and loss-from-care both considered competing risks, with robust estimates at site level. In evaluating HIV RNA suppression, we assessed the proportion with HIV RNA <400 c/mL at 24 months. We allowed for a window of 3 months earlier or later (21–27 months) and only included patients with at least one HIV RNA result during that window. We used the minimum HIV RNA value if multiple results were available during the window. We used a random-effects logistic regression for HIV RNA suppression accounting for the random effect of workplace versus community HIV program. We also assessed CD4 change over time on ART up to 24 months on ART. We only included participants with more than 12 months of follow-up to maintain consistency with the HIV RNA suppression analysis. We used mixed effects linear regression to estimate the slope in CD4 count by NRTI agent and HIV RNA suppression including ART initiation year, ART clinic setting, and patient, as random effects in all models . We additionally assessed for interactions with NRTI that could affect CD4 slope. We completed a time to event analysis for loss-from-care, using a competing risk framework, using death as the competing risk and using robust estimates at site level. We used a random effects Poisson model accounting for site-level effects to calculate incident rates for loss-from-care, adjusted for site-level differences. For mortality, we calculated hazard ratios and adjusted hazard ratios using Cox proportional hazard modeling, controlling for site level effects by using robust estimates with a fixed effect for program (workplace or community). For both single drug substitution and loss-from-care competing risk analyses, we included ART initiation year and ART clinic setting (workplace or community) to adjust for secular and site level effects.In univariable analysis we assessed for associations between the outcomes and NRTI agent, sex, age, NNRTI agent, WHO clinical stage at ART initiation, CD4 count at ART initiation, HIV RNA at ART initiation, and ART initiation year. Except in the CD4 change analysis, we chose not to use time up-dated data for CD4 count and HIV RNA as these could potentially be affected by NRTI agent characteristics  and hence could be on the causal pathway from exposure to outcome.Because of the considerable imbalance between characteristics between workplace and community programs, we completed sensitivity analyses by repeating all adjusted analyses restricting to either workplace or community sites. We also explored single-drug substitutions in a sensitivity analysis restricting to comparisons between d4T and AZT for the period prior to TDF registration in South Africa, so as to exclude patients who may have been switched from another agent to TDF, not because of adverse events, but because the provider perceived TDF to be superior.3  met inclusion criteria. There were 2,668 patients excluded because they were not ART naïve at study entry , <17 years old (n = 266), or were initiated on a regimen other than the allowed regimens n = 200; . The medOver the first two years of ART, 10 patients had a single drug substitution from TDF , 691 from d4T , and 95 from AZT . However, the rate of single drug substitution varied by time on ART. During the first 6 months of ART, the rate of single drug substitution was lowest for TDF  and was similar for d4T  and AZT . However, from 6 months onward, the rate was highest for d4T  and lower for TDF  and AZT .In multivariable modeling, the differences in single agent substitution by time on ART remained . During When restricting to workplace sites for a sensitivity analysis, the adjusted hazard ratio compared to TDF, was 13.8 (95% CI: 3.5–55.1) for d4T and 4.7 (95% CI: 1.3–17.0) for AZT during the first 6 months of ART; after 6 months, the hazard ratio was 9.2 (95% CI: 2.2–38.5) for d4T and 4.7 (95% CI: 1.4–15.8) for AZT. In the community program, when compared to TDF, during the first 6 months of ART, the adjusted hazard ratio for d4T was 0.9 (95% CI: 0.3–3.0) and for AZT was 1.5 (95% CI: 0.4–5.1) while after 6 months the adjusted hazard ratio for d4T was 7.3 (95% CI: 1.8–29.9) and for AZT was 4.2 (95% CI: 1.1–17.8).We also repeated the analysis restricting to the time period prior to TDF availability, limiting single drug substitutions to AZT from d4T or to d4T from AZT. During the first 6 months, the rate of substitution from d4T was 5.9 per 100 PYRs and from AZT was 5.8 per 100 PYRs. After 6 months the rate from d4T was 4.5 per 100 PYRs and from AZT was 0.5 per 100 PYRs. The hazard ratio for single agent substitution from d4T (compared to AZT) during the first 6 months was 1.2 (95% CI: 0.8–2.0), and from d4T (compared to AZT) from 6–24 months was 7.8 (95% CI: 4.1–14.8).The distribution of patients with HIV RNA data at 24 months was 46% of TDF recipients, 36% of d4T recipients, and 42% of AZT recipients. Where HIV RNA data were available, HIV RNA suppression (<400 copies/ml) was achieved in 74% on TDF, 83% on d4T, and 70% of patients on AZT. In the random-effects logistic regression model, when compared to TDF, the odds ratio for virological suppression on d4T was 1.1 (95% CI: 0.7–1.8) and on AZT was 0.8  for d4T and 0.9 (95% CI: 0.6–1.6) for AZT. Restricting to the community program, the adjusted hazard ratio with TDF as the referent agent was 1.5 (95% CI: 0.4–5.6) for d4T and was 1.2 (95% CI: 0.3–4.8) for AZT.3/year, respectively  for AZT (p<0.001).The distribution of patients with over 12 months of CD4 data for inclusion in assessing CD4 change was 74% of TDF recipients, 62% of d4T recipients, and 73% of AZT recipients. In mixed linear regression, with NRTI agent as the only fixed effect, NRTI agent was significantly associated with CD4 increase with an annual increase for TDF, d4T, and AZT of 67.0 , 79.2 , and 53.1  cells/mmp<0.001; . In adju3/year. In the community program, the association was no longer statistically significant (p = 0.3): TDF, 65.2 ; d4T, 82.7 ; and AZT, 78.4 .In a sensitivity analysis, when restricting to workplace sites, we continued to observe a statistically significant association between NRTI agent and CD4 slope (p<0.001) with a slope for TDF of 85.3 , for d4T of 90.0 , and for AZT of 69.0  cells/mmDuring the 24 month follow-up period, the rate for loss-from-care appeared similar for TDF  and AZT  and was highest for patients on d4T . In the adjusted model, compared to TDF, the hazard ratio for d4T was 1.5 (95% CI: 1.1–1.9) and for AZT was 1.2  and for AZT was 1.1  when compared to TDF. When restricting to community sites, the hazard ratio for d4T was 2.0  and for AZT was 1.4  when compared to TDF. Thus, when analyzed separately by workplace and community clinics, NRTI agent was not significantly associated with loss-from-care. There were no significant interactions between loss-from-care and duration on ART, nor were there interactions between NRTI and any of sex, age, or NNRTI in predicting loss-from-care.The overall adjusted mortality rate for TDF was 9.2 per 100 PYRs , for d4T was 17.8 per 100 PYRs , and for AZT was 11.1 per 100 PYRs . In adjusted proportional hazards modeling, the hazard ratio was 2.7  for d4T and 1.4  for AZT when compared to TDF .In a sensitivity analysis, when restricting to workplace sites, the adjusted hazard ratio for d4T was 1.5 (95% CI: 1.1–2.2) and for AZT was 1.2 (95% CI: 0.9–1.4) with TDF as a referent. In the community program the adjusted hazard ratio for d4T was 1.5 (95% CI: 1.3–1.8) and for AZT was 1.0 (95% CI: 0.7–1.5) with TDF as the referent agent. Thus d4T was associated with higher mortality in both program types.During the first 24 months of ART, use of TDF as part of a first-line regimen was associated with lower rates of single drug substitution than either 30 mg of d4T or AZT. In addition, mortality appeared lower for TDF and AZT than for d4T. However, among patients alive and in-care, HIV RNA suppression was similar and loss-from-care was not consistently different by NRTI agent while CD4 count recovery was attenuated for AZT.An important strength of our analysis is that our study population received care in a large routine HIV care program. Furthermore, we had robust death ascertainment through linkage to a national vital status registry. However, use of data from routine service delivery programs also has limitations, including missing data, the potential for secular trends, and possible indication bias. For example, we had incomplete data on suspected adverse reactions. Thus we did not attempt an analysis based on these data and instead used the proxy of single-drug substitution to indicate a possible adverse event. Although single-drug substitutions may have occurred for other reasons, we believe the majority of these substitutions occurred as a result of suspected side effects, as the only reason for a single drug NRTI substitution provided in the treatment guidelines was for side effects. Missing HIV RNA data may have affected our virologic suppression analysis; however, we do not suspect a bias in HIV RNA enumeration by NRTI agent. Another limitation is lack of adherence data; however, such data are difficult to accurately and consistently collect in routine care settings.Most importantly, confounding by indication is an inherent problem when analyzing cohort data gathered from multiple sites with changes in care over time. We have attempted to address the potential of time-effect bias in our results through adjusting for ART initiation year by either stratification in the Cox proportional hazards analysis or including it as a random effect in the mixed effects models. More importantly, there is a substantial risk of bias due to inclusion of patients from workplace and community settings as ART regimens and patients differed considerably between the settings. To address this limitation, we adjusted for program or clinic in all analyses. In addition, we further assessed for marked variation in results through sensitivity analyses limited to either the workplace or community setting. We are reassured by our findings of overall similar direction of effect when restricting the analyses; however, the effect size varied. We believe that our sensitivity analyses help to validate the overall direction of associations, although the specific effect sizes may be imprecise and population dependent. The notable discrepancy was with loss-from-care in which NRTI agent was statistically associated with loss-from-care in the full analysis but lost association in the sensitivity analyses. The reason for this remained unclear even after review of clinic chart abstractions to identify reasons for loss-from-care that may not have been indicated by routinely abstracted data. Further studies of loss-from-care, by ART regimen, from other programs would be of value.Our study findings were similar, in terms of single drug substitutions, to a study from Zambia comparing TDF, d4T, and AZT Neither the Zambian nor the Lesotho studies identified differences in loss-from-care by regimen. Drug tolerability is a reported reason for discontinuation of care Our mortality findings were also similar to the Zambian and Lesotho studies in which d4T was associated with an increased mortality hazard. Both AZT and d4T were associated with a higher mortality in the Lesotho study We did not observe a difference in HIV RNA suppression by NRTI at 24 months. The comparable studies from Zambia and Lesotho did not assess this outcome as HIV RNA enumeration was not part of routine ART care in those countries. This finding suggests that among patients remaining in-care and attending clinic sessions, agent efficacy was similar. In addition, there was no evidence that the worse outcomes with d4T were mediated through a lower rate of HIV RNA suppression (or adherence as estimated by HIV RNA suppression).3Although HIV RNA suppression did not differ by NRTI, CD4 count slope was slightly less for patients receiving AZT. This is consistent with prior AZT experience, although the absolute difference in slope is less pronounced than reported from several clinical trials in which the differences were approximately 30 cells/mmIn our cohort, TDF appeared to outperform d4T at the 30 mg dosing and AZT in terms of need for drug substitution and all-cause mortality. From a public health standpoint, fewer drug substitutions may be important for program success and controlling costs. We believe that our results add to the data supporting the public health use of TDF as part of a first-line regimen, as recommended by the WHO. Our findings also suggest that, even at the current lower dose of d4T, the agent continues to have adverse effects leading to single-drug substitutions and may be contributing to increased losses from care and mortality. Longer-term evaluations of these regimens are needed.


HIV-1/HAART-associated lipodystrophy syndrome (HALS) has been associated with exposure to stavudine (d4T) through mitochondrial dysfunction. We performed a 48-week study to assess the effects of switching from d4T to raltegravir (RAL) on metabolic and fat molecular parameters of patients with HALS. Forty-two patients with HALS and a median exposure to d4T > 7 years were switched to RAL and followed for 48 weeks. Fasting metabolic tests, HIV RNA, CD4 cell count, and fat measured by DEXA were obtained at baseline and week 48. mtDNA and gene transcripts for PPAR gamma, adiponectin, cytochrome b, Cox IV, TNF alpha, MCP-1 and CD68 were assessed in paired subcutaneous fat tissue biopsies. Lipid parameters, fasting glucose, insulin, and HOMA-IR did not change significantly. Whole body fat (P = 0.0027) and limb fat mass (P<0.0001) increased from baseline. Trunk/limb fat ratio (P = 0.0022), fat mass ratio (P = 0.0020), fat mass index (P = 0.0011) and percent leg fat normalized to BMI (P<0.0001) improved after 48 weeks. Relative abundance of mtDNA, expression of PPAR gamma, adiponectin, Cyt b, and MCP-1 genes increased, whereas Cox IV, TNF alpha, and CD68 did not change significantly from baseline. Switching from d4T to RAL in patients with HALS is associated with an increase in limb fat mass and an improvement in markers of adipocyte differentiation and mitochondrial function in SAT. The landscape of HIV-1 infection has been changed forever by the availability, in developed countries, of highly active antiretroviral therapy (HAART) since 1996. However, the doubtless efficacy of HAART is still shadowed by significant toxicity, especially long-term toxicity Management strategies for HALS have included diet modifications, switches from NRTI to other drugs and pharmacological interventions Raltegravir (RAL) is the first integrase inhibitor and it has exhibited a benign safety profile with no known deleterious effects on fat content in clinical trials Hospital de la Santa Creu I Sant Pau in Barcelona, and Hospital Clínico and Hospital de la Princesa, the latter two in Madrid. They attend a population of 3800 HIV-1-infected patients on active follow-up. Patients recruited for this study were those with HIV infection, treated with a d4T-based antiretroviral regimen who had clinically-evident HALS. Exclusion criteria included previous use of RAL, including failure with a RAL-based regimen, and the absence of HALS. At baseline, d4T was switched to RAL whereas the other two drugs of the regimen remained unchanged until week 48.All patients for this observational study were recruited through three HIV-1 infection clinics, between July 2008 and December 2009, at the At the time of study entry no patient used any other drug known to influence glucose metabolism or fat distribution such as anabolic hormones or systemic corticosteroids, uridine, recombinant human growth hormone, or appetite stimulants. The diagnosis of AIDS was based on the 1993 revised case definition of the Centers for Disease Control and Prevention Subjects were weighed on calibrated scales after removing shoes, outdoor clothing, and other heavy items. Body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters. Waist circumference was measured to the nearest millimeter using anatomical landmarks as defined for the Third National Health and Nutrition Evaluation Survey Whole body dual energy X-ray absorptiometry (DEXA) scans  were conducted by a single operator at each site on all the patients. Instruments were calibrated daily. The percentage fat at the arms, legs and central abdomen  as well as total lean body mass in kilograms was recorded. All DEXA scans were centrally read by the same evaluator , blind to patient therapy. We measured fat symmetry distribution, the following fat ratios were analyzed, trunk/limb fat ratio by dividing trunk fat by limb fat Lipoatrophy was identified by patient self-reporting and/or clinician observation of relevant changes to the face (loss of cheek and/or preauricular fat pads), arms, legs, and subcutaneous abdominal tissue, and confirmed clinically by qualitative physical examination. Lipohypertrophy was diagnosed by the presence of central adiposity which was defined by a waist-hip ratio (WHR) of >0.90 in men, and >0.80 in women) Visual aspects of HALS were assessed with the Lipodystrophy Severity Grading Scale (LSGS) based on that reported by Lichtenstein et al. All laboratory investigations were performed after a 12 hour overnight fast and at least 15 minutes after the placement of a peripheral intravenous catheter. Patients were seated during blood drawing and abstained from smoking at least 15 minutes before sampling; tourniquet use was avoided when possible and, if not, was maintained for less than 1 minute. All lipid measurements were performed in a Hitachi 911 system from Roche . Serum total cholesterol and triglycerides were measured by fully enzymatic standard method and high-density lipoprotein (HDL) cholesterol by a direct method using polyethylene glycol modified enzymes (PEGME) Fat tissue samples were retrieved at baseline and 48 weeks after switching. They were obtained from SAT abdominal depot through a small surgical biopsy performed by an 8 mm punch under local anesthesia with mepivacaine. The SAT obtained was immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction (see below). The amount of mitochondrial DNA was determined as previously reported Data are expressed as median with inter-quartile range (IQR) or as frequencies and percentages or otherwise specified. Continuous variables were assessed with the Wilcoxon signed-rank test and the Mann-Whitney test for dependent and independent data, respectively. Categorical data were compared by use of the Fisher's exact test. Differences between baseline and 48-week data were assessed with paired t test. The level of significance was established at 0.05 and all reported P values are two-sided. All analyses were performed with the SAS version 9.2 software . A logistic regression analysis was used to examine the association of baseline parameters with limb fat gain ≥800 grams over 48 weeks; variables associated with a P<0.1 in the bivariate analyses were included in the multivariate stepwise analysis.Forty-two Caucasian patients were recruited. There were 32 men (76.2%) and 10 women (23.8%), with a mean age of 45.3±7.43 years (median: 45.6 years [IQR: 41.5–50.0]). Demographic, viro-immunological, metabolic and fat parameters of these patients were not different with respect to recruitment center. Prior AIDS-defining conditions were present in more than 60% of the patients. The demographics, virological and immunological parameters are shown in Most of the patients were highly antiretroviral-experienced with a median prior exposure to 5 (IQR: 3-6) different combination antiretroviral regimens. The d4T-based regimen was a salvage regimen for 62% of patients. Antiretroviral drug exposure is summarized in Changes in metabolic and fat parameters are summarized in All the patients had clinically-evident HALS. All the patients had lipoatrophy, but in addition 20 (47.6%) presented also with lipohypertrophy. No correlation between appendicular fat at baseline and prior exposure to d4T both in months and grams  was found. Seventeen patients (40.5%) at baseline and 11 (26.2%) at week 48 had <3 kg of limb fat . The median LSGS score at baseline was 8 (IQR: 8-12) and 8 (IQR: 7-10) at week 48 (P = 0.0070). The median facial score was 2 (IQR: 2-3) at 2 (IQR: 2-2) at week 48 (P = 0.0049). The median limb fat gain at 48 weeks was 800 grams (IQR: 270-1400). Whole body fat and limb fat mass increased significantly . The medA logistic regression analysis was performed taking increase in limb fat (< or ≥800 g) as the dependent variable and as independent all those baseline variables with a P<0.1 in univariate analyses , and basPaired fat biopsies were available for 18 patients. Demographic, viro-immunological, metabolic and fat parameters of these patients were not different from the whole study group. PPAR gamma and adiponectin gene expression significantly increased from baseline . The relOur study shows that patients with long-lasting exposure to d4T, who have developed HALS, experienced a significant limb fat gain after switching to RAL. Consequently, indexes of body fat distribution symmetry also showed improvement after switching. Furthermore, fat gains were accompanied by improvements in gene markers of adipocyte differentiation and mitochondrial function at SAT level. Unfortunately, the objective fat gain did not translate into a benefit perceived by the patient, as occurs in other studies In vitro studies do not show a complete absence of toxicity on mitochondria for either ABC or TDF ex vivo sub-study of ACTG 5224s showed a decrease in mtDNA fat content in patients on ABC- and TDF-based regimens Switching from thymidine analogues to mitochondrially-friendly NRTI has been a popular strategy in recent years for lipoatrophy reversal. The rationale for this strategy is the well-known mitochondrial toxicity of thymidine analogues and the apparent lack of this toxicity of ABC and TDF In our study two baseline variables were independently associated with a greater limb fat recovery, these being the length of HIV infection and baseline left leg fat. This suggests a concerted action of both the deleterious effects of HIV infection and prolonged exposure to antiretrovirals on the ability of SAT to recover. This is in line with the fact that HIV and antiretroviral drugs are able to affect biology and function not only of adipocytes but also of adipocyte precursor cells There have been two switching clinical trials with RAL We did not see any improvement in terms of dyslipidemia or insulin sensitivity. The use of d4T has been associated with hypercholesterolemia, hypertriglyceridemia and insulin resistance in vitro models and in ex vivo fat samples The molecular findings of our study are in line with known drug side-effects and also with the observed fat changes. The well-known mitochondrial toxicity of d4T is exemplified by the significant increase in mtDNA and Cyt b, a subunit of the respiratory mitochondrial chain encoded by mtDNA Inflammation is a powerful force in the pathogenesis of HALS Our study has inherent limitations. First, it is an observational study and therefore no causal relationship should or must be drawn. The lack of control arm is an important limitation of the study, but the evidence linking d4T exposure and HALS was so overwhelming when the study was designed, that having a control arm was not considered for ethical reasons In summary, switching from d4T to RAL in patients with HALS induces a remarkable increase in limb fat mass with and improvement of molecular markers of adipocyte differentiation and mitochondrial function in SAT, without beneficial effects on dyslipidemia or insulin sensitivity.