Determinants of residual viraemia during combination HIV treatment: Impacts of baseline HIV RNA levels and treatment choice.

OBJECTIVES: Effective HIV therapy reflects suppression of plasma HIV RNA levels below assay detection thresholds, although lower levels of "residual viraemia" have also been demonstrated over extended periods of effective antiretroviral treatment. Here we examine the determinants of HIV RNA suppression below the standard assay threshold (40 HIV-1 RNA copies/mL) as well as factors associated with detectable HIV RNA below this reported detection limit. METHODS: Between 2007 and 2010, 11 575 consecutive viral load (VL) tests were obtained from 1540 patients, including 356 on effective antiretroviral therapy followed since initiation (1996-2001: n = 165; 2002-2009: n = 191). Analyses modelled the probability of an undetectable VL given successful suppression to < 200 copies/mL, and the probability of residual viraemia given an undetectable result. RESULTS: Detectable HIV RNA amplification was demonstrated in 20% of samples with a VL result < 40 copies/mL. Longitudinal analyses from 356 patients revealed that the likelihood of achieving results < 40 copies/mL was increased with current nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy [odds ratio (OR) 2.0; P < 0.05] and reduced with prior virological rebound (OR 0.5; P < 0.05). In contrast, the presence of detectable HIV RNA < 40 copies/mL was strongly associated with pretreatment HIV RNA levels among those on current protease inhibitor (PI) treatment (OR 1.5 per log10 copies/mL increase; P = 0.02) as well as those on NNRTIs (OR 1.7; P = 0.002). CONCLUSIONS: While HIV treatment history was associated with plasma HIV RNA levels below the detection limit, residual viraemia results were dominantly determined by pretreatment VL. These findings support the concept of a stable, long-lived reservoir of latently infected cells as a source of residual viraemia despite effective HIV treatment.

Association between 25-OH vitamin D and insulin is independent of lipoatrophy in HIV.

OBJECTIVE: Vitamin D deficiency (VDD) is prevalent in HIV, and following antiretroviral therapy (ART), increased rates of lipoatrophy and metabolic abnormalities are described. We investigated the relationships between 25-hydroxyvitamin D [25(OH)D] and other metabolic parameters in a group of HIV patients with and without lipoatrophy to examine whether lipoatrophy could explain the high prevalence of VDD and metabolic abnormalities. BACKGROUND: Vitamin D receptors are expressed in adipose tissue implicating vitamin D, through paracrine/autocrine mechanism, in exerting effects on fat metabolism. HIV patients frequently suffer from VDD, and those treated with thymidine analogues frequently suffer from lipoatrophy so we investigated whether lipoatrophy could explain these associations. DESIGN AND PATIENTS: Cross-sectional study of HIV-infected male patients (n = 107; 39 with lipoatrophy) from the West Australian cohort with measurements of 25(OH)D, adiponectin, insulin, lipids and leg fat as a percentage of mass. RESULTS: Reduced 25(OH)D levels were common and significantly associated with higher serum insulin in the entire cohort (P = 0·006), but there was no difference in 25(OH)D between untreated and antiretroviral-treated patients with or without lipoatrophy. Treated patients with lipoatrophy were more likely to take thymidine analogue therapy, were older and on therapy longer than treated patients without lipoatrophy. Adiponectin levels did not correlate with 25(OH)D, but lipoatrophic-treated patients had lower levels of adiponectin compared with nonlipoatrophic-treated patients. CONCLUSIONS: Lower 25(OH)D is associated with higher serum insulin but not lipoatrophy or hypoadiponectinemia in HIV-infected patients. The association between VDD and insulin resistance is likely to be mediated by independent mechanisms.

Evaluation of IL12B as a candidate type I diabetes susceptibility gene using data from the Type I Diabetes Genetics Consortium.

As part of its efforts to identify genes affecting the risk of type I diabetes (T1D), the Type I Diabetes Genetics Consortium commissioned an extensive survey of variants associated with genes reported earlier to have an association with disease susceptibility. In this report, we present the analysis of a set of single-nucleotide polymorphisms (SNPs) within and flanking the IL12B gene, which encodes the p40 subunit of the cytokines interleukin (IL)-12 and IL-23. No SNP showed individually significant association in the population as a whole. Nevertheless, subjects stratified according to genotype at the earlier reported SNP in the IL12B 3'UTR, rs3212227, confirmed small, but significant, differences in age of disease onset with a relative hazard=0.88 (P=0.005). The protective effect of rs3212227 allele 2 was gender specific (P=0.004 overall and P=0.0003 when unaffected siblings were considered). Among females, the 2.2 genotype was more protective, with relative hazard=0.75. We conclude that while there was no major effect of IL12B polymorphisms on T1D susceptibility in the entire study group, they have an impact on a subset of at-risk individuals.

Effect of linkage status of affected sib-pairs on the search for novel type 1 diabetes susceptibility genes in the HLA complex.

AIM: Type 1 diabetes susceptibility is influenced by a number of genes, of which those with the strongest effects map to the human leucocyte antigen (HLA) complex. Evidence for linkage of non-HLA genes in several affected sib-pair analyses was increased if the HLA or gender status of the sibs was considered independently. We investigated whether linkage status at the HLA complex differentially affected transmission of alleles to sibs depending on their HLA or gender linkage status. METHODS: Genotypes of 2437 markers typed on 11 279 individuals from 2363 families, stratified according to HLA and gender, were examined using the transmission disequilibrium test. RESULTS: Several significant single nucleotide polymorphisms (SNPs) loci were found near the class II genes; no significant SNPs were found by these analyses near the class I or class II genes. Other significant effects were found when the gender of the sibs or the parents was considered. There was not a significant difference in HLA-DRB genotypes between the stratified sets. CONCLUSIONS: These results suggest the presence of novel recessive susceptibility gene(s) within the HLA complex.

Association of MHC SNP genotype with susceptibility to type 1 diabetes: a modified survival approach.

AIM: The Major Histocompatibility Complex (MHC) is a highly polymorphic region on chromosome 6 encompassing the human leucocyte antigen (HLA)-DQ/DR loci most predictive of susceptibility to type 1 diabetes (T1D). To assess the contribution of other MHC genes, in this exploratory analysis of Type 1 Diabetes Genetics Consortium (T1DGC) family data we characterize association between susceptibility and MHC single nucleotide polymorphism (SNP) genotype, with an emphasis on effects of genetic variation additional to carriage of predisposing or protective MHC haplotypes. METHODS: We use Cox regression analyses of age of onset, stratified by family, to jointly test both linkage and association. Analysis is restricted to children from families having both affected and unaffected siblings and is conducted with and without adjustment for known HLA class I and II effects. Model fits provide scores for each individual that are based on estimates of the probability of being affected by the age of 35, given the individual's SNP genotype. The mean within-family variation in these scores provides a measure that closely reflects the relative size of the likelihood ratio test statistics, and their covariation provides a means of mapping patterns of association that incorporate both effect size and commonality of effect that is attributable to the strong linkage disequilibrium (LD) extending across the region. RESULTS: Univariate analyses yielded strong associations with T1D susceptibility that are dominated by SNPs in the class II HLA-DR/DQ region but extend across the MHC. Similar effects are frequently observed across SNPs within multiple genes, sometimes spanning hundreds of kilobases. SNPs within a region at the telomeric end of the class II gene HLA-DRA yielded significant associations with and without adjustment for carriage of the predictive DR3, DR4, DR2 and DR7 HLA haplotypes, and remained highly prominent in a secondary analysis that was restricted to 66 families in whom at least one of the affected siblings carried neither the DR3 nor DR4 haplotype. CONCLUSIONS: While many of the associations can be attributed to LD between the SNPs and the dominant HLA-DRB/DQA/DQB loci, there is also evidence of additional modifying effects.

Missingness in the T1DGC MHC fine-mapping SNP data: association with HLA genotype and potential influence on genetic association studies.

AIM: The absence or 'missingness' of single nucleotide polymorphism (SNP) assay values because of genotype or related factors of interest may bias association and other studies. Missingness was determined for the Type 1 Diabetes Genetics Consortium (T1DGC) Major Histocompatibility Complex (MHC) data and was found to vary across the region, ranging up to 11.1% of the non-null proband SNPs, with a median of 0.3%. We consider factors related to missingness in the T1DGC data and briefly assess its possible influence on association studies. METHODS: We assessed associations of missingness in the SNP assay data with human leucocyte antigen (HLA) genotype of the individual and with SNP genotypes of the parents. Within-cohort analyses were combined (over all cohorts) using (i) Mantel-Haenszel tests for two-by-two tables or (ii) by combining test statistics for larger tables and regression models. Mixed effect regression models were used to assess association of the SNP genotypes with affected status of the offspring after adjustment for parental SNP genotypes, cohort membership and HLA genotypes. Log-linear models were used to assess association of missingness in the unaffected sib assays with SNP genotypes of the probands. RESULTS: Missingness of SNP values near the HLA class I (A, B and C) and class II (DR, DQ and DP) loci is strongly associated with carriage of corresponding HLA genotypes within these groups. Similar associations pertain to missing values among the microsatellite data. In at least some of these cases, regions of missingness coincided with known deletion regions corresponding to the associated HLA haplotype. We conjecture that other regions of associated missingness may point to similar haplotypic deletions. Analysis of association patterns of SNP genotypes with affected status of offspring does not indicate strong informative missingness. However, association of missingness in proband data with parental SNP genotypes may impact transmission disequilibrium test (TDT)-type analyses. Comparisons of affected and unaffected siblings point to possible susceptibility regions additional to the classical HLA-DR3/4 alleles near BAT4-LY6G5B-BAT5 and NOTCH4. CONCLUSIONS: Potentially informative missingness in SNP assay values in the MHC region may impact on association and related analyses based on the T1DGC data. These results suggest that it would be prudent to assess the degree to which missingness may abrogate assessed SNP disease markers in such studies. Initial analyses based on comparison of affected and unaffected status in offspring suggest that at least these may be little affected.

Evidence-based, multifactorial approach to addressing non-adherence to antiretroviral therapy and improving standards of care.

BACKGROUND: Near-perfect adherence to antiretroviral therapy over time is critical to achieve viral suppression and recovery of functional immunity in individuals infected with HIV. The concept of adherence as a dynamic behaviour influenced by multiple biopsychosocial factors motivated us to implement an integrated, multifactorial programme in our hospital-based setting. The aims of this study were to survey the scope and determinants of non-adherence in patients attending the Ambulatory HIV Service at Royal Perth Hospital, to develop a method for longitudinal monitoring and to implement measures tailored to support individuals. METHODS: The US Adult AIDS Clinical Trials Group self-report baseline adherence, follow-up and side-effect questionnaires were used to survey 247 patients at two time-points between September 2002 and February 2003. A longitudinal monitoring method was developed and the WA HIV Cohort Study database used to collate results with clinical markers up to December 2005. RESULTS: Adherence was associated with viral suppression and CD4 T-cell recovery and improved over the 3-year period under observation (all P < 0.001). Diminishing adherence was associated with younger age (P = 0.002), substance use (P < 0.01), perceived stress (P = 0.04) and indicators of depression (P = 0.03). The analyses showed relationships between personal experience of side-effects and the depression indicator scale in patients on antiretroviral therapy. CONCLUSION: The programme resulted in an improvement in adherence in our cohort even after adjusting for pill burden, dosing frequency and highly active antiretroviral therapy regimen and has enhanced focus on patients vulnerable to non-adherence while supporting those not currently at risk.

Influence of inhibitory killer immunoglobulin-like receptors and their HLA-C ligands on resolving hepatitis C virus infection.

An estimated 2%-3% of the world's population is chronically infected with hepatitis C virus (HCV) and this is a major cause of liver disease worldwide. Following acute infection, outcome is variable with acute HCV successfully resolved in some individuals (20%-30%), but in the majority of cases the virus is able to persist. Co-infection with human immunodeficiency virus has been associated with a negative impact on the course of HCV infection. The host's immune response is an important correlate of HCV infection outcome and disease progression. Natural killer (NK) cells provide a major component of the antiviral immune response by recognising and killing virally infected cells. NK cells modulate their activity through a combination of inhibitory and activatory receptors such as the killer immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen (HLA) Class I molecules. In this workshop component, we addressed the influence of KIR genotypes and their HLA ligands on resolving HCV infection and we discuss the implications of the results of the study of Lopez-Vazquez et al. on KIR and HCV disease progression.

Prospective genetic screening decreases the incidence of abacavir hypersensitivity reactions in the Western Australian HIV cohort study.

Abacavir therapy is associated with significant drug hypersensitivity in approximately 8% of recipients, with retrospective studies indicating a strong genetic association with the HLA-B*5701 allele. In this prospective study, involving 260 abacavir-naive individuals (7.7% of whom were positive for HLA-B*5701), we confirm the usefulness of genetic risk stratification, with no cases of abacavir hypersensitivity among 148 HLA-B*5701-negative recipients.

The effective life of ivermectin on Western Australian sheep farms--a survival analysis.

A mail survey of 235 Western Australian sheep farmers who had performed faecal egg count reduction tests for anthelmintic resistance in 1999 or 2000 was conducted, with some telephone follow-up. A response of 56% was achieved. Resistance to ivermectin, a member of the macrocyclic lactone class of anthelmintics, had developed on 44% of the farms surveyed. We used time to occurrence of resistance to ascertain factors that contributed to extending the time ivermectin remained an effective drench on these farms (median time=10.5 years). This time was significantly longer when farmers implemented more worm control practices on their farms (P=0.003). We developed a multivariable survival model that contained the following main effects: reduced winter drenching frequency, 0-2 flock treatments in 5 years (hazard ratio (HR) 0.52); availability of alternative effective anthelmintic classes on the farm (HR 0.30); always using safe pastures (HR 0.23); and veterinarians as the primary source of worm control advice (HR 0.58). The relationship of these findings to the understanding of anthelmintic resistance is discussed.

Killer immunoglobulin-like receptors and HLA act both independently and synergistically to modify HIV disease progression.

Variation in the host response to infection by pathogens including HIV-1 may be conferred by polymorphic genetic factors such as HLA and killer immunoglobulin-like receptors (KIR) genes. Here, we examined KIR and HLA genotype effects on pretreatment viral load, rate of CD4(+) T-cell decline and progression to AIDS among adult HIV-1-infected patients within the Western Australian HIV Study Cohort. In this study, carriage of KIR genes within the 'B' haplotype (eg KIR2DS2) was specifically associated with a more rapid CD4(+) T-cell decline over time and progression to AIDS. In contrast, KIR gene repertoire had no effect on pretreatment viral load while selected HLA alleles (eg HLA-B*5701, HLA-B*2705) demonstrated significant protective effects on viremia. Furthermore, interactions between specific HLA and KIR genes did appear to influence HIV disease progression. The results suggest that host genetic variation within the HLA and KIR gene complexes have clinically relevant effects on the course of HIV-1/AIDS, acting independently as well as synergistically to modify disease progression at multiple levels.

Sorption and mobility of 14C-labeled imazaquin and metolachlor in four soils as influenced by soil properties.

Aqueous batch-type sorption-desorption studies and soil column leaching studies were conducted to determine the influence of soil properties, soil and suspension pH, and ionic concentration on the retention, release, and mobility of [14C]imazaquin in Cape Fear sandy clay loam, Norfolk loamy sand, Rion sandy loam, and Webster clay loam. Sorption of [14C]metolachlor was also included as a reference standard. L-type sorption isotherms, which were well described by the Freundlich equation, were observed for both compounds on all soils. Metolachlor was sorbed to soils in amounts 2-8 times that of imazaquin, and retention of both herbicides was related to soil organic matter (OM) and humic matter (HM) contents and to herbicide concentration. Metolachlor retention was also related to soil clay content. Imazaquin sorption to one soil (Cape Fear) increased as concentration increased and as suspension pH decreased, with maximum sorption occurring in the vicinity of pK(a1) = (1.8). At pH levels below pK(a1) imazaquin sorption decreased as hydronium ions (H3O+) increased and competed for sites. NaCl was more effective than water in desorption of imazaquin at pH levels near the pK(a1). Mechanisms of bonding are postulated and discussed. The mobility of imazaquin through soil columns was in the order Rion > or = Norfolk > Cape Fear > or = Webster, whereas for metolachlor it was Rion > or = Norfolk >> Webster > or = Cape Fear. Imazaquin was from 2 to 10 times as mobile as metolachlor.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on primitive hematopoietic stem cell (PHSC) function and numbers, after chemotherapy.

OBJECTIVE: Primitive hematopoietic stem cell function was assessed after cyclophosphamide with granulocyte-macrophage colony-stimulating factor (GM-CSF), with or without preadministration of interleukin-1, using competitive repopulation. METHODS: C57B6/J mice injected with one or four biweekly intravenous injections of cyclophosphamide, 200 mg/kg, received granulocyte-macrophage colony-stimulating factor, 1 microg, subcutaneously for 5 days, beginning 24 hours after cyclophosphamide. Alternatively, mice were injected with interleukin-1, 1 microg, 20 hours before administration of drug or drug and cytokine. Marrow obtained from mice sacrificed 4 weeks after the last dose of drug or drug and cytokine was used in competitive repopulation. RESULTS: Significant reductions in marrow repopulating ability occurred after a single dose of cyclophosphamide or multiple injections. Repopulating units (RU) were calculated, and both binomial and Poisson models for estimation of primitive hematopoietic stem cell (PHSC) numbers were used. RU were significantly diminished for all treatment groups when compared to controls. PHSC numbers were not significantly affected by either regimen of cyclophosphamide given alone. Addition of GM-CSF to cyclophosphamide, whether the latter was given in single or multiple doses, led to further, although insignificant, declines in repopulating ability, as well as PHSC and RU numbers. Interleukin-1 usage exacerbated the observed repopulating defect. There was evidence of replicative failure in individual cells, indicating a qualitative defect also. SUMMARY: Additive stem cell depletion and qualitative replicative defect occur after chemotherapy-cytokine usage. However, the replicative defect of PHSC seen after addition of GM-CSF is not significantly worse than that seen with cytotoxic drug use alone.

Analysis of the stem cell sparing properties of cyclophosphamide.

OBJECTIVES: Cyclophosphamide was examined for its ability to spare the most primitive hematopoietic stem cell (PHSC). METHODS: C57BL6/J mice (Groups A and B) were sacrificed 24 h and 4-6 wk, respectively, after a single or second injection of low-dose cyclophosphamide (90 mg/kg) on days 1, 3, 7, or 15. A competitive repopulation assay was then performed, using B6-HbbdGpi-1a competitor cells, to determine the repopulating ability of exposed PHSC. RESULTS AND CONCLUSIONS: PHSC function was preserved after a single injection of cyclophosphamide and after a second injection on days 7 and 15 in both groups. In Group A, PHSC repopulating ability declined after a second injection on days 1 and 3 (p<0.05 only for day 1), as did repopulating units [RU]; PHSC numbers did not. In Group B, an insignificant decrease in repopulating ability and RU numbers was observed after a second injection on days 1 and 3, suggesting different etiologies for losses in the 2 groups, or correction of drug-induced defects within 1 month of cyclophosphamide administration. Total RU increased in single, day 1, 7 and 15 treatment groups. A significant number of marrow cells entered the S phase after cyclophosphamide dosing on day 3, and it is possible that a relationship exists between cell cycling and replicative damage. DNA damage was also increased 1 and 3 d after cyclophosphamide administration, although the significance of differences from controls was not definitive. CONCLUSION: Low-dose cyclophosphamide can spare stem cells, depending upon the timing of subsequent doses.

Stable or increasing bone mineral density in HIV-infected patients treated with nelfinavir or indinavir.

BACKGROUND AND OBJECTIVES: To determine the factors contributing to changes in bone mineral density (BMD) over time in HIV-infected patients receiving highly active antiretroviral therapy (HAART). METHODS: Analyses of lumbar spine BMD in 183 male Caucasian participants in the Western Australian HIV Cohort study, comprising a longitudinal analysis of data from 54 patients on stable HAART regimens, and a cross-sectional analysis comparing data from 131 protease inhibitor (PI)-treated patients and 52 PI-naive (including 28 antiretroviral treatment-naive) patients. RESULTS: Average lumbar spine BMD remained stable or increased over the time frame considered. Although there was no evidence of a change of average BMD over time in patients receiving nelfinavir (P = 0.92), there was evidence of increasing bone density in the indinavir group (average increase, 0.31 z-score per year; P < 0.001). Lower initial z-scores in the longitudinal analysis were significantly associated with lower pre-HAART BMI (P = 0.003), consistent with results of the cross-sectional analysis in which lowest BMI prior to initial dual X-ray absorptiometry scan was associated with decreased BMD (P = 0.02, overall group). Although PI therapy was also associated with decreased BMD in a univariate analysis of the cross-sectional data (P = 0.04), this effect was abrogated in a multiple linear regression analysis (P = 0.11) with lowest BMI remaining significant (P = 0.04). CONCLUSIONS: We found no evidence, overall, of accelerated bone loss in patients treated with nelfinavir- or indinavir-containing HAART regimens, and propose that indinavir therapy may be associated with an increase in bone mineral density over time. Pre-HAART BMI was an independent and powerful determinant of an individual's initial z-score in the longitudinal analysis, and adjustment for this effect in a cross-sectional analysis abrogated the association between PI therapy and decreased lumbar spine z-score.

Chronic hyperlactatemia in HIV-infected patients taking antiretroviral therapy.

OBJECTIVE: To determine the prevalence, course and risk factors for hyperlactatemia in HIV-infected patients. DESIGN: A prospective, longitudinal study of venous lactate concentrations over an 18-month period in 349 participants of the Western Australian HIV Cohort Study. RESULTS: In 516 patient-years of observation, two patients experienced severe fulminant lactic acidosis (lactate > 5 mmol/l) and hepatic steatosis attributable to nucleoside analogue reverse transcriptase inhibitors (NRTI). A further five patients with lesser elevations of lactate (2.8-4.1 mmol/l) but with symptoms of nausea or abdominal discomfort and evidence of hepatic steatosis had NRTI therapy revised, with relief of symptoms and a fall in lactate levels. Most remaining patients on highly active antiretroviral therapy (HAART) had mild, chronic, asymptomatic hyperlactatemia, with mean lactate level between 1.5 mmol/l and 3.5 mmol/l most commonly. Longitudinal data was analysed in a non-linear mixed effects growth model which indicated that average lactate levels rose after the start of HAART but tended to stabilise at low-grade elevation, with an average 0.23 mmol/l greater long term level in stavudine users compared with zidovudine users (p < 0.01). A multiple linear regression model showed that the association between stavudine and higher lactate level was not confounded by longer duration of total NRTI exposure. Risk of hyperlactatemia was not significantly associated with use of other NRTIs, protease inhibitors, non-nucleoside analogue reverse transcriptase inhibitors or multiple immunological and virological factors in multivariate analyses. CONCLUSIONS: Chronic, compensated, asymptomatic hyperlactatemia is common in patients taking HAART. Decompensated, life-threatening lactic acidosis/hepatic steatosis is rare. Treatment with stavudine appears to be the predominant risk factor for development of chronic hyperlactatemia.

Contribution of nucleoside analogue reverse transcriptase inhibitors to subcutaneous fat wasting in patients with HIV infection.

BACKGROUND: Progressive subcutaneous fat wasting, fat accumulation, dyslipidaemia and insulin resistance in HIV-infected patients on antiretroviral therapy has been attributed to the long-term toxicity of HIV protease inhibitors (PI). More recently, fat wasting has been observed in patients who have never taken a PI, implicating an independent effect of nucleoside analogue reverse transcriptase inhibitor (NRTI) therapy. OBJECTIVES: To determine the relative contribution of NRTI and PI, as well as any other factors, to fat wasting in HIV-infected patients. DESIGN: Longitudinal cohort study involving 277 participants of the Western Australian HIV Cohort Study. METHODS: The time to onset of clinically apparent fat wasting in patients receiving different antiretroviral regimens was compared using standardized clinical criteria. Regional fat measured by dual energy X-ray absorptiometry (DEXA) in 161 patients was also compared. The average rate of percentage fat reduction was estimated in 70 patients who had consecutive DEXA scans at approximately 6-monthly intervals. Multiple confounding factors were considered in the analyses. RESULTS: Progressive subcutaneous fat wasting, indistinguishable from that described in PI-treated patients, does occur in PI-naive, NRTI-treated patients. In patients taking triple combination antiretroviral therapy, age (relative risk = 1.052 per year; P < 0.0001), white race (relative risk = 3.9; P = 0.023), longer duration of dual NRTI therapy prior to addition of PI (relative risk = 1.021 per month; P = 0.0046) and increased cumulative time on stavudine-containing regimens compared with time on zidovudine-containing regimens (relative risk = 1.085 per month; P < 0.0001) are associated with increased risk of fat wasting. Stavudine increases the risk of fat wasting by 265% per year compared with zidovudine. However PI therapy is associated with faster progression to clinically apparent wasting compared with dual NRTI therapy without PI. The results of DEXA scanning supports these clinical data and suggest a non-linear decline in fat over time. CONCLUSIONS: NRTIs do have an independent contribution to fat wasting, but PI are the predominant influence and may act synergistically with NRTIs. NRTIs appear to predispose individuals to slowly progressive fat loss, which is markedly accelerated when a PI and NRTIs are combined. Of the NRTIs, stavudine leads to an earlier onset of clinically apparent fat wasting compared with zidovudine. Fat wasting associated with NRTI use may be a manifestation of mitochondrial toxicity, which may be exacerbated by PI use.