High rates of mortality and morbidity have been described in sub-Saharan African patients within the first few months of starting highly active antiretroviral therapy (HAART). There is limited data on the causes of early morbidity on HAART and the associated resource utilization.A cross-sectional study was conducted of medical admissions at a secondary-level hospital in Cape Town, South Africa. Patients on HAART were identified from a register and HIV-infected patients not on HAART were matched by gender, month of admission, and age group to correspond with the first admission of each case. Primary reasons for admission were determined by chart review. Direct health care costs were determined from the provider's perspective.6 cells/L (IQR 15-115). The primary reasons for admission in the HAART group were more likely to be due to adverse drug reactions and less likely to be due to AIDS events than the no-HAART group . Immune reconstitution inflammatory syndrome was the primary reason for admission in 10% of the HAART group. Lengths of hospital stay per admission and inpatient survival were not significantly different between the two groups. Five of the 15 deaths in the HAART group were due to IRIS or adverse drug reactions. Median costs per admission of diagnostic and therapeutic services  were higher in the HAART group compared with the no-HAART group , but the more expensive non-curative costs  were not significantly different .There were 53 in the HAART group with 70 admissions and 53 in the no-HAART group with 60 admissions. The median duration of HAART was 1 month (interquartile range 1-3 months). Median baseline CD4 count in the HAART group was 57 × 10Causes of early morbidity are different and more complex in HIV-infected patients on HAART. This results in greater resource utilization of diagnostic and therapeutic services. Highly active antiretroviral therapy (HAART) has dramatically improved the prognosis of HIV infection in high-income countries by decreasing mortality, AIDS illnesses, and hospitalization -3. In lo6 cells/L and median 290 × 106 cells/L) than is typical for sub-Saharan African programmes. A South African study included patients more typical of the region, but did not include IRIS as a diagnostic category [Few studies have assessed the causes of early morbidity on HAART in sub-Saharan African ART programmes. Most studies report only HIV-related morbidity ,9 or on category ,9.We conducted a cross-sectional study to determine whether the reasons for hospitalization , outcomes, and resource utilization during hospital admissions were different in HIV-infected patients on HAART compared with HIV-infected patients not on HAART in South Africa, a country with a very high HIV prevalence.The study was conducted at G.F. Jooste Hospital, a secondary-level hospital providing care for indigent patients referred from primary health-care providers and clinics in the poor urban communities of metropolitan Cape Town.6 cells/L or with clinical AIDS [A cross-sectional study was conducted to assess the spectrum of disease and resource utilization of medical admissions to G.F. Jooste Hospital among HIV-infected patients on HAART (HAART group) compared with those not on HAART (no-HAART group) during a nine-month period from July 2003 through March 2004. The patients on HAART accessed antiretroviral therapy predominantly from donor-funded programs in G.F. Jooste Hospital's catchment area, because the national public sector HAART roll-out program only commenced in 2004. The donor-funded programs included Médecins sans Frontières (MSF) at Khayelitsha ; Hannan cal AIDS .Patients in the HAART group were identified from a registry of HIV-infected patients on HAART who were admitted to the medical wards. The registry was maintained by two resident infectious disease physicians. Patients in the no-HAART group were matched by gender, month of admission, and age group  to correspond with the first admission of each patient in the HAART group. Subsequent admissions of patients in both groups during the study period were also captured.The study was approved by the University of Cape Town's Research Ethics Committee.The following data were extracted by retrospective chart review: demographic information, WHO clinical stage prior to admission, tuberculosis treatment or prophylaxis, cotrimoxazole prophylaxis, HAART regimen and date of commencement, treatment administered in hospital , investigations during hospitalization (cross-checked with the laboratory's electronic database), CD4+ cell count within six months of admission , length of stay, discharge diagnoses, discharge disposition , and survival. The data were recorded in a spreadsheet  and checked for illogical or missing entries by the database analyst. Data were analyzed with EpiInfo, version 6. Barlett's test was used for homogeneity of variance, which was shown to differ between the two groups, therefore the Kruskal-Wallis test was used to analyse differences between the outcomes and costs of the two groups.The study physician classified the primary reason for admission (identified from the discharge diagnoses and a chart review) into the following diagnostic categories: AIDS-defining events, adverse drug reactions, HIV-related conditions, and other medical conditions not necessarily related to HIV. In the WHO clinical staging classification, pulmonary tuberculosis is defined as stage 3, whilst extrapulmonary tuberculosis is stage 4 (AIDS). Because it was not always clear from discharge diagnoses which form of tuberculosis patients had, we did not separate tuberculosis by site and included all forms of tuberculosis as AIDS illnesses. HIV-related conditions were recorded primarily according to WHO clinical stages 1 to 4 . IRIS waCosting took the perspective of the health-care provider. The median cost per inpatient day was calculated separately for non-curative care costs and for patient-specific diagnostic and therapeutic costs. Patient-specific laboratory and therapeutic costs were calculated using a standard ingredients approach, which involves linking the usage of resources to the cost of each resource . As the Non-curative care costs, including recurrent overhead and hotel costs, capital costs, and clinical staff costs, were calculated by the study economist using a standard step-down approach . Hotel cClinical staff costs included salaries of doctors  and nurses . The clinical staff cost per inpatient day was calculated by establishing the full cost of employment (including benefits) of doctors and nurses together with the number of full time equivalent staff working in the medicine wards. Multiplying these two allowed us to estimate the annual cost of these clinical inputs. The clinical staff cost per inpatient day was then calculated by the formula:Annual clinical staff cost/(number of beds × average bed occupancy × 365 days per year).Patient-specific diagnostic and therapeutic costs included laboratory investigations, radiology, intravenous fluids and blood, and non-ART medications. Laboratory costs were provided by the National Health Laboratory Service. Drug costs were obtained from the hospital pharmacy price list, which is derived from the 2003 Provincial Tender Price List. Radiology and special investigations were costed from the central government's Uniform Fee Schedule (UPFS) 2002, inflated to the 2003 level using the consumer price index, less mortgage bonds ,20.All costs were converted from South African Rands to U.S. dollars at the average bank rate at the midpoint of the study (November 2003): U.S. $1 = 6.73231 Rand . The totBetween July 2003 and March 2004, 72 admissions of HIV-infected individuals on HAART met the inclusion criteria, but 2 patients' folders were lost, leaving 70 admissions analyzed . Two-hundred thirty-seven HIV-infected individuals not on HAART who had been admitted to G.F. Jooste during the study period were identified, and 204 patient folders from those admissions were found. Fifty three patients in the no-HAART group were matched with the first admission of the 53 patients in the HAART group representing 60 admissions . Five patients in the no-HAART group were matched by season of admission as no exact month match could be made.Ninety-seven percent of patients admitted on HAART were on a non-nucleoside reverse transcriptase inhibitor (NNRTI) regimen with dual NRTI. One patient was on self-funded dual NRTI antiretroviral therapy, but was included in the study in the HAART group. The median duration of antiretroviral therapy was 1 month .6 cells/L in 4 admissions . AvailaTable Table The total cost of hospitalization of all of the admissions, 1657 patient days, was US$257,033. In both groups, non-curative costs constituted the greatest proportion of total hospitalization costs . Within non-curative costs, overheads constituted the largest share of the costs . The costs of hospitalization of both groups are depicted in Table We analysed diagnoses, outcomes and resource utilization in 130 secondary hospital HIV admissions comparing patients who recently started HAART with those not on HAART in a high HIV prevalence area. We found that the primary reasons for admission were more likely to be due to adverse drug reactions and less likely to be due to AIDS events in patients on HAART. IRIS emerged as an important cause of early morbidity, accounting for 10% of the primary causes of admission in the HAART group. IRIS together with adverse drug reactions accounted for a third of all deaths in patients on HAART.The patients in our study were severely immune suppressed, as assessed by WHO clinical staging and CD4 cell counts, which is typical of patients starting HAART in Africa. We found that tuberculosis was by far the commonest diagnosis in both the HAART and no-HAART admissions. In the West African studies tuberculosis was the second commonest diagnosis ,9, but iHospitalization costs are a major component of costs for HIV care in developed countries, particularly in patients with more advanced disease . The hosOur findings have several implications. First, starting HAART at higher baseline CD4 counts would reduce the incidence of IRIS, many adverse drug reactions, and HIV-related morbidity. This would result in a shift from inpatient to outpatient HIV care, which would free substantial resources for the many other competing health care priorities in Africa. Second, the use of safer antiretroviral drugs (e.g. tenofovir or efavirenz instead of stavudine or nevirapine) will reduce morbidity as adverse drug reactions were the primary reason for admission in 34% of our HAART group. The World Health Organization has recommended this strategy in resource-limited settings, but recognizes that agents with better safety profiles are currently more costly . Third, Our study has a number of limitations. We collected data retrospectively, which is always less reliable and complete than in prospective studies. We could only detect large difference between patients in the HAART and no-HAART groups given our relatively small sample size and comparisons between the two groups should be interpreted with caution. Our study was also only limited to inpatients and should not be extrapolated to outpatients. However, detailed costing was done on all 130 admissions, which involved a considerable amount of data collection. Limitations of our cost analysis include lack of a detailed assessment of clinical staffing costs, and exclusion of the indirect and direct non-health care costs borne by patients and their families. Importantly, future cost savings from HAART, notably reduction in rates of hospitalisation, were not assessed. Our study was conducted early on in the roll out of antiretroviral therapy when the numbers of patients on HAART in our catchment area was considerably lower than it is currently, but CD4 counts at HAART initiation were lower than they are currently, which should result in lower hospitalization rates and possibly different reasons for hospitalisation. CD4 counts were discouraged on inpatients as a cost-saving measure, but were done as outpatients. Because only a minority of patients in the no-HAART group was in HIV care while all those on HAART were, we had access to CD4 counts in most of the HAART group and few of the no-HAART group. Finally, although access to diagnostic and therapeutic interventions was similar for both groups, we could not exclude the possibility that clinicians looking after patients on HAART might have been more active in investigating and treating them due to a perceived better prognosis.In conclusion, our study provides disease and economic information that may assist with the planning and delivery of comprehensive HIV/AIDS care in Africa. A high proportion of admissions for patients on HAART is due to adverse drug reactions or IRIS. Further large prospective cohort studies should be conducted to identify the nature of the early morbidity on HAART in Africa more accurately, and to identify strategies to prevent, diagnose and manage these morbidities.The authors declare that they have no competing interests.GM, KR, and GAM conceived the study. TKS assisted with the study design, collected the data, developed the database, and wrote the paper. SC, the study health economist, calculated the non-curative costs and contributed to writing the costing section. JHS analyzed the database. GM supervised the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/205/prepub


Breakdown of the gut mucosal barrier during chronic HIV infection allows translocation of bacterial products such as lipopolysaccharides (LPS) from the gut into the circulation. Microbial translocation also occurs in inflammatory bowel disease (IBD). IBD serological markers are useful in the diagnosis of IBD and to differentiate between Crohn's disease (CD) and ulcerative colitis (UC). Here, we evaluate detection of IBD serological markers in HIV-infected patients with advanced disease and their relationship to HIV disease markers.IBD serological markers  were measured by ELISA in plasma from AIDS patients (n = 26) with low CD4 counts (<300 cells/µl) and high plasma LPS levels, and results correlated with clinical data. For meta-analysis, relevant data were abstracted from 20 articles.IBD serological markers were detected in approximately 65% of AIDS patients with evidence of microbial translocation. An antibody pattern consistent with IBD was detected in 46%; of these, 75% had a CD-like pattern. Meta-analysis of data from 20 published studies on IBD serological markers in CD, UC, and non-IBD control subjects indicated that IBD serological markers are detected more frequently in AIDS patients than in non-IBD disease controls and healthy controls, but less frequently than in CD patients. There was no association between IBD serological markers and HIV disease markers  in the study cohort.IBD serological markers may provide a non-invasive approach to monitor HIV-related inflammatory gut disease. Further studies to investigate their clinical significance in HIV-infected individuals are warranted. CD4 T-cells in gut-associated lymphoid tissue (GALT) are primary target cells of HIV, and GALT is an important site for HIV replication and pathogenesis Microbial translocation is also a noted feature in inflammatory bowel disease (IBD) Saccharomyces cerevisiae antibody) E.coli) Saccharomyces cerevisiae cell wall The role of enteric microflora in IBD pathophysiology is highlighted by the presence of antibody reactivity to microbial antigens. These serological markers can be helpful to distinguish between CD and UC as well as aiding in the diagnosis of IBD along with clinical history, endoscopy, and physical examination . Small intestinal villous atrophy and enterocyte defects have been described in HIV-infected patients. Because these defects usually occur in the absence of enteric pathogens, the term HIV enteropathy has been used A number of parallels are observed between HIV and IBD in terms of gut disease We previously demonstrated that elevated circulating LPS levels correlate with monocyte activation during HIV infection, and may thereby contribute to chronic immune activation AIDS patients with CD4 counts <300 cells/µl were recruited at the Lemuel Shattuck Hospital (n = 20), or at 3 sites in the National NeuroAIDS Tissue Consortium (NNTC)  (n = 6) with written informed consent and IRB approval at each study site. Patients with active bacterial or opportunistic infections were excluded. There was no available radiographic, endoscopic, or histopathologic data for the gut. All plasma samples were stored at −80°C until analyzed.Frozen plasma samples were shipped on dry ice to Prometheus Laboratories  and analyzed in a blinded fashion; Prometheus staff did not have access to clinical information except for HIV/HCV status. Prometheus IBD Serology 7 is the most comprehensive IBD test available, utilizing several proprietary markers and incorporating computer-based Smart Diagnostic Algorithm pattern recognition technology to aid in the diagnosis of IBD as well as differentiate between CD and UC. Prometheus IBD Serology 7 tests include the following assays: ASCA IgA and IgG, anti-OmpC IgA antibodies, IgG anti-CBir1, and pANCA autoantibody by ELISA, and immunofluorescence assay (IFA) to determine the perinuclear pattern of neutrophils and DNAse sensitivity. The reference cut-off for each assay was defined by Prometheus laboratories . Positive samples showing patterns consistent with IBD were reported based on analysis by Prometheus Smart Diagnostic Algorithm Technology.β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-α, IFNγ, TNF-α granulocyte-monocyte colony stimulating factor (GM-CSF), monocyte chemoattractive protein (MCP-1/CCL2), macrophage inflammatory protein (MIP-1β/MIP-1α), IP-10, MIG, Eotaxin and RANTES. Briefly, plasma was diluted 1∶2 and incubated with antibody-coupled beads. Complexes were washed and then incubated with biotinylated detection antibody followed by streptavidin-phycoerythrin prior to assessing titers of cytokine concentration. Recombinant cytokines were used to establish standard curves. Cytokine levels were determined using a multiplex array reader from Luminex™ Instrumentation System . Analyte concentration was calculated using Bioplex Manager Software.A multiplex immunoassay , consisting of fluorescent microspheres conjugated with a monoclonal antibody specific for a target protein, was used according to the manufacturer's instructions to measure levels of the following cytokines IL-1Data were analyzed using the Mann-Whitney U test and Spearman rank correlation coefficient. Differences were considered significant at p<0.05.Limulus amebocyte lysate (LAL) assay. The study cohort consisted of 26 AIDS patients with relatively high plasma viral loads  and low CD4 counts , together with a high frequency of intravenous drug abuse (IVDU) (65%), HCV co-infection (50%), and HIV-associated dementia (HAD) (50%). Eleven subjects were both IVDU and HCV positive. Demographic and clinical characteristics of the study cohort are shown in To investigate the frequency and pattern of IBD serological markers in AIDS patients, we used the Prometheus IBD Serology 7 test to detect IBD serological markers in plasma samples. Subjects were selected from a larger cohort of 119 AIDS subjects with CD4 counts <300 cells/µl described in a previous study In the study cohort (n = 26), ASCA IgA, pANCA, and anti-OmpC were detected in 31% (8/26), while ASCA IgG and anti-CBir1 were detected in 15% (4/26) . PromethPrevious studies reported that AIDS patients with heroin IVDU, HCV co-infection or HAD have higher plasma LPS levels compared to respective control groups Higher levels of ASCA IgG , anti-OmpC , and anti-CBir1  were detected in subjects with a positive IBD pattern as compared to those with a negative pattern . In contIn IBD, the dysregulated inflammatory response is associated with upregulation of mucosal and systemic levels of cytokines  Previous data suggested that the magnitude of antibody responses to microbial antigens is associated with increased risk of complicated CD In this study, we examined IBD serological markers in AIDS patients with high plasma LPS levels and detected an IBD-like serological pattern in 46%. Among subjects with a positive IBD pattern, 75% had a CD-like pattern and 25% had a UC-like pattern. The association of pANCA with CD markers has been described as a UC-like Crohn's pattern Our original hypothesis was that high plasma LPS levels or HIV disease markers would be associated with detection of IBD serological markers. However, we found no difference in LPS levels between AIDS subjects with versus without an IBD-like serological pattern, and no association between the frequency or magnitude of IBD serological markers and HIV RNA levels, CD4 cell counts, plasma sCD14 or LPS levels. We found a higher magnitude of antibody responses to 2 antigens in AIDS patients with low CD4 counts or high viral loads as compared to other clinical subgroups, but this difference did not reach statistical significance. pANCA antibodies were previously detected in 18% to 41.9% of patients with symptomatic HIV infection To assess the diagnostic precision of IBD serological antibodies and their ability to distinguish between CD, UC, inflammatory and non-inflammatory non-IBD disease subjects, and healthy controls, we performed meta-analysis of serological data from 20 published studies IBD serological markers alone are not recommended for diagnosis and monitoring of IBD because they are not robust enough for routine use Several cytokines with proinflammatory activities  are upregulated in IBD, and are likely to play an important role in clinical and immunopathological manifestations of the disease Limitations of this study are its cross-sectional design and small sample size, which may have decreased the power to detect significant associations between IBD serological markers and HIV disease markers. Another limitation is the narrow selection criteria used to define the study cohort, limited to AIDS subjects with CD4 counts <300 cells/µl and high plasma LPS levels. These narrow selection criteria may in part explain our inability to detect a significant difference in HIV disease markers, plasma LPS levels, or plasma cytokine/chemokine levels between patients with and without an IBD-like pattern. In view of these limitations, we recognize the need for further studies to examine the detection of IBD serological markers and frequency of an IBD-like pattern in patients with acute or chronic HIV infection before progression to AIDS and in relation to gut disease documented by endoscopic or pathologic exam. Despite these limitations, this study opens the door for new opportunities to explore and validate the detection of IBD serological markers in peripheral blood samples as novel markers of gut disease in HIV infection.In summary, we detected at least one IBD serological marker in approximately 65% of AIDS patients with high LPS levels, and an IBD-like serological pattern in 46%. Detection of these markers, particularly ASCA, anti-OmpC, and anti-CBir1, could provide a potential non-invasive approach to monitor HIV-related gut disease. Further studies are warranted to understand the clinical significance of IBD serological markers in HIV infection and their utility as tools for studies of HIV-related gut disease and monitoring therapeutic responses.Figure S1Differences in levels of IBD serological markers  in subjects with versus without IBD pattern are shown. Lines indicate median levels for each group. Mann Whitney test was used to assess the difference in antibody levels between the two groups, *p<0.05 was considered statistically significant. (EPS)Click here for additional data file.Figure S2IL-6 levels correlated positively with levels of anti-CBir1 . (EPS)Click here for additional data file.Table S1Meta-analysis of 10 studies using Prometheus laboratories IBD Serology 7 (DOC)Click here for additional data file.


Clinical, immunologic and virologic outcomes at large HIV/AIDS care clinics in resource poor settings are poorly described beyond the first year of highly active antiretroviral treatment (HAART). We aimed to prospectively evaluate long-term treatment outcomes at a large scale HIV/AIDS care clinic in South Africa.Cohort study of patients initiating HAART between April 1, 2004 and March 13, 2007, and followed up until April 1, 2008 at a public HIV/AIDS care clinic in Johannesburg, South Africa. We performed time to event analysis on key treatment outcomes and program impact parameters including mortality, retention in care, CD4 count gain, virologic success and first line regimen durability.3 after 6 months of treatment in almost all patients. By the fourth year of HAART, the majority  of patients had at least one first line drug (mainly stavudine) substituted. Women were twice as likely to experience drug substitution . By 6 months of HAART, 90.8% suppressed virus below 400 copies. Among those with initial viral suppression, 9.4% (95% CI 8.5-10.3%) had viral rebound within one year of viral suppression, 16.8% (95% CI 15.5-18.1) within 2 years, and 20.6% (95% CI 18.9-22.4) within 3 years of initial suppression. Only 10% of women and 13% of men initiated second line HAART.7583 HIV-infected patients initiated care and contributed to 161,000 person months follow up. Overall mortality rate was low , but high in the first three months of HAART . Long-term on-site retention in care was relatively high . CD4 count was above 200 cells/mmDespite advanced disease presentation and a very large-scale program, high quality care was achieved as indicated by good long-term clinical, immunologic and virologic outcomes and a low rate of second line HAART initiation. High rates of single drug substitution suggest that the public health approach to HAART could be further improved by the use of a more durable first line regimen. Infection with human immunodeficiency virus (HIV) affects over 33 million people globally. Global Antiretroviral treatment roll-out clinics in sub-Saharan Africa have achieved clinical, immunologic and virologic outcomes in the first year of HAART comparable to those observed in developed countries -14. LittIn this study of a large  and rapidly expanding (> 200 new patients each month since 2004) clinic in Johannesburg, South Africa, we evaluated the outcomes of the first four years of activity  of a South African Department of Health program by comprehensively assessing five key parameters: mortality, loss to follow-up, CD4 count gain viral suppression, and durability of first line HAART regimen.The Themba Lethu Clinical Cohort is a prospective clinical cohort of adults initiating HAART in Johannesburg, South Africa. The program is funded by the South African National and Gauteng Department of Health, with support from Right to Care funded by USAID and PEPFAR. The Themba Lethu Clinic at the regional Helen Joseph Hospital in urban Johannesburg has over 17,000 patients in care and is currently the largest single clinic providing HAART in South Africa, and one of the largest HAART clinics worldwide.Patients were included in this study if they initiated HAART between 1 April 2004 and 31 March 2007. Patients were censored at death, loss to follow-up or on 31 March 2008, whichever came first.Patients are referred to the clinic from voluntary counseling and testing clinics, hospitals, prenatal care facilities, other ART clinics or by self-referral. HAART-eligible patients attend educational and adherence sessions, and are assessed by a physician prior to initiating treatment. After HAART initiation, patients are scheduled for monthly pharmacy visits, clinical visits at month 4 and every 6 months thereafter, and additional visits whenever needed. Patients receive all antiretroviral medications free of charge but were required to pay 35 Rand (about $5 US) per clinic visit prior to October 2006, after which all fees were eliminated. Patients more than three months late for a scheduled clinic or pharmacy visit are actively traced  to ascertain the reason for loss to follow-up.As per the South African national guidelines, the majority of patients receive a first-line HAART regimen of stavudine (d4T), lamivudine (3TC), and either efavirenz (EFV) or nevirapine (NVP). Most pregnant women receive lopinavir boosted with ritanovir (LPVr) in place of EFV or NVP. Second-line HAART consists of AZT, didanosine (DDI), and LPVr.Clinical and laboratory  data are collected at all scheduled visits, except for viral load which is not collected at baseline. All data were stored in the TherapyEdge-HIV™  database, and analyzed in SAS v9.1.3 .Results were regularly controlled for quality by a senior nurse or data quality assurance manager. Duplicate entry of CD4 counts, viral loads, and hemoglobin from the National Health Laboratory Service database into the TherapyEdge-HIV database demonstrated high quality of data entry as 98.8% of values matched between the two databases.lost to follow-up. Retention into care refers to patients known to be alive and receiving HAART at Themba Lethu Clinic at the end of follow up . Viral suppression was defined as achieving viral load below 400 copies per ml. Initial virologic success was defined as achieving a viral suppression within six months of HAART initiation in patients with a viral load measured between 30 days and 6 months of HAART. Viral rebound was defined as two consecutive  viral load measurements greater than 400 copies/ml, or one viral load greater than 5000 following initial virologic success. Body mass index (BMI) was measured in kg/m2 and categorized as underweight (BMI < 18.5), normal (18.5 ≤ BMI < 25), overweight (25 ≤ BMI < 30), and obese (BMI ≥ 30) [Anemia was defined as a hemoglobin (Hb) value below 13.0 g/dl (men), 12.0 g/dl (women), or 11.0 g/dl (pregnant women)[Baseline covariates were those collected closest to time of initiation of HAART, and no more than four months before initiation of HAART or one month after initiation.Patients more than three months late for a scheduled visit, and on whom the tracing team was unable to gain information were categorized as MI ≥ 30) . Anemia nt women), Hb valuht BMI < .5, normaBaseline demographics were characterized using standard descriptive statistics. Missing baseline height (n = 48) or weight (n = 20) was assigned using median height and weight by gender. Chi-square tests were used to compare categorical variables and Wilcoxon rank-sum tests for continuous variables by category. Rates were estimated using crude Poisson models.To examine the effect of CD4 count on mortality and loss to follow-up, we created Kaplan-Meier curves stratified by baseline CD4 counts, and used competing-risks Cox proportional hazards models ,20 to esCD4 gain was assessed using two linear generalized estimating equations models with an unstructured correlation matrix. To allow the slope of CD4 increase to change over time, the first model included the main variable of months since HAART initiation, indicator variables for baseline CD4 count, and static changepoints at months 4, 10, and every 6 months thereafter (corresponding to routine clinic visits) until end of follow-up. To allow the slope to change both in time and by initial CD4 count, the second model included all terms from the first model as well as interaction terms between baseline CD4 count categories, continuous time and all static changepoints.Time to drug substitution, initiation of second-line HAART or virologic failure was evaluated using Kaplan-Meier curves with right censoring of the latter at time of last viral load assessment and censoring at time of death and loss of follow up. Rates of changes in drug regimens and drug toxicities were assessed using Poisson models and simple descriptive statistics.All analyses were intent-to-treat with regards to HAART, ignoring treatment interruptions. Time to loss to-follow-up was set at the last observed visit plus half the mean time between clinic visits of any kind while in care (20 days). Where CD4 counts or viral load measurements were missing, complete case analysis was performed. Multiple imputation (10 imputations) for missing CD4 counts was performed to check the assumption that complete case analysis was appropriate.This study was reviewed by the Institutional Review Boards of the University of the Witwatersrand and the University of North Carolina at Chapel Hill.Between 1 April 2004 and 31 March 2007, a total of 7583 (about 210 new patients each month) initiated first-line HAART at the clinic. Data from 7536 (99.4%) patients was available for analysis (Table 3 (IQR 31-158), one third (34.4%) of patients initiated HAART at CD4 < 50 cells/mm3, and19.1% of patients were underweight. Men presented at an older age and with more advanced disease  compared to non-pregnant women, and non-pregnant women presented with more advanced disease compared to pregnant women.Patients presented with advanced HIV disease, with median baseline CD4 count of 87 cells/mmDuring 161,000 person-months of follow-up , 385 (5.1%) patients died and 1234 (16.4%) patients were lost to follow up. More than one in three deaths  and losses to follow-up  occurred in the first 90 days of treatment. The crude rate of death was 2.9 deaths per 100 person-years (95% CI 2.6-3.2) overall. Early death rate was higher: 8.4 (95% CI 7.2-9.9) per 100 person-years in the first three months and 6.8 (95% CI 6.0-7.7) per 100 person-years in first six months of HAART.3 compared to those with baseline CD4 count above 200 cells/mm3. In contrast, baseline CD4 was only a weak predictor of loss to follow up . Inverse probability of censoring weighted estimates of the effect of CD4 count on mortality produced qualitatively very similar results. Multiple imputation analyses indicated that a complete case analysis did not affect the estimated effect of CD4 count on hazard of death during follow-up.Crude survival curves for death and loss to follow-up are shown in Figures The Kaplan-Meier estimates of retention into care were 82.8% (95% CI 81.9-83.6) at 1 year, 77.5% (76.5-78.5) at 2 years, 75.2% (74.1-76.3) at 3 years and 74.4% (73.2-75.6) at 4 years. This corresponds to monthly attrition rates of 1.43 per month in the first 12 months of HAART, 0.94 per month in the first two years, 0.69 per month in the first three years, and 0.53 in the first four years of HAART.3. The observed mean four-month CD4 gain was 117 cells/mm3 and differed by baseline CD4 count: 71 CD4 cells/mm3 for individuals with a baseline CD4 > 200 cells/mm3 compared to 122 CD4 cells/mm3 for those with baseline CD4 ≤ 200 cells/mm3 (p < 0.0001). The estimated mean CD4 count during the first three years of HAART by baseline CD4 count is shown in Table A first follow up CD4 count was available for 5601 (74.3%) patients after approximately 4 months . The observed median and mean CD4 count at four months of treatment was 206 and 227 cells/mmAmong individuals who had viral load measured between 30 days and six months after HAART initiation (n = 5263 or 69.8%), 90.8% suppressed virus to ≤ 400 copies/ml. Probability of initial viral suppression did not differ by baseline CD4 count. Of the 485 patients who failed to achieve initial virologic success, 294 (60.6%) achieved viral suppression during follow-up of which about half (n = 144 or 49%)) suppressed on their first line HAART regimen.Among those with initial virologic success, the Kaplan-Meier estimate of the proportion of patients with documented viral rebound was 9.4% (95% CI 8.5-10.3%) within one year of initial suppression, 16.8% (95% CI 15.5-18.2) within 2 years, and 20.6% (95% CI 18.9-22.4) within 3 years of initial suppression  by gender is illustrated in Figure Peripheral neuropathy, lactic acidosis or symptomatic hyperlactatemia, and lipodystrophy were the most frequent toxicities with incidence rates of 8.7 cases per 100 person-years of follow-up (95% CI 8.1-9.2) for peripheral neuropathy, 5.1 (95% CI 4.7-5.5) per 100 person-years for lactic acidosis or symptomatic hyperlactatemia, and 4.9 (4.5-5.3) cases per 100 person-years for lipodystrophy. Compared to men, women experienced peripheral neuropathy at a lower rate , but lipodystrophy and lactic acidosis or symptomatic hyperlactatemia at higher rates than men .Second-line HAART (AZT-DDI-LPVr) was initiated by 420 individuals after a median time of 507 (IQR 309-721) days. The crude rate of initiation of second-line therapy was 3.2 (95% CI 2.9-3.6) per 100 person-years. Women were less likely to initiate second-line HAART than men . The proportion of patients initiating second-line HAART within the first four years of HAART was 10% for female patients and 13% for male patients  at smaller roll-out clinics  -14,22.3) was similar. For example, the death rate observed in a clinic-based ART in Lusaka, Zambia was 16.1 per 100 person years overall, and 26 per 100 person years and in the first 90 days of HAART [3 or less, absolute risk of death was very small among patients in care at the Themba Lethu Clinic, with only 120 deaths among 11,436 person-months in which such a CD4 count was observed. As a result, mortality rates did not change when accounting for loss to follow-up using inverse probability of censoring weights [We observed a low overall mortality, with a crude overall death rate of 2.9 deaths per 100 person years, comparable to that observed in the Swiss HIV cohort study , but a rof HAART . The morof HAART . The low weights .The four-year 74.4% retention into care was high compared to the rather pessimistic two-year 60% retention estimate in a recent systematic review of African ART clinics by Rosen et al . The disWe observed good virologic responses to treatment and relatively infrequent first line treatment failure. By month 6 of HAART, 90.8% of patients achieved viral suppression. Viral rebound within the first 3 years of achieving suppression (corresponding to approximately the first 3.5 years of HAART) was observed in an estimated 20.6% of patients experiencing viral rebound, which is slightly lower than the 27% estimate of viral rebound observed within the same time period in treatment naïve patients in the EuroSIDA study . An estiDespite good clinical, immunologic and virologic outcomes, the durability of the first line regimen in this population was poor, with an estimated 59.6% of patients experiencing at least one drug substitution in their first line regimen (mostly d4T) in the first four years of treatment. This observation is especially alarming taken into account the public health approach, where a limited number of standardized HAART regimens are available, in contrast to the more than 20 antiretroviral drugs in industrialized countries. Female patients were twice as likely to experience drug substitutions, in part due to the higher rates of some drug toxicities and to a lesser extent to switching of antiretroviral drugs for reasons of pregnancy. These estimates for first line substitution are higher than those reported in another South African cohort, where only 28% of patients experienced substitution of at least one drug in the first 3 years of treatment . The reaOur analysis has many strengths including a large sample size, resulting in robust estimates, the prospective standardized data collection, and the use of time-to-event analysis instead of cross sectional assessment, thus overcoming the limitation of overrepresentation of patients with short follow-up. Our analysis also has a number of limitations, chiefly related to the observational nature of the cohort, missing data  and use of routine data collected from a high workload clinic. Some deaths may have been misclassified as loss to follow-up, despite the implementation of active contact tracing of patients . The obsIn this large patient cohort in Johannesburg, South Africa, we observed good long-term retention and excellent clinical, immunologic and virologic outcomes. The relatively high early mortality rate and high loss to follow up in the first months of treatment presented an important challenge to achieving the best possible HAART outcomes, and suggests the need to strengthen strategies that promote early HIV diagnosis, early access to care, and rapid initiation of HAART in the very ill patients. The high rate of single drug substitution in this population strongly suggest that the formulation of an easy-to-use, non-toxic and more durable first line regimen will be fundamental in the success of the public health approach to antiretroviral treatment in resource poor settings.The authors declare that they have no competing interests.AVR had full access to the all the data in the study and takes responsibility for the integrity and the accuracy of the data analysis.Study concept and design: IMS, DW, APM, AVR.Acquisition of data: IMS, DR, PM.Analysis and interpretation of data: DW, AVR.Drafting of the manuscript: DW, AVR.Critical revisions for important intellectual content: IMS, DW, APM, DR, PM.Obtaining funding: IMS.Supervision of database: APM.Administrative, technical or material support: DR, PM.All authors read and approved the final manuscript.


The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort.Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008). A genotypic resistance result was obtained for 1,276/1,312 (97.3%). The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml.wilson 10.7–14.3; p for trend = 0.25). NRTI resistance was predominant (7.5%) but decreased significantly over time . NNRTI resistance tended to increase over time , whereas PI resistance remained stable . Resistance to all drug classes was frequently caused by singleton resistance mutations . The majority of NRTI-resistant strains (79.8%) carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains . The majority of patients in both groups were treated successfully within the first year after ART-initiation .Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (N = 158/1,276; 12.4%; CIOverall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected with HIV might be related to the introduction of novel antiretroviral drugs and a wider use of genotypic resistance analysis prior to treatment initiation. Testing for HIV resistance to antiretroviral drugs in plasma is a rational supplement to guide clinicians in their therapeutic choice for patients receiving antiretroviral therapy, ART. Problems of drug failure related to drug resistance as a consequence of individual pharmacogenetics and adherence problems rendering drug levels suboptimal remains a major obstacle for clinicians to choose an effective antiretroviral regime. Once resistant strains of HIV are selected in the host population, they can be transmitted to new hosts and spread in the population at risk. Resistance-associated mutations of HIV have been documented for each of the different classes of antiretroviral drugs Studies of the impact of primary resistance on treatment response The study was approved by the local ethical committee of the Charité, University Medicine, Berlin, Germany. Written informed consent was given by all patients. The HIV-1 Seroconverter Study is a nationwide multicentre observational study based on an open cohort of HIV-1-infected persons for whom the date of seroconversion is known (1. acute seroconverters) or can be estimated (2. documented seroconverters).Acute seroconverters: laboratory diagnostic criteria for an acute seroconversion were i) detectable HIV-1 RNA (or p24 antigen) combined with a negative or indeterminate ELISA result or ii) reactive HIV-1-ELISA combined with a negative or indeterminate immunoblot-result followed by confirmation of complete seroconversion within 6 months. In persons recruited during seroconversion the date of the first reactive test was used as an approximation for the infection date.Documented seroconverters: individuals with a last negative and a first positive HIV-antibody test with a maximum three year interval were recruited for the study. In documented seroconverters the mid-point between the dates of the last negative and the first positive HIV-antibody test was used to estimate the time of infection.As we performed a long term observational study, various commercial kits to determine viral load (VL) with different detection limits were used over time. In this analysis viral suppression was determined as treatment success in case of viraemic suppression to at least 500 copies/ml including any category of low-level viral load measurement . Two consecutive viral load measurements within 5 to 12 months after initiation of first line therapy were mandatory for inclusion into the analysis of treatment success. Two or more consecutive viral load measurements <500 copies/ml were categorised as virological failure. We defined 3 months as a minimum duration of first line treatment to analyse for treatment success.pol fragments encoding the complete protease (99 amino acids) and reverse transcriptase (1–296 amino acids) were directly sequenced using in-house primers. This method has already been described elsewhere At enrolment baseline blood samples were collected from each individual. Genotypic resistance testing was only performed for antiretroviral-naïve patients. The ViroSeq™ HIV-1 Genotyping System was applied according to the instructions of the manufacturer . Alternatively, viral RNA was extracted from plasma using the viral RNA Kit  and reverse transcribed  with Superscript II  and amplified with the Expand high fidelity PCR system . The 1.5-kb Proportions were calculated with a 95% Wilson score confidence interval based on a binomial distribution. Median values were given with the interquartile ranges (IQR). –Due to low numbers Fishers's exact test was used to compare proportions. The Mann-Whitney-U-test (MWT) was used to compare age, viral loads and CD4-cell-counts. Trends in the prevalence of TDR were calculated by logistic regression equivalent to the Cox-Armitage trend test. Kaplan-Meier analysis was used for the comparison of duration of first line regimen. Two consecutive viral load measurements within 5 to 12 months after initiation of first line therapy were mandatory for inclusion into the analysis of treatment success. Two or more consecutive viral load measurements above 500 copies/ml were categorised as virological failure. Therapeutic success was defined as viral suppression below the detection limit of at least ≤500 copies/ml. We defined 3 months as a minimum duration of first line treatment. All p-values are two sided, and a p-value of <0.05 was considered significant. All data were analysed using SPSS 17.0.Of 1,564 HIV-positive patients with a known or estimated date of seroconversion 1,312 primary samples from drug-naïve patients were available and included into the analysis. All study participants seroconverted between 01.01.1996 and 31.12.2007. Genotyping was performed in 1,276/1,312 available samples of treatment naive patients (97.3%). There was no bias between the seroconverters genotyped and the group of patients not included . Nearly two thirds of patients were documented seroconverters . Concordantly with all reported HIV-1 cases in Germany, the predominant route of transmission in the German HIV-1 Seroconverter cohort was sex between men, MSM (85.2%). Hence, our study has a bias towards MSM comparing to the national HIV surveillance data. However, the proportion of the transmission route is comparable to the national surveillance data.10/ml , respectively  and 5.17 logectively . AntiretMost patients who used their first-line regimen less than 49 days changed substances within one day . Changes within drug classes appeared in 39.7% of these patients (25/63). Unfortunately, reasons for regimen switches were not systematically documented by the practitioners. However, 24 of these 63 patients with a prescribed 2NRTI/1NNRTI regimen and 23 of 63 patients with a 2NRTI/1PIr regimen changed to another drug class. 30.3% (n = 33) of the 109 patients dropped out and no further information was available. The first-line regimen was censored by the date of data extraction for this manuscript in 11 cases (10.1%). For the remaining 1.8% (n = 2) no further treatment information was available.Wilson: 10.7–14.3) during the period of observation ( for trend over time 1996–2007p = 0.25) (Wilson: 5.0–7.8). Followed by 2.4% NNRTI resistance  and 2.1% PI resistance . Dual- and multi-class resistance was only seen in 1.4% and 0.2% of patients with TDR, respectively. NRTI resistance reached a mean prevalence of 7.5% (CIWilson: 6.2–9.1), if the numbers were cumulated from all categories of transmitted resistance classes, e.g. mono-, dual- and multi-resistant HIV . Prevalence of transmitted NRTI resistance dropped between 1999 and 2000 resulting in an overall declining trend (NRTI:  for trend over time 1996–2007p  = 0.02) ( for trendp = 0.07). Overall PI resistance remained stable among the study population genotyped over the time  . NRTI as = 0.02) . In contvs. 33.0; IQR 28.0–38.0; pMWT  = 0.7) (Fisher Exact = 0.9). Being infected with subtype B virus was associated with a significantly higher risk to acquire resistant HIV strains  and the exposure category unknown is significantly more frequently reported in patients with resistant HIV strains . Men acquired TDR more frequently than women , respectively. The median age at seroconversion did not differ between patients with or without TDR  . In a unTDR was mostly related to viruses carrying single resistance mutations . The majThe majority of the NRTI resistant strains (79.8%) carried thymidine analogue resistance mutations (TAMs) selected by zidovudine and stavudine. Among the TAMs, revertant amino acid substitutions at position 215 of the reverse transcriptase were most prevalent (74.7%). Despite the significant decrease of transmitted NRTI resistance there is no clear cut decrease of either the prevalence of TAMs or the T215 revertant substitions . T215 reAnalysis of treatment and treatment response was performed on the basis of resistance data according to the SDRM list of mutations. First-line therapy was initiated in 382/1,118 (34.2%) patients with susceptible strains and in 61/158 (38.6%) patients with TDR associated mutations.2Log Rank  = 0.004; p = 0.95). Time between seroconversion and ART initiation was 327 days  and Kaplan-Meier showed no significant difference in comparison to patients with resistant HIV strains . CD4 cell count measurements reported at the start of therapy were 335 cells/µl  and plasma HIV-RNA measurement of 5.3 log10/ml . Any PI containing first line therapy was prescribed in 60.5% (231/382) of the patients with susceptible genotype. Predominantly, a 2 NRTI/1 PIr regimen was used as first-line therapy in 45.3% (173/382) of the patients. 34.3% (131/382) of individuals with susceptible strains received a NRTI/NNRTI first-line therapy. Any NNRTI comprising first-line therapy was initiated in 38.7% (148/382) of the patients. Efavirenz was preferred to nevirapine .First-line therapy was initiated in 382 patients with susceptible HIV strains and lasted for 182.0 days . A Kaplan-Meier analysis revealed no significant difference in duration of first-line therapy comparing susceptible and resistant strains (χMWT = 0.48). Also, plasma viral load was not significantly lower in patients with resistant strains compared to initial viral load measurements in the susceptible group . Comparing VL between acute seroconverters with and without TDR, no significant difference was observed. First-line regimen containing any PI were used in 37/61 (60.7%) patients with resistant HIV. Any NNRTI was used in 24/61 (39.3%) patients in the TDR group. Efavirenz was again the preferred NNRTI compared to nevirapine in patients with TDR .First line therapy was initiated in 61 patients (61/158) with TDR. Time between seroconversion and start of first-line therapy was 448 days . The durability of the first-line regimen in this group was 144 days . CD4 cell counts measured at time of antiretroviral treatment initiation were 312 cells/µl median and did not differ significantly to the susceptible group  of the patients with susceptible genotype. After applying the minimum duration of first-line therapy, e.g. 3 months, the patients included were 184/252 (73%). Consecutive viral load measurements within 5 to 12 months after treatment initiation were scarce. Data eligible for the analysis of treatment success were available only from 37.5% (69/184) of antiretroviral treated patients in the cohort. The majority of these patients with available viral load data  were treated successfully. Only 2.9% (2/69) of individuals did not reach the viral load limit (≤500 copies/ml) between 5–12 months after start of therapy. 5/69 (7.2%) patients were reported to have had two to three detectable viral load measurements after viraemic suppression .Data about any viral load measurement data during first year of first-line therapy – taking the minimum duration of first line therapy into account - were available for 9/40 (22.5%) of patients with resistant HIV . TherapeReferring to the subpopulation entered into the analysis of treatment success, we performed an analysis on time trends in response to therapy solely in the group of patients infected with susceptible HIV strains (69 patients). We excluded the group of patients infected with resistant HIV due to small numbers .We used a logistic regression equivalent to the Cox-Armitage trend test. In this logistic regression analysis we applied as dependent variable binary coded treatment success and as covariant ‘Year of start of first line therapy’ . The result indicates that with the higher the category the more likely to have treatment success . However note, that the result is not significant at the alpha = 0.05 level.In contrast to trends of TDR reported in other European cohorts The significant decrease of NRTI-related resistance over time (p = 0.02) in this population might have been caused by several factors. The reduction of the intensive use of mono- and dual therapies in previous years and the reduced replication capacity of viruses harbouring NRTI and multi drug resistance are associated with the reduced prevalence of NRTI resistance in vitro but are able to acquire T215Y more rapidly than the wild type under the selective pressure of zidovudine The overall prevalence of viruses with genotypic resistance in recently HIV-infected individuals did not vary over time Many treatment options are available for the initial antiretroviral regimen of patients infected with HIV It was previously assumed, that TDR detected at HIV-diagnosis would not impact the course of infection as fitter wild-type strains would become the dominant quasispecies. It is now well known, that TDR can persist for years as dominant quasispecies More than half of the patients in both groups  were reported to be under detection limit within 5–12 months (≤500 copies/ml) after start of antiretroviral treatment. A long-term observation of the changes in the composition of individual antiretroviral regimen and monitoring of the treatment outcome is necessary to assess treatment success adequately. As reported in other studies, the presence of revertants, which were identified at a high prevalence in this study, seems to have a negative impact on virological response Our findings reinforce that a broad and representative HIV resistance surveillance network between virologists, practitioners, clinicians and patients has to be installed to intensify the epidemiological knowledge about the transmission of resistant HIV, genotypic resistance testing frequencies, and treatment response in patients carrying resistant strain.Figure S1Prevalence of TAMs and NRTI resistance mutations by year of seroconversion. The prevalences of TAMs and NRTI resistance mutations (SDRM list) were calculated by year of seroconversion.(0.23 MB TIF)Click here for additional data file.Figure S2Prevalence of NRTI resistance mutations by year of seroconversion. The prevalences of each of TAMs and other NRTI resistance mutations (SDRM list) were calculated per year of seroconversion. A TAMs. Never observed: K219R. B NRTI resistance mutations other than TAMs. Never observed: K65R, T69i, L74V, V75AMTS, F116Y, Q151M.(0.88 MB TIF)Click here for additional data file.Figure S3Prevalence of NNRTI and PI resistance mutations by year of seroconversion. The prevalences of each of the NNRTI and PI resistance mutations (SDRM list) were calculated by year of seroconversion. A NNRTI resistance mutations. Never observed: L100I, K101E, V106M, Y181I, G190EQ, M230L (P236L not included). B PI resistance mutations. Occurred once: L24I, V32I, G48V, G73ACST.(0.73 MB TIF)Click here for additional data file.Figure S4Prevalence of TDR caused by one or more resistance mutations in the HIV genome by year of seroconversion.(0.51 MB TIF)Click here for additional data file.


A key advantage of monitoring HIV viral load (VL) in persons receiving antiretroviral therapy (ART) is the ability to detect virologic failure before clinical deterioration or resistance occurs. Detection of virologic failure will help clarify the need for enhanced adherence counseling or a change to second- line therapy. Low-cost, locally performable alternates to expensive VL assays are needed where resources are limited.10 copies/mL). However, the limits of agreement between the methods exceeded the biologically relevant change of 0.5 log copies/ml. Therefore, the RT assay cannot be used interchangeably with the other assays to monitor individual patients. The RT assay was 100% sensitive in detecting viral loads of ≥400 copies/ml compared to gold-standard assays. After 24 weeks of treatment, viral load measured by the RT assay was undetectable in 95% of 65 patients with undetectable RNA PCR VL (<400 copies/ml), 90% of 67 patients with undetectable bDNA VL, and 96% of 57 patients with undetectable VL in both RNA PCR and bDNA assays. The negative predictive value of the RT assay was 100% compared to either assay; the positive predictive value was 86% compared to RNA PCR and 70% compared to bDNA.We monitored the response to 48-week ART in 100 treatment-naïve Kenyan adults using a low-cost VL measurement, the Cavidi reverse transcriptase (RT) assay and gold-standard assays, Roche RNA PCR and Bayer Versant HIV-1 RNA (bDNA) assays. In Altman-Bland plots, the mean difference in viral loads between the three assays was small (<0.5 logThe RT assay compared well with gold standard assays. Our study highlights the importance of not interchanging viral load assays when monitoring an individual patient. Furthermore, the RT assay may be limited by low positive predictive values when used in populations with low prevalence of virologic failure. An estimated 3 million HIV-infected patients in low- and middle-income countries were receiving antiretroviral therapy at the end of 2007 ® reverse transcriptase assay, a commercially available kit that is already used in some African countries. While gold-standard viral load assays such as Roche Amplicor HIV-1 Monitor (RNA PCR) and Bayer Versant HIV-1 RNA (bDNA) may cost ∼$80 per test, the RT assay costs approximately $21 dollars in Kenya. The equipment required for the RT assay is minimal and the assay can be conducted in a district- or provincial-level laboratory http://www.cavidi.se/Templates/Cavidi/FileService.axd?id=38&v=1).One alternative to directly measuring the number of HIV RNA copies is to estimate it by measuring the activity of the HIV virus-encoded reverse transcriptase enzyme (RT) using the Cavidi ExaVirThe RT assay has not been evaluated in a longitudinal field trial for monitoring patients on ART in RLS. Therefore, we monitored HIV-infected Kenyan adults initiating ART for 48 weeks using the RT assay, the RNA PCR, and the bDNA assays.The study was conducted at the Bomu Medical Center (BMC), a non-governmental outpatient clinic located in an urban slum in Mombasa, Kenya. Since 2004, New York University School of Medicine has received funding from the Presidential Emergency Plan for AIDS Relief (PEPfAR) to provide free HIV care to patients attending BMC. ART treatment initiation criteria were CD4 count <200 cells/µL or WHO stage III or IV, irrespective of CD4 T-cell count One hundred consecutive ART-naïve patients were enrolled into the study if they were eligible to start ART and all provided written informed consent. The institutional review boards at Kenyatta National Hospital and at New York University School of Medicine approved this study.Study visits occurred from 2004 through 2006, at baseline (before first ART dose) and weeks 4, 12, 24, 36 and 48. Each patient reported the number of doses of ART missed since the last visit. Perfect adherence was defined as reporting no missed doses since the last visit.10) copies/ml and 50 (1.7 log10) copies/mL, respectively. The VERSANT HIV-1 RNA 3.0 assay  was performed at BMC according to the manufacturer's instructions by a technician trained by Bayer Diagnostics 10) copies/ml, and the LLQ is 75 (1.9 log10) copies/mL. Due to a technical error in one run, nine samples for the bDNA assay were excluded from the analysis.Viral load measurements were batch tested at the end of the study on plasma samples stored at −80°C. Viral load measurements were conducted on a single plasma sample that underwent a single thaw prior to testing. The Roche Amplicor HIV-1 Monitor Test, v1.5.  procedure was performed, in blinded fashion, at the Kenya Medical Research Institute, according to the manufacturer's instructions ® Load Reverse Transcriptase Assay, version 2 assay was conducted in a blinded fashion according to the manufacturer's directions by Chem-Labs in Nairobi, the reference laboratory for Cavidi. Briefly, interfering enzymes in plasma were inactivated, virus was captured on a separation gel, drugs and other inhibitors washed away, and virions lysed to recover virion-associated RT enzyme. RT activity was measured by the enzymatic incorporation of bromdeoxyuridine (BrdU) into DNA captured on a poly-A coated enzyme immunoassay (EIA) plate, and the amount of incorporated BrdU was quantified by colorimetric detection. The results were calculated using the ExaVir® Load Analyzer software and expressed in femtogram/ml (fg/ml) and equivalent RNA copies/ml (eq copies/ml). The ULQ for this assay varied in each run and ranged from 400,000 to 970,000 copies/mL, and the LLQ is 400 copies/ml.The Cavidi ExaVirLaboratory measurements included CD4 T-cell count, complete blood count, blood electrolytes, and liver function tests. CD4 cell counts were measured using Partec-GmBH  and FACSCount . No external quality assurance program was in place for either type of CD4 cell count instrumentation because this technology had been only recently introduced to treatment programs in Kenya. Twenty-seven patients did not have a CD4 cell count determination made after 24 weeks for the following reasons: 17 patients either died, were lost to follow-up or transferred to another institution and 10 patients either did not have CD4 counts ordered by the physician or there was insufficient blood volume. There was no statistically significant difference in terms of baseline CD4 cell count, baseline viral load, age, sex, or virologic failure between patients who did and did not have week 36 or 48 CD4 count.Although the lower limit of detection is different in the three viral load assays, we defined a “detectable viral load” as ≥400 copies/ml and “undetectable viral load as <400 copies/ml for all three assays. Sensitivity of the RT assay was the proportion of patients at baseline with detectable viral loads using the RT assay among those with detectable viral loads using RNA PCR or bDNA assays. Specificity of the RT assay was the proportion of patients after week 24 with undetectable RT activity among those with undetectable viral loads using RNA PCR or bDNA assays. The RT and RNA PCR or bDNA assays were compared using the Spearman correlation coefficient and the Altman-Bland plot 10 transformed for analysis.At 48 weeks, we defined the status of each patient as “in study,” “deceased,” “lost to follow-up” (missed 2 or more consecutive visits after week 24) or “transferred” to another facility. “Immunologic failure” was defined as a) a fall of CD4 count to pre-therapy level or b)>50% fall from the on-treatment peak, or c) a persistent CD4 level below 100 cells/ml Χ2 and Fisher's exact tests were used to compare categorical data and Student's t test was used to compare continuous variables. P values of <0.05 were considered to be statistically significant. Logistic regression was used to model variables associated with undetectable RNA PCR viral load. Patients who died, were lost to follow-up or transferred to another institution before the end of the study were not included in this analysis. Analyses were conducted using SPSS, version 11.P = 0.03). Otherwise, baseline characteristics  did not differ between patients who remained in the study and those who did not. Among the seven patients who died, five died within 12 weeks after initiating therapy and two between 12 and 36 weeks.The median age of the 100 patients was 35 years ; 69 (69%) were women  and the At baseline, 97 (97%) of 100 persons had detectable viral load by RNA PCR and 88 (99%) of 89 persons had detectable viral load by bDNA assay. Data from nine persons were excluded because of technical error in one bDNA assay run and two persons did not have sufficient sample volume to conduct all three assays at baseline . The senThe specificity of the RT assay was 95% compared to the RNA PCR assay  and 90% compared to the bDNA assay . Among the 83 patients tested after 24 weeks of treatment, in eight, viral load was undetectable using the RT assay but detectable using either the RNA or bDNA assays. Five of the eight samples were RNA positive but bDNA negative, one was RNA negative but bDNA positive and two were negative in both RNA and bDNA assays. Therefore, the specificity of the RT assay was 96% compared to both RNA and bDNA assays .P<0.00001, and 0.66, P<0.00001, respectively). The measure of agreement between the RT assay and RNA PCR or bDNA assays is shown in the Altman-Bland plots in Correlations between RT and RNA PCR assays and RT and bDNA assays were statistically significant  for identifying patients with virologic failure using the RT assay. The optimal inflection point was located at an RT viral load cut-off of ∼750 copies/ml .10 copies). Six patients did not have a follow-up viral load after the baseline visit, and eleven did not have a follow-up viral load after week 24. At week 48 of treatment, 18% of patients had a single detectable viral load (≥400 copies/ml) using the bDNA and RT assays and 22% using the RNA PCR assay (P = 0.03) and were more likely to have imperfect adherence   .3  (The median increase in CD4 T-cell count was 130 cell/mm 34–247) . SeventyWe describe a longitudinal study of virologic treatment response to ART among antiretroviral-naïve Kenyan adults. Many received HIV testing late in their disease as demonstrated by the fact that 74% of those requiring ART were diagnosed within the prior year. The virologic failure rate, defined as a detectable RNA viral load on two consecutive measurements after 24 weeks of treatment, was 12%, with younger age and imperfect adherence being significant risk factors for failure. A wide range of virologic failure rates are reported from RLS, ranging from 4%–36%, in part because of the use of inconsistent definitions of virologic failure 10 copies/ml) and did not vary systematically over the range of the assays. However, because the limits of agreement between the methods exceeded what is considered a biologically relevant change of 0.5 log copies/ml The mean difference in viral loads between the RT assay, RNA PCR and bDNA assays as shown in the Altman-Bland plot was small (<0.5 log® RT V.2 assay to gold standard viral load assays, however none was longitudinal Several studies have compared the ExaVirCost-effectiveness studies have shown that adding viral load monitoring is not cost-effective when second-line therapy is not availableMany cost-effectiveness studies assume that a single high viral load would automatically trigger a switch to the more expensive second-line regimenAside from cost, another advantage of the RT assay is that it does not require specialized laboratories or equipment and therefore can be conducted in a district or provincial laboratory, rather than a central laboratory There were several limitations to this study. Two different instruments in different laboratories were used to measure the CD4 cell count. This compromises the estimation of immunologic failure through changes in CD4 cell counts. However, previous studies substantiate our finding that using immunologic criteria to predict virologic failure is problematic Clearly defined strategies on the use of viral load monitoring technologies in RLS are not currently available and many questions remain, including whether viral load monitoring is even necessary


Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia.10 difference, P = 0.0015) in patients who experienced subsequent cART failure than in successfully treated patients. In multivariate analysis, adjusted for baseline CD4+ counts, prior ART experience, and particular cART regimens, the maximal usRNA level under therapy was the best independent predictor of subsequent therapy failure . The only other factor significantly associated with cART failure was prior ART experience . Levels of usRNA under cART inversely correlated with baseline CD4+ counts (P = 0.0003), but did not correlate with either baseline usRNA levels or levels of prDNA under therapy.Here, we report the results of a retrospective longitudinal study of twenty-six HIV-infected individuals who initially responded to cART by having plasma viremia suppressed to <50 copies/ml. Eleven of these patients remained virologically suppressed, whereas fifteen experienced subsequent cART failure. Using sensitive methods based on seminested real-time PCR, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the peripheral blood mononuclear cells (PBMC) of these patients at multiple time points during the period of undetectable plasma viremia on cART. Median under-therapy level of usRNA was significantly higher (0.43 logOur data demonstrate that the level of HIV-1 usRNA in PBMC, measured in cART-treated patients with undetectable plasma viremia, is a strong predictive marker for the outcome of therapy. Combination antiretroviral therapy (cART), the standard of care for human immunodeficiency virus type 1 (HIV-1) infection + counts, HIV-1 DNA level in PBMC) were reported to be associated with the risk of VR In initial cART responders, previous ART experience and specific drug regimens are associated with subsequent therapy failure  Whether HIV-1 continues to replicate in patients on cART is a matter of considerable debate et al., manuscript in preparation). This finding indicates that in untreated patients, HIV-1 usRNA load in PBMC may be a more sensitive prognostic indicator than viral RNA load in plasma. Here, we used these methods to study the predictive value of intracellular levels of viral RNA and DNA for the virological outcome of therapy.We have recently developed sensitive seminested real-time PCR methods based on TaqMan chemistry for quantitation of intracellular HIV-1 proviral (pr) DNA and both unspliced (us) and multiply spliced (ms) RNA forms Cellular HIV-1 load  was quantified in PBMC at baseline and at multiple time points during the period of undetectable HIV-1 plasma viremia on cART (“the eclipse phase”) in patients who were successfully treated with cART (“successes”) and those who experienced VR on cART after achieving undetectable HIV-1 load in plasma (“failures”). UsRNA and prDNA were detected and quantified in all baseline PBMC samples and in 94% of the under-therapy PBMC samples. MsRNA could be detected and quantified in 83% of the baseline samples, but only in 16% of the under-therapy samples , precludP = 0.015) and baseline CD4+ count (P = 0.019), was significantly associated with subsequent VR . Among the antiretroviral drugs received by the patients, only the use of nelfinavir was significantly associated with VR (P = 0.024), confirming earlier reports None of the baseline characteristics, except for prior ART experience , whereas the corresponding drop in usRNA level was 1.05 (0.90–1.50) log10 (P<0.001). The difference between the drops in prDNA and usRNA levels was significant (P = 0.002). Subsequently, we used repeated measures analysis of variance (RM-ANOVA) to compare the under-therapy levels of HIV-1 prDNA, usRNA, and CD4+ count between the patient groups. Baseline values were excluded from this analysis. No significant differences were observed between the failures and successes in the under-therapy level of prDNA and CD4+ count (P = 0.0015), the median usRNA level of failures being 0.43 log10 higher than that of successes. This difference was established early after the start of cART and observed throughout the eclipse phase , with prDNA declining on average by 0.12 log10/year, and CD4+ T cell number increasing by 68 cells/mm3 blood/year. Levels of usRNA did not significantly change over time in all patients , indicating the ability of the maximal level of usRNA under therapy to correctly predict VR in 80% of random cases, and the cutoff value of usRNA was 3.43 log10 copies/µg total RNA . As shown by the Kaplan-Meier plot  curve was 0.800 . Subsequently, maximal level of usRNA under cART, along with other factors associated with the VR in univariate analyses with significance levels <0.1 , were included in the multivariate logistic regression analysis. Maximal level of usRNA under cART was independently predictive of the therapy outcome (adjusted OR for VR (95% CI), 24.4 (1.5–389.5), P = 0.024). The only other factor significantly associated with VR was prior ART experience (adjusted OR (95% CI), 12.3 (1.1–138.4), P = 0.042). Area under curve of the logistic regression was 0.882 , meaning that the predictive power of the multivariate model was 88.2%.Next, we stratified the patients into two groups based on the determined cutoff value of usRNA. Of the eight patients whose usRNA level under cART did not reach the cutoff value, only one experienced VR, whereas VR was observed in 14 out of 18 patients whose maximal usRNA level was higher than the cutoff value (unadjusted odds ratio (OR), 24.5; 95% CI, 2.3–262.7; + count under therapy  and, even more strongly, with baseline CD4+ count . Of note, no significant correlation was observed between the baseline and under-therapy levels of usRNA, whereas baseline and under-therapy levels of prDNA strongly correlated between each other . As subsequently assessed by Pearson analysis, 40% of the variance of usRNA level under cART could be explained by the baseline CD4+ count, but only 6% of its variance could be explained by the baseline usRNA level. By contrast, 59% of the variance of prDNA level under cART could be explained by the baseline prDNA level.Finally, correlation tests were performed between pairs of the under-therapy levels of the studied parameters  and comb+ under-therapy PBMC samples was 0.48 log10 higher than usRNA level in the msRNA− samples , suggesting that the correlation between levels of usRNA and msRNA, observed at baseline, exists also under cART. The levels of msRNA in PBMC before the start of cART were, on average, 1.5 log10 lower than those of usRNA  detectability of msRNA.Due to the low detectability of msRNA in the under-therapy PBMC samples, we could not assess correlations of this marker with the other parameters. However, median level of usRNA in the msRNAof usRNA . Interes samples . If thisThe main clinical objective of cART is suppression of HIV-1 plasma viremia to below the lowest existing detection limit of commercial assays. In most patients on cART, this objective is achieved, and therefore plasma viremia in these patients cannot be informative about the subsequent outcome of therapy. Hence, additional markers have to be identified that are associated with therapy outcome in patients with fully suppressed plasma viremia. In this study, we demonstrated that the level of HIV-1 usRNA in PBMC from such patients is predictive of subsequent VR. To the best of our knowledge, this is the first report showing that a viral parameter–measured in a patient on cART during the period of undetectable plasma viremia–is predictive of the outcome of therapy. Several reports have indicated the presence of HIV-1 mRNA in PBMC from patients on cART + T cells are considered to be an extremely stable viral reservoir in patients on suppressive cART, and a major barrier to HIV-1 eradication + T cells, but we assume that the majority of usRNA was derived from activated CD4+ T cells, productively infected with HIV-1. This is supported by the observed differences in longitudinal trends between prDNA and usRNA after the start of cART. Whereas levels of prDNA gradually diminished throughout the whole follow-up period, those of usRNA first sharply declined after the start of therapy and reached the plateau during the eclipse phase , and not simply by the size of viral reservoir (reflected in the levels of prDNA). Consequently, level of HIV-1 usRNA in PBMC under cART, which we found to be (i) strongly inversely associated with baseline CD4+ count, and (ii) strongly predictive of subsequent VR, provides a missing link explaining the previously observed inverse association between the baseline CD4+ count and the risk of therapy failure + count at baseline. Although baseline CD4+ count could explain 40% of the variance of usRNA level under cART, other parameters known to be associated with VR, such as adherence to cART, may also influence the levels of usRNA.Latently infected resting CD4se phase . These dasma RNA . Converset al.There are certain limitations to this study. First, our usRNA assay does not distinguish between genuine intracellular HIV-1 usRNA and cell-associated virion RNA. However, Fischer In summary, our findings demonstrate that the level of HIV-1 unspliced RNA in PBMC is a strong predictive marker for the outcome of therapy in cART-treated patients. Use of this quantitative assay in the standard laboratory practice could aid in monitoring the course of cART and facilitate the early detection of drug-resistant escape mutants before the actual failure of the therapy.We have used archival PBMC samples from HIV-1 infected individuals who were participating in the Amsterdam Cohort Studies (ACS) on HIV infection and AIDS. The ACS have been conducted in accordance with the ethical principles set out in the Declaration of Helsinki, and written informed content has been obtained prior to sample collection. The study has been approved by the ACS committee. The ACS have been approved by the Medical Ethical Committee of the Academic Medical Center.We have selected 26 HIV-1 infected individuals who received cART between 1996 and 2002, and have initially responded to cART by showing undetectable HIV-1 RNA loads in plasma (<50 copies/ml). cART was defined as at least a triple-therapy regimen, consisting of two nucleoside reverse transcriptase inhibitors (NRTI) and at least one protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI). All patients were men infected by HIV-1 subtype B strains. Two groups of patients were selected, matched by median calendar year of cART initiation: successfully treated patients (n = 11) and patients who experienced VR after initially responding to therapy . Individuals treated with cART, in whom HIV-1 plasma viremia dropped to the undetectable levels (<50 copies/ml) and remained undetectable for the whole period of the therapy (with the minimum of one year), were considered to have virological success. Occasional “blips” (transient episodes of detectable plasma viremia) of <500 copies/ml, preceded and followed by measurements of <50 copies/ml, were allowed. Individuals treated with cART for at least six months, in whom HIV-1 plasma viremia has dropped to the undetectable levels after the start of therapy, has been undetectable for at least three months after that, and subsequently became detectable (>50 copies/ml in two consecutive measurements or >500 copies/ml in any measurement), were considered to experience VR (cART failure). No difference was observed in the frequency of blips between the patient groups.P = 0.35). For every PBMC sample used in this study, concurrent measurements of plasma viremia and CD4+ count were available.Baseline was defined as the date of start of cART. The follow-up period was calculated from the start of therapy until the date of first measurement of plasma viremia above the detection limit (for failures), or until the date of last measurement of plasma viremia under the current therapy regimen (for successes). Median  follow-up periods were 2.30  years for all patients, 2.16  years for failures, and 2.48  years for successes. During the follow-up period, plasma viremia was monitored at least every four months, and cellular HIV-1 load was quantified at multiple time points . The num6 PBMC was used for one extraction) according to the isolation method of Boom et al. 6 PBMC for prDNA, or as number of copies per µg total RNA for usRNA and msRNA. The sensitivity of all three assays was four copies per reaction, which translated into approximately 40 copies/106 PBMC for the prDNA assay and 100 copies/µg total RNA for RNA assays , and the linear range was at least five orders of magnitude. The reproducibility and accuracy of these assays have been demonstrated earlier 10-transformed values, were 5.31%  for the prDNA assay, and 2.29%  for the usRNA assay.For quantitation of cellular HIV-1 RNA and DNA load, PBMC were isolated by standard Ficoll-Hypaque density gradient centrifugation and frozen in aliquots in liquid nitrogen. Total cellular nucleic acids were extracted from PBMC samples , according to manufacturers' recommendations. For those patients who were treated with cART before the year 2000 (when ultrasensitive assays for plasma HIV-1 RNA quantitation were introduced in our laboratory), HIV plasma viremia was remeasured using the modern ultrasensitive assays.pol gene of HIV-1 were amplified in the nested PCR with the following primers: 5′Prot I (5′-AGGCTAATTTTTTAGGGAAGATCTGGCCTTCC-3′) and 3′ET-21 (5′-AGCTGGCTACTATTTCTTTTGCTACTACAGGTGG-3′) (pol-PCR I), and 5′Prot II (5′-TCAGAGCAGACCAGAGCCAACAG-3′) and 3′RT20 (5′- CTGCCAGTTCTAGCTCTGCTTC-3′) (pol-PCR II). PCR products were sequenced directly using the primers of the pol-PCR II. The sequenced fragments contained all 99 codons of protease and the first 284 codons of reverse transcriptase. Analysis of drug-resistance mutations was carried out using the Geno2pheno software , which includes the algorithm for the determination of resistance for every antiretroviral drug individually by using the Z-score (number of standard deviations above the mean of drug-naïve patients) produced by a particular pol genotype.To determine the presence of drug-resistant HIV-1 strains at baseline, we used prDNA extracted from the baseline PBMC samples as described above. Regions corresponding to the gag gene of HIV-1 and containing the primer and probe binding sites were amplified in the nested PCR with the following primers: 5′GAG-1 (5′-CATGCGAGAGCGTCAGTATTAAGCGG-3′) and SK39 (5′-TTTGGTCCTTGTCTTATGTCCAGAATGC-3′) (gag-PCR I), and GAG-2I-SP6 (5′-CGATTTAGGTGACACTATAGGGGAAAAAATTCGGTTAIGGCC-3′) and GAGAE-3-T7 (5′-TAATACGACTCACTATAGGGACTATTTTATTTAATCCCAGGAT-3′) (gag-PCR II). The PCR products were sequenced directly with the primers of the gag-PCR II. Subsequently, the effects of mismatches in the primer and probe binding regions on the efficiency of real-time PCR were determined by performing the seminested real-time PCR with the patient-derived PCR amplicons that contained all the primer and probe target sites, as templates. The concentrations of the template amplicons were determined spectrophotometrically and equalized by dilution before real-time PCR. Patient-specific mismatch-related quantification errors (MRQE) were calculated as the differences between the log10-transformed output copy numbers of the individual mismatched templates and the log10-transformed median output copy number of the templates without mismatches. The presence of mismatches did not substantially influence the real-time PCR quantification, as the difference between the 10% and 90% percentiles of the MRQE values was 0.26 log10, and the total range, except for one outlier, was 0.34 log10 , and between the patient groups .To account for the possible effects of mismatches in the primer and probe binding regions on the efficiency of real-time PCR, prDNA extracted from patients' PBMC samples was used for sequence analysis of the target regions of prDNA and usRNA real-time PCR assays . Regions corresponding to the 34 log10 . There w10-transformed values. All quantified amounts of prDNA and usRNA in the baseline and under-therapy PBMC samples were normalized to the individual MRQE values (see above), and statistical analyses were based on the normalized values. For the four PBMC samples in which prDNA and/or usRNA were undetectable, the prDNA and usRNA levels were left-censored at the corresponding detection limits, normalized to the MRQE values. For the comparisons of the baseline parameters between the patient groups, we used Mann-Whitney test for continuous variables and Fischer's exact test for discrete variables. For the comparisons of the levels of usRNA and prDNA and CD4+ count in patients on cART, under-therapy PBMC samples were divided in four groups based on sampling time from cART initiation: group I, 0.1–0.6 years; group II, 0.7–1.5 years; group III, 1.9–2.5 years; group IV, 2.9–6.0 years. Any of these groups did not contain more than one value from any patient. In three cases, patients were sampled twice in the period corresponding to the groupings (two cases in group IV and one in group I). In these cases, mean values per patient were used for the statistical analysis. The parameters were compared between baseline and time on cART-grouped PBMC samples by using paired Wilcoxon test and between failures and successes by using RM-ANOVA. Baseline values were excluded from the latter analysis. Longitudinal trends of the studied parameters under cART were estimated by fitting the linear mixed models, taking into account correlations of repeated measurements within the individual patients. Subsequently, linear mixed models were used to compare the virological parameters between the patient groups. The cutoff value of usRNA for the risk of VR was determined by the ROC curve analysis with therapy outcome as the classification variable and maximal value of usRNA under therapy as the diagnostic variable. Correlations of virological parameters and CD4+ count were assessed by using Spearman tests. All parameters, except for CD4+ count under therapy, were normally distributed , and Pearson r2 values of pairwise correlations were calculated for these parameters. RM-ANOVA was performed by using XLSTAT (http://www.xlstat.com), logistic regression and ROC curve analysis by using MedCalc 10.4 , and linear mixed model analysis by using SPSS 16.0 (http://www.spss.com/). All other statistical tests were performed by using GraphPad Prism 5.01 (http://www.graphpad.com). All statistical tests were two-sided. P values<0.05 were considered statistically significant.For cellular and plasma HIV-1 load, statistical analyses were performed on logFigure S1Sequences of primer and probe target regions of seminested real-time PCR assays for usRNA and prDNA detection.(0.07 MB PDF)Click here for additional data file.Table S1+ counts.HIV-1 load in PBMC and plasma and CD4(0.37 MB PDF)Click here for additional data file.