The HIV virus is known for its ability to exploit numerous genetic and evolutionary mechanisms to ensure its proliferation, among them, high replication, mutation and recombination rates. Sliding MinPD, a recently introduced computational method , was useAnalysis based on the predicted networks suggests the following:: significantly stronger recombination signals (p = 0.003) for the inferred ancestors of the X4 strains, recombination events between different lineages and recombination events between putative reservoir virus and those from a later population, an early star-like topology observed for four of the patients who died of AIDS. A significantly higher number of recombinants were predicted at sampling points that corresponded to peaks in the viral load levels (p = 0.0042).Our results indicate that serial evolutionary networks of HIV sequences enable systematic statistical analysis of the implicit relations embedded in the topology of the structure and can greatly facilitate identification of patterns of evolution that can lead to specific hypotheses and new insights. The conclusions of applying our method to empirical HIV data support the conventional wisdom of the new generation HIV treatments, that in order to keep the virus in check, viral loads need to be suppressed to almost undetectable levels. Some of the remarkable properties of within-patient HIV quasispecies are their large population size, high replication rate, and short generation time. HIV has a high mutation rate (0.2 errors per genome per cycle) and an even higher recombination rate (3 events per genome per cycle) [One of the earliest and most striking observations made about HIV was the extensive genetic variation that the viral population exhibits within individual hosts, particularly in the hypervariable regions of the r cycle) . Recent r cycle) . The curin vivo [Conventional phylogenetic programs are constrained to produce simple branching trees that can lead to misinterpretations of the phylogenetic relationships if the data set contains recombinants. One main assumption of most phylogenetic methods is that there is only one phylogeny underlying the evolution of the taxa under study. Recombination violates this assumption by generating mosaic genes, where different regions have different phylogenetic histories -8. Furthin vivo  and its in vivo ,20. A bein vivo HIV evolution was published [env gene collected at recurring time intervals from nine patients over a span of 8 to 12 years. The study provided new insights [in vivo evolution of HIV. Henceforth we will refer to the HIV data set as the Shankarappa99 data set. We used Sliding MinPD to analyze the Shankarappa99 data set using data for eight of the nine patients for which hand-curated alignments were made available.In 1999, a comprehensive study of ublished . The stuinsights , but didIn this paper, we explore the discovery power of our computational tool by taking a closer look at the generated serial evolutionary networks. We present eight evolutionary networks obtained by applying Sliding MinPD to the Shankarappa99 data set and describe how the network information facilitates the study of viral evolutionary relationships, evolutionary patterns, splitting and merging of lineages, and how it helps to determine how these correlate with the disease status of the patient. Our analysis provides insight into within-host viral evolution and helps to find patterns that may explain the emergence of harmful mutants associated with disease progression.In a 1992 study, Holmes et al. created an "evolutionary framework" to express the inferred ancestor-descendent relationships in HIV sequence data that was serially sampled from a single patient . The autenv gene sampled serially from patients with a moderate or slow rate of disease progression. All PCR amplifications were done with procedural safeguards to prevent recombination from occurring during PCR [, where it is available as supplementary material for a study on immune-mediated positive selection driven by HIV-1 molecular variation [env gene of the reference sequences. The data sets of the 8 patients were then analyzed using Sliding MinPD. Duplicate sequences from the same sampling point were removed as they represented the same ancestral sequence, retaining just one copy, but marked with an x followed by the number of duplicate copies. Thus, the sequence labeled "11 × 2" from Figure The Shankarappa99 data set is available for download from GenBank and the HIV Los Alamos database. It consists of viral DNA and plasma RNA sequences from the C2-V5 region of the ring PCR . Two hanariation . Both veariation . The lenThe sequences in the Shankarappa99 data set from the same time point had a relatively low level of diversity (average genetic distance: 0.03) and a low level of divergence relative to an initial founder sequence from the first sampling period (average genetic distance: 0.05). The exceptions were the data sets for patient 9 at all sampling points, and the sequences for patients 2, 3, and 7 for the last sampling times . Low divWe provided the alignment files, two for each patient, as input to Sliding MinPD, which ran each file using the three different recombination detection options: B-RIP (Bootscan RIP), SB (standard Bootscan) and RIP, producing 6 result files for each patient. In a final step, a consensus file was manually created by combining information from the 6 result files using a consensus strategy and a keen awareness of the strengths and weaknesses of the three methods used .The settings for each option of the program were the same as the default options presented previously , with thA consensus file of ancestor descendant relationships was created manually from the 6 result files by applying the following guidelines in sequential order :▪ Majority rule: The relationship chosen by a majority (at least four) of the six methods was chosen as the consensus.▪ Consensus by bootstrap support: The result with the highest bootstrap value was chosen.Consensus by priority: The results using the B-RIP option were given highest priority because of its superior performance and because it outputs the statistical bootstrap support for each estimate . The resA serial evolutionary network was constructed for each of the 8 patients from the consensus file containing the ancestor-descendant relationships. A short summary of the guidelines used for interpreting the networks is given below:▪ The sampling times in units of "months" from the time of seroconversion are shown at the top of each network. Thus, all sequences from the same sampling time are aligned vertically under the corresponding time.▪ Full lines indicate branch lengths. Thus longer full lines indicate greater evolutionary distance. Dotted lines indicate linking relationships. Finally, dashed blue lines indicate recombination-linking relationships between donors and recombinants.▪ Recombinant sequences are underlined in blue.env gene, it is necessary to know the coordinates of the gapped sequences with regards to the env gene map. The approximate start coordinates {within env gene| within HIV genome} of the env regions covered by the sequences are: pos 1 – C2:{799|7023}, pos 98 – V3:{891|7115}, pos 254 – C3:{995|7219}, pos 443 – V4: {1150|7374}, pos 579 – C4:{1243|7467}, pos 712 – V5:{1367|7591}, pos 772 – C5:{1403|7627}.▪ Breakpoint positions appear after a slash at the point were the lines intersect. If the left donor is at the top  end of the recombination-linking dashed line, this is indicated by a forward  slash followed by the breakpoint position. To locate a breakpoint position along the C2-C5 region of the x from Figure ▪ X4-mutants appear in red and are also marked with a top-right elevated small x, as in the taxon 10(79)▪ An equal sign "=" next to a node indicates that the sequence is identical to an ancestral sequence from a previous sampling period. For example, the sequence 18(100) of patient 2 at 91 months is identical to sequence 18(82) from the 80 months sampling period.▪ Statistical bootstrap support values, which provide statistical significance scores for the ancestor-descendant predictions are interpreted as follows:◦ The bootstrap values are added in parenthesis after the sequence ID.◦ The bootstrap values correspond to the ancestor-descendant relationship and not to the clades or topological position in the subtrees. The bootstrap value represents the support given to the choice of linking a specific query sequence to the ancestor at the root of the immediate subtree. Thus, the taxon labeled 43(97) at 61 months in Figure ◦ Bootstrap values of recombinant sequences were obtained from computations involving only a few sequences from the pool of donor candidates.◦ Unlike the bootstrap values of recombinant sequences, the bootstrap values of non-recombinant sequences were calculated by using all sequences up to the previous sampling point of the query sequence.The sequence IDs have been shortened to decrease the amount of clutter and to make space for the bootstrap values. In their shortened form, they are unique within a sampling point, but not across the whole serial evolutionary network. For example, the GenBank ID of p2c005-03 is shortened as 03 in the network of patient 2 under the sampling time 5 monthsEvolutionary networks for three of the eight patients who survived after the end of the analysis were discussed in detail. The statistical analysis section below discusses results based on all eight data sets and networks. In a previous study , the net+ T-cells and the start of the antiretroviral drug therapy at the 103rd month period. The patient took three different drugs that included Zidovudine (ZDV), between 97 and 120 months, dideoxycytidine (ddC), between 109 and 125 months, and Stavudine (d4T), after 114 months. An interesting recombination event was suggested for sequence 126 m-19(93) with a putative left-side donor of 68 m-19(83), which was sampled almost 5 years earlier (a possible reservoir virus), and which was from a different lineage that did not carry the X4 mutation. This suggests the existence of dormant viruses that escaped latency and recombined with viruses from the dominant population. Putative dormant strains might enter the blood stream through the low degree of ongoing viral replication that is thought to occur in latently infected, resting CD4+ T cells [The Sliding MinPD analysis resulted in the following inferences and observations: Three different lineages were inferred up to the 30-months sampling time point. At that point, the lineage of 5 m-03 (with a 5 months start) became extinct. At 51 months the 5 m-09 lineage ceased to exist with sequence 51 m-25(97) as its last sampled sequence. Of more relevance is the splitting of the 5 m-11 lineage into several lineages, with strain 20 m-17(47) inferred as an early ancestor of the large X4 population that emerged after the 51 months period. All other arms of the 5 m-11 lineage disappeared within 68 months with the last sampled representative being 68 m-14(76). The evolutionary network made it convenient to understand how widespread the X4 genotype is in each sampling period. In particular, the most recent common ancestor of all post-61-months sequences carrying the X4 mutation was identified as 51 m-91(93), a recombinant sequence. The network suggests an increase in recombination activity in the last sampling period, which coincided with a decrease in the number of CD4 T cells .+ T-cells, but no increased recombination effect could be observed.The network of patient 8 as shown in Figure As with patient 8 and 2, the network of patient 9 , and has predicted donors from two separate lineages, one that ceased to exist at 111 months and the other that persisted in recombination events through 134 months. Yet another feature of interest is that many recombinant sequences in patient 9 are themselves putative parental donors contributing to other recombination events. Patient 9 was on Zidovudine (ZDV) therapy since a doctor's visit at 57 months and was the only patient (among the ones considered in this work) with data spanning a long period of therapy of over 6 years.Statistical analysis was performed by considering the data on all 8 patients Figures , 7 and 8The networks revealed that the ancestors of the X4 strains were either recombinant or displayed strong recombination signals. A recombination signal is defined as an indication that different parts of a viral sequence may descend from different ancestors. In order to determine the significance of this pattern a score was devised to measure the recombination signal (RS) based on the average bootstrap support (BS) of the non-recombinant inferences, the number of recombinants (R) and the number of different ancestors (A) from the 6 results produced by analyzing two alignments  by the 3 methods previously described. While only the consensus predictions are considered in the network construction, conflicting results may still indicate support for a recombination event, based on those three parameters. The strength of this support is measured by the recombination signal. The score is given by the following formula:A low bootstrap support can indicate that either there is more than one possible ancestor, signaling a possible recombination event, or the determination of the ancestor is inconclusive. Results that differ in their ancestor prediction may also indicate a recombinant origin. A t-test confirmed that the ancestors of the X4 strains had a significantly higher recombination signal than the overall average recombination signal in all 8 patients (p = 0.003).In a further effort to investigate the relationship between clinical factors and evolutionary parameters we analyzed possible associations between the proportion of recombinants, the proportion of X4 mutants, the peaks in the viral load levels and the administration of antiretroviral drug therapy as shown in Figure In this study, a consistent relationship was noted among the 8 patients between time variables involving clinical markers and proportion of recombinants as shown in Figure We next asked if the star-like evolutionary pattern observed in the network of the five patients who died at the end of the study was associated with a faster disease progression. A star-like pattern is seen when many descendants share the same ancestor. A slight negative correlation was found between CD4+ T cell counts and the proportion of ancestors for each time period among 6 of the eight patients (with a mean slope of -0.19). A one sample t test comparing the mean slopes to a slope of zero returned a p-value of 0.0566.env gene. The average breakpoint position for the non-X4 recombinant sequences was 373, while for the recombinant X4 sequences it was 427. A 1-tailed paired t-test returned a p-value of 0.0475. The analysis included predictions from the 6 result files per patient, far more than the few consensus recombinant sequences shown in the evolutionary networks. For example, for patient 5, there are many recombinant predictions but little consensus in most of them. No significant difference was found between breakpoint positions before and after drug therapy. The scatter plots of the breakpoint positions in relationship to the sampling time showed considerable diversity from patient to patient. The plots indicated a preference for the 300 to 500 bp region in all patients, but no particular in vivo hotspot for recombination could be observed. The distribution of recombination sites for all predicted recombinant sequences did not show discernible patterns across the C2 to C5 region with the exception of patient 1, for whom the breakpoint sites moved further to the left as the sampling time increased .Analysis of the sequences predicted as recombinants revealed that the average breakpoint position of X4 recombinant sequences was further to the right towards the V4 region of the env region of eight patients. The network representation facilitated the locating of viral strains due to the temporal organization within the network and allowed for a better understanding of the evolutionary relationships of within-patient HIV strains. The evolution of HIV within-host environments is very different from between-host HIV evolution. There is strong evidence that natural selection is the driving force of within-host evolution. Within-host HIV phylogenies have a strong temporal structure, reflecting the successive fixation of advantageous mutations and the extinction of unfavorable lineages [The Sliding MinPD method was used to analyze and represent the evolutionary relationships of HIV-1 sequence data sampled serially over a period of many years from the HIV-1 lineages . This caA few of the recurring patterns observed in the evolutionary networks of the eight patients are summarized below and will be discussed in more detail in the next section:▪ The network of the five patients who died at the end of the study displayed a starlike evolutionary pattern (approximate time of death in months appears in the network next to the symbol †).▪ The founder strains of the lineage containing the X4 strains displayed significantly stronger recombination signals.▪ Recombination events with early viral quasispecies suggest the resurgence of reservoir virus.▪ Recombination events linking different major lineages were observed in the three surviving patients.It is important to mention the role that the visualization of the results plays in understanding the different patterns of within-host viral evolution. The evolutionary networks made it easy to locate any viral strain due to the temporal positioning within the network, where strains sampled at the same sampling point were vertically aligned. The network representation can also encourage the pursuit of new research directions or generate new lines of inquiry. For example, knowing that five of the eight patients passed away after the analysis period, it was possible to ask whether any marked differences existed between the networks for the surviving and the deceased patients. As discussed in the results section, the networks of patients 2, 8 and 9 shared very similar characteristics such as the persistence of different lineages, unbalanced networks , recombination events between lineages and recombination events with much earlier viral strains. On the other hand, with the exception of patient 5, the networks of the other patients lacked these characteristics, with a distinctive star-like evolution of one single lineage from the onset of the study. Explosive growth generates characteristic 'star-like' phylogenies in which a single strain, the 'master sequence' in quasispecies theory, gives rise to a large population of diverse quasispecies with a spectrum of small mutational changes from the master sequence ,29. No l+ T-cells, and therefore, a more rapid progression to AIDS [env region) of crossover sites for other recombinant sequences.The network representation allowed us to better understand the evolutionary roadmap of the X4 strains. Each of the patients had X4 strains, which are known to be associated with a higher rate of decline of CD4 to AIDS . TraditiThe role of co-infection and recombination has received some attention in recent years and these findings may support the recombinant origin of the X4 strains -32. It hThe serial evolutionary network representation allowed us to look for patterns present in the networks of the three surviving patients and absent in the other networks. As discussed earlier patients 8 and 9 have very similar network characteristics: separate lineages with linking recombination events and recombination events with strains from a much earlier time point. Patient 2 also displayed separate lineages and similar but less obvious patterns of recombination. In contrast to those three, Patient 5's network displays two long-lasting lineages with no linking consensus recombination events.The presence of different types of HIV reservoir cells that harbor dormant virus has been discussed in many studies ,27,33,34In a recently proposed controversial model , it was Figure The current study did not estimate the Ka/Ks ratio of selection pressure, as it is known that recombination can affect the estimation of that measure. Current sitewise methods for detecting positive selection on gene sequences assume no recombination. When this assumption is violated it can lead to incorrect detection of sites undergoing positive selection . A recenThe serial evolutionary networks of the 8 patients revealed different aspects of the within-host evolution of the HIV virus. Among the networks for the 8 patients we found several consistent patterns that are of evolutionary significance with potential impact on the development of new anti-retroviral drugs. The visualization of the results played an important role in understanding the different patterns of within-host viral evolution. The network representation suggested new avenues of research and lead to statistical analysis that connected our observations with known facts and previously published results on the evolution of the HIV-1 virus. The serial evolutionary network proved to be an effective and useful tool, able to produce noteworthy results, as well as generate important and productive new lines of inquiry in the study of viral evolution.The main line of inquiry was aimed at studying the origin of the disease associated X4 strains. As previously discussed, the putative founder strains of the X4 population are most likely a result of recombination events in large and diverse viral populations. Our results suggest that evidence of recombination increases with higher viral loads. On the other hand, recombination between strains from different lineages or between reservoir viruses and the viruses from the dominant population were more prevalent in the asymptomatic patients. A star-like topology could be observed in the networks of four of the five patients who died of AIDS. As mentioned in the earlier study , it is nRecent sequencing technologies such as that from 454 Life Sciences have made it possible to quickly and cheaply generate a large number of short clonal transcripts (~100–400 bp). The inevitability of large-scale HIV within-host sequences data in the near future makes it critical to develop analytical methods of the kind presented in this paper. We expect an abundance of new clonal sequences from longitudinal studies, which can be used in the reconstruction of serial evolutionary networks for a much larger number of patients and can contribute to the development of tools for HIV patient surveillance and prognosis. Sliding MinPD, which is the most efficient and accurate programs for this kind of data, should prove extremely useful in such analyses. Although modern HIV combination drug therapy has been successful at suppressing viral replication to <50 c/ml in patients who adhere to treatment, viral replication has been shown to persist in viral reservoirs and inconsistent adherence to treatment can lead to drug resistance. Moreover, in developing countries, infected individuals with limited access to drugs face an increased risk of developing AIDS. It therefore continues to be of foremost importance to understand the evolutionary process of HIV within a patient to aid in the design of effective drug treatments and in the evaluation of patient prognosis.PB designed the study, carried out the statistical analysis and drafted the manuscript. GN participated in the design of the study and writing of the manuscript. All authors read and approved the final manuscript.


To evaluate the safety, tolerability, and efficacy of ritonavir-boosted saquinavir 1000/100 mg twice daily administered as a 500 mg film-coated tablet in HIV-1-infected patients.In this open-label, observational, 24-week survey conducted in 8 European countries, eligible HIV-infected participants had been prescribed saquinavir/ritonavir in combination with other nonprotease inhibitor (PI) antiretroviral agents as part of their HIV treatment regimen. The safety (grade 3 or 4 adverse events [AEs]), tolerability (by an investigator-reported subjective rating system), and efficacy (the percentage of participants with <50 and <400 copies/mL HIV RNA and change from baseline in mean CD4+ cell count) were analyzed for the overall study population and 7 subpopulations.The enrolled population included 2122 participants with 1908 completing the study; 44 (2.1%) withdrew prematurely because of AEs, including 7 nontreatment-related deaths. There were 33 grade 3 or 4 AEs in 29 (1.4%) participants; 7 AEs in 7 (0.3%) participants were considered treatment-related. Tolerability was reported to be “very good” or “good” in 42% and 25% of participants, respectively. From baseline to week 24, the proportion of participants with HIV RNA <50 copies/mL increased from 31.2% to 47.6% and the proportion with <400 copies/mL increased from 42.5% to 61.4%; the mean CD4+ cell count increased by 75 cells/µL. In the subpopulation analysis, the greatest efficacy benefits occurred in participants who were treatment-naïve and in those not having received prior PI therapy.Treatment with the saquinavir 500 mg film-coated tablet resulted in few grade 3 or 4 AEs and was well tolerated and effective in a broad population of patients. The current U.S.  and EuroSaquinavir mesylate  is a potent inhibitor of the HIV protease. SQV has been approved by the European Medicines Agency (EMEA) and the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection in combination with other antiretroviral (ARV) agents. Available for oral administration as a 200 mg hard gelatin capsule and a 500 mg film-coated tablet , the 500The efficacy and safety of SQV/r 1000/100 mg twice daily in combination with other ARVs have been established in randomized clinical trials –10. HoweThis was an open-label, multicenter, phase 4 observational cohort study of HIV-infected patients 18 years of age or older who had been prescribed SQV/r 1000/100 mg twice daily with NRTIs or NNRTIs as part of their HIV treatment regimen. Excluded were patients who had received previous treatment with SQV, who had simultaneous treatment with a PI other than SQV/r, who had SQV/r at doses other than 1000/100 mg twice daily, or who had nonadherence or poor compliance to SQV/r that may have resulted in survey data being deemed unreliable.All participants were prescribed SQV 1000 as 2 × 500 mg tablets twice daily. Survey data were transcribed by the investigator from the patient chart into an internet-based CRF system TRI@L-IT. In cases in which participants were being treated prior to enrollment, data were collected both retrospectively and prospectively. Baseline (before the first dose of SQV/r) demographic characteristics, hepatitis status, ARV history, laboratory data , and clinical data were recorded as well as year of HIV diagnosis and current clinical disease stage and concomitant medications including lipid-lowering agents. The following data were collected from any visits occurring up to 24 weeks after initiation of therapy and were classified into the applicable time frame : HIV RNA viral load, CD4+ cell count, dates of these measures, assays used and lower limit of detection, changes in concomitant medications including changes in or introduction to lipid lowering agents, unexpected and grade 3 and 4 adverse events (AEs) and relationship to the study specific medication, adherence information, and results of hematology and biochemistry tests. Safety analyses included the monitoring of changes in the following laboratory parameters from baseline to last available value: fasting lipids, bilirubin, glucose, alanine transaminase, aspartate transaminase, gamma-glutamyltransferase, and the percentage of patients receiving lipid-lowering agents.This study was conducted in accordance with the Declaration of Helsinki in its revised edition  and the guidelines of current Good Clinical Practice. Local ethics committees approved the study protocol and documents relevant to their local regulatory requirements prior to recruitment to the survey. Written informed consent was obtained before any observational data were collected for inclusion in the survey. The right to request withdrawal of data from the study was reserved at any time.The primary endpoint was the percentage of participants with a grade 3 or 4 AE. Secondary endpoints included the percentage of participants with <50 copies/mL and <400 copies/mL HIV RNA at week 24 and the increase in mean CD4+ cell count (cells/µL) from baseline to week 24. The efficacy analysis was based on the intent-to-treat population. Overall tolerability and efficacy  were assessed by the investigator at week 24. Analysis of safety and efficacy was also undertaken for the following subpopulations: treatment-naïve; treatment-experienced but naïve to PIs; switched from an alternative PI regimen  due to toxicity; switched from a prior non-PI regimen due to toxicity; infected with hepatitis C virus (HCV); prior treatment at any time with a regimen that included a PI ; and prior treatment at any time with a regimen that did not include a PI. Participants could be included in more than one subpopulation. Note that these categories are not mutually exclusive, ie, a participant could have received a prior treatment regimen that included a PI as well as one that did not include a PI. In addition, AEs were analyzed according to gender, race/ethnicity, and country of residence. Adverse events, reported diseases, and medications were not coded but provided as listings of the original terminology. Participants who discontinued SQV/r treatment for any reason prior to week 24 were included in the survey as treatment failures.A planned sample size of 2000 was selected as sufficient to gain representative information about treatment experience. Descriptive summary statistics were used in the analysis of this survey.A total of 119 investigators at centers in Austria, Germany, Latvia, the Netherlands, Poland, Spain, Switzerland, and the United Kingdom enrolled 2309 participants between May 3, 2005 and September 20, 2007. Of those enrolled, 2122 were eligible for analysis and were included in the “all participants” population . The 187The number of participants in each subpopulation is presented in There were 33 reports of grade 3 or 4 AEs in 29 (1.4%) participants in the “all participants” population . Of thesThe incidence of AEs was comparable between men and women (6.7% vs. 7.4%) and across ethnicities. The highest occurrence of AEs by country was reported for participants from Spain while the lowest incidence (0.8%) was reported in those from Poland. The investigators rated the tolerability of SQV 500 mg as “very good” in 42% of participants, “good” in 25%, “sufficient” in 4%, and “insufficient” in less than 2% . No infoMean increases from baseline to 24 weeks in fasting plasma lipids are shown in Mean fasting blood glucose levels were elevated at baseline in three treatment groups : PI-naïve, treatment-experienced , toxicity switch from prior non-PI regimen , and HCV coinfected . All had mean reductions resulting in normal levels by 24 weeks: PI-naïve, treatment-experienced , toxicity switch from prior non-PI regimen , and HCV co-infected .The percentages of participants in the intent-to-treat population with HIV RNA viral loads of <50 and <400 copies/mL at baseline and at week 24 for each of the subpopulations are shown in An increase in CD4+ cell count was also seen during the survey period in 523/734 (71%) of participants in the overall population having CD4+ cell count determinations . The larThis observational survey demonstrated that treatment with the 500 mg film-coated tablet of SQV was well tolerated and effective in HIV-infected participants. Not only was there a very low incidence of grade 3 or 4 AEs, investigators rated the tolerability of the regimen as “very good” or “good” in 67% of their participants. A comparison of the efficacy across subpopulations showed that the majority had a favorable response to treatment, with those who were treatment- and PI-naïve deriving the most benefits. This is to be expected since these subpopulations would be most susceptible to a new regimen or to the addition of a new class of agent.There were certain limitations to this survey, including its open-label design and its subjective assessment of tolerability (which could have biased the results) and the short duration of follow-up of 24 weeks. Still, the lack of blinding or of a control regimen was unlikely to bias the laboratory-based virologic efficacy endpoints. Furthermore, it was not possible to control for the variability in ARV regimens and populations across study sites. However, the value of this survey is its “real-world” clinical setting, which increases the level of heterogeneity of the enrolled participants and the concomitant ARV therapies. These results compare favorably with those of a recent study that showed that at 24 weeks, approximately 60% of trial nonparticipants  achieved a viral load <500 copies/mL when initiating treatment with one of three PI-based regimens . In contP < 0.012); triglyceride levels were significantly higher in the LPV/r group at week 48. The present study showed that in both treatment-naïve and treatment-experienced patients, oral SQV 500 mg was effective and well tolerated with a more convenient and practical dosing regimen than prior SQV-containing regimens.The GEMINI study , an open


Protease inhibitors (PIs) have been associated with metabolic complications. There is a trend to switch to simpler therapy to improve these disturbances. We report a case-series describing the effects in metabolic abnormalities in seven HIV-infected children, previously treated with protease inhibitor (PI) after switching to nevirapine.Seven children with stable PI-containing regimen and a long lasting HIV-1 RNA < 50 copies/ml were switched to nevirapine. All patients were naïve to non nucleoside reverse transcriptase inhibitor. PIs were switched to nevirapine. Preentry nucleoside reverse transcriptase inhibitors were maintained. The substitution of PIs with nevirapine was made when the patient showed hyperlipidemia or lipodystrophy or the physician and/or the patient's willingness to simplify. Clinical, laboratory data and anthropometric parameters were assessed every 3 months. Dual-energy X-Ray absorptiometry scans (DXA) was performed at baseline and at 12 months.Seven HIV-infected children were enrolled. Median age: 130 months . Median baseline CD4%: 32%. All had HIV-1 RNA < 50 copies/ml. Median length of preentry PI-therapy was 47 months . Median age at the beginning of nevirapine was 120 months . Median decrease in cholesterol in 7.2 mmol/L was observed (P = 0.09), from baseline to 12 months. HDL-cholesterol increased in 5.1 mmol/L (P = 0.03) throughout the study period. No significant changes were observed in DXA with regard to body fat, but changes in total body bone mineral content and lean body content were significant. CD4% remained stable. All patients but one maintained viral load < 50 copies/ml at 12 months. The patient with virologic failure referred bad adherence. Children referred to take medication more easily.PI substitution with nevirapine improved lipid profile in our patients, although this strategy did not show significant changes in body fat or lipodystrophy. Metabolic complications are prevalent in HIV-infected children treated with highly active antiretroviral therapy (HAART). Nowadays, lipodystrophy and osteopenia had also been reported in HIV-infected children . LipodyChildren's regimens were changed if they fulfilled these criteria: all were older than 12 months; they were receiving a stable PI-containing regimen (2 nucleoside reverse transcriptase inhibitor (NRTI) plus 1 or 2 PIs) for at least 6 consecutive months; they were naive to non nucleoside reverse transcriptase inhibitor (NNRTI). All patients had plasma HIV-1 RNA viral load < 50 copies/ml at baseline and for a median of 6 months before study entry and a stable clinical and immunological situation. Then, PIs were switched to nevirapine and preentry NRTI were maintained. The substitution of PIs with nevirapine was made if the patient showed hyperlipidemia and/or lipodystrophy or if the physician or the family wanted to simplify the antiretroviral regimen. Hyperlipidemia was considered if cholesterol was ≥ 6.5 mmol/L and/or LDL-cholesterol was ≥ 4 mmol/L and/or triglycerides were ≥ 5.5 mmol/L. Glucose metabolism was evaluated and was considered abnormal if fasting glucose was between 100 mg/dl and 126 mg/dl. Insulin levels and C-peptide were also performed and were considered abnormal if insulin was > 15 UI/ml and C-peptide was > 2 ng/ml. Lipodystrophy was diagnosed if patients presented at least one of the following features: peripheral loss of adipose tissue: sunken cheeks, thinning extremities, hips or buttocks (lipoatrophy), central gain in adiposity: dorsocervical and/or abdomen fat accumulation (lipohypertrophy) or both features (mixed syndrome).Children were excluded if they suffered from hepatitis C virus infection or if they showed aminotransferases elevations or if they were receiving lipid-lowering drugs. Standard clinical and laboratory data were performed every 3 months. Laboratory data included: CD4 cell count, plasma HIV-1 RNA viral load , blood counts and blood chemistries . All data were obtained after 8–12 hours of fasting. No dietary or exercise history was recorded.Metabolic evaluation and anthropometric measurements were performed every 6 months including C-peptide, insulin, lipid profile, body weight, height, mid-arm circumference, triceps and subescapular skin folds. Skin folds were measured using a manual trackball. Body weight was measured on a balance beam scale, and height was measured using the same wall-mounted stadiometer. Measurements were performed by the same person. Dual-energy X-Ray absorptiometry scans (DXA) was used to estimate whole-body composition. The same scanner (Norland XR-26HS densitometer) was used for evaluations on any patient. The entire body was scanned. Variables analysed including: bone mineral density (BMD), expressed in g/cm2, lean body mass (LBM) expressed in grams and body fat expressed in grams and as a percentage of total body weight. All compartments were analysed in the arms, trunk, legs and face. Z score for lumbar BMD and BMC-age related changes were estimated based on Zachetta's report . In thisThe statistical software Staview Mc Intosh was used. Quantitative data were expressed in terms of means and range. Qualitative variables were expressed as percentages. Wilcoxon rank sum test for continuous data was used to determinate changes over time and statistical significance. P < 0.05 were considered statistically significant.Written consent was obtained from six of the patients.Seven patients were enrolled (2 girls and 5 boys), median age was 120 months . All were Caucasians. Six children acquired infection by vertical transmission. One girl had HIV infection of unknown origin. At baseline, median CD4 percentage was 33%(29–42) and all had viral load under 50 copies/ml at baseline and at least 6 months before. Related to CDC category, 2 patients were on A1, 2 on C3, 2 on B2 and one on A3. At the beginning of the follow up, 4 children were prepuberal. A girl reached stage IV at 12 years. Other 2 patients reached Tanner stage II.  at baseline to 37%  at 12 months, (P = 0.7).At the beginning, cholesterol was ≥ 6.5 mmol/l in 3 patients, LDL-cholesterol was ≥ 4 mmol/l in 3/6 patients and HDL-cholesterol was ≤ 1.16 mmol/l in 4/6 patients. At study entry, in all patients, triglyceride levels, fasting glucose, insulin, C-peptide, amylase or lipase were normal and did not change throughout the study period , (data not shown). Lipodystrophy was clinically detected at the beginning in 2 patients, one subject showed clinical signs of peripheral lipoatrophy and another patient had central lipohypertrophy. At 12 months, there were no changes in patients with lipoatrophy, but there was a decrease in abdominal fat accumulation in the patient with central hypertrophy, this child were receiving indinavir before changing.Patients were asked about compliance and difficulty in taking medication. All of them referred a sense of less difficulty in taking nevirapine compared with the PI-regimen.A 13-year-old girl who was receiving d4T + 3TC + amprenavir boosted with ritonavir was simplified to d4T + 3TC + nevirapine because she suffered from hypercholesterolemia. A decrease in total cholesterol, LDL cholesterol and triglycerides was observed. She remained with VL < 50 copies/ml but her lumbar BMD Z score showed a decrease from -1.05 to -3.34.A 9-year-old girl who was receiving d4T + 3TC + amprenavir was simplified to d4T + 3TC + nevirapine because she wanted to do her regimen easier to take. She referred that she was happier after this change. She remained with undetectable viral load. She did not show osteopenia along the follow up.An 8-year-old-boy who was receiving ZDV + ddI + ritonavir was simplified to ZDV + ddI + nevirapine because he suffered from hypercholesterolemia. A light decrease in total cholesterol was observed but not in LDL-cholesterol. He remained with undetectable viral load and without osteopenia.A 9-year-old girl who was receiving d4T + 3TC + nelfinavir was simplified to d4T + 3TC + nevirapine because she suffered from hypercholesterolemia. A decrease in total cholesterol and in LDL-cholesterol was observed. She showed undetectable viral load at 12 months and the osteopenia remained.A 10-year-old boy who was receiving d4T + abacavir + indinavir was simplified to d4T + abacavir + nevirapine because he wanted to do his regimen easier to take and to avoid renal damage associated to indinavir.A 14 year-old adolescent who was receiving ZDV + ddI + indinavir was simplified to ZDV + ddI + nevirapine because he wanted to do his regimen easier to take and to avoid renal damage associated to indinavir. At 12 months he showed a decrease in total cholesterol and LDL-cholesterol but had detectable viral load because of bad adherence. He suffered from facial lipoatrophy and it remained without changes after the substitution (we have to take into account that NRTIs were not removed and these drugs are implied in fat distribution).An 8-year-old boy who was receiving ZDV + 3TC + indinavir was simplified to ZDV + 3TC + nevirapine because he wanted to do his regimen easier to take, to avoid renal damage associated to indinavir and to improve his abdominal lipohypertrophy. He showed an important decrease in abdominal fat accumulation at the end of the year. His viral load remained undetectable.We report seven cases in which the substitution of PIs  with nevirapine resulted in a mild improve in total cholesterol and HDL-cholesterol. Virological maintenance was achieved in all patients but one during the 12 months. The patients that showed virological failure at 12 month referred bad adherence although the child's adherence at baseline was considered good and the new regimen was less complex. Nevertheless, all children maintained CD4 cell count and good clinical and growth parameters.PIs may have a strong association with dyslipidemia ,3,5,9-12-129-12, Some PIs including indinavir, have been associated with insulin resistance and an improvement in insulin sensitivity has been observed in some studies when PIs were switch to NNRTI or abacavir ,30. In oWith regard to anthropometric measurements, there was an increase in weight, height and in all the skin folds from baseline to 12 months in all patients. Nevertheless, only the increases in weight, height and mild-arm circumference were significant. These changes could have been due to normal growth in these children, one of the parameter that we are not able to evaluate due to lack of a control arm.About DXA follow up, significant changes in TBMC, LBM have been observed, but no in fat content. Therefore, in all patients there was an increase in TBMC and LMB and there was a decrease in total fat in 3 children, but changes were not significative and all gained weight with regard to baseline. Lumbar BMD Z score and BMC – age related changes did not change significantly during the study period.Physical examinations did not show a clinical improvement in lipoatrophy. This was expected due to the continuous use of zidovudine plus didanosine in this boy. In one patient a decrease in central obesity was observed. He had been receiving Indinavir before nevirapine-regimen and this PI had been associated to central lipohypertropy . These cAlthough adherence questionnaires were not performed, our patients referred a feeling of an easier to take medication and all their new regimens contained fewer pills than the previous therapy.This case-series includes only seven patients and 12 months may not be sufficient for a correct follow up to detect significant changes in lipid and mainly in anthropometrics parameters, specially in children and adolescents, who are experimenting continuous changes in body composition and therefore, the observed changes in these cases could be related to others factors like growth, puberty and normal physical development. Therefore, this strategy could be an option in selected children but more studies are necessary.The substitution of PI with nevirapine may be an option in selected HIV-infected children that might lead to improvement in some lipid abnormalities as other studies have showed ,13-17.HAART: Highly active antiretroviral therapy; PIs: Protease inhibitors; LD: Lipodystrophy; BMD: Bone mineral density; NRTI: Nucleoside reverse transcriptase inhibitor; NNRTI: Non nucleoside reverse transcriptase inhibitor; DXA: Dual-energy X-Ray absorptiometry scans; TBMC: Total body bone mineral content; LBM: Lean body mass.This study has been performed with the support of Boehringer Ingelheim Pharmaceuticals. Boehringer Ingelheim Pharmaceuticals is the manufacturer of Nevirapine.All the authors contributed to design the study, included patients and performed the follow up. JTRA was the principal investigator, MLNG, MJMP, PMF and PRC, also participated in the design of the study and in the follow up of the patients. All of them also contributed to write the article and have approved the final manuscript.The pre-publication history for this paper can be accessed here:


This is a prospective study evaluating the efficacy of botulinum toxin A injection in benign prostatic hyperplasia (BPH) patients. Twenty-one men with benign prostatic enlargement on chronic indwelling catheter for at least 3 months who were not candidates for surgery because of poor general condition received 200 U botulinum toxin A in the transition zone by transrectal approach under ultrasound guidance. This was done as an outpatient procedure without any anesthesia. Mean patient age was 80 ± 2 years. No significant local effects occurred. Patients were reevaluated at 1 and 3 months post-treatment. Baseline prostate volume of 70 ± 10 ml decreased to 57 ± 10 ml at 1 month and to 47 ± 7 ml at 3 months. At 1 month, 16 patients (76%) could resume voiding with a mean Qmax OF 9.0 ± 1.2 ml/s. At 3 months, 17 patients (81%) voided with a mean Qmax of 10.3 ± 1.4 ml/s. Residual urine was 80 ± 19 ml and 92 ± 24 ml at 1 and 3 months, respectively. Mean serum total PSA decreased from 6.0 ± 1.1 ng/ml at baseline to 5.0 ± 0.9 ng/ml at 3 months. The authors conclude that botulinum A injection into the prostate swiftly reduces prostate volume and may be a promising treatment for refractory urinary retention in patients with benign prostatic enlargement who are unfit for surgery.The effects of botulinum toxin A on the prostate has been studied in different species, including rats, dogs, and humans. The exac


The majority of antiretroviral treatment programmes in sub-Saharan Africa are scaling up antiretroviral treatment using a fixed dose first-line antiretroviral regimen containing stavudine, lamivudine, and nevirapine. One of the primary concerns with the use of this regimen is nevirapine-associated hepatotoxicity.Study participants were 1809 HIV-infected, antiretroviral naïve adults initiating nevirapine-based antiretroviral therapy between November 2002 and December 2006. The primary outcome was early hepatotoxicity. Secondary outcomes were associations with hepatotoxicity and mortality at six months. The cumulative proportion of early hepatotoxicity ranged from 1.0–2.0% giving an incidence-rate at 102 days of 3.6–7.6 per 100 person-years. Median time to hepatotoxicity was 32 (IQR 28–58) days. At 12 weeks, only 8% of patients had alanine aminotransferase monitoring at all the time-points recommended by national guidelines. No association was found between age, gender, baseline CD4 count, concurrent tuberculosis infection, prior participation in a prevention of mother-to-child-transmission program, or baseline weight and early hepatotoxicity. There was no association between early hepatotoxicity and mortality.The cumulative proportion of early hepatotoxicity in nevirapine based antiretroviral therapy was low in this resource-constrained setting. Hepatotoxicity was not associated with mortality. Frequent routine monitoring of alanine aminotransferase proved difficult to implement in this public sector primary care programme. Focused monitoring in the first month may be a more cost-effective and pragmatic option in settings with limited resources. Correlation with clinical signs and symptoms may allow future alanine aminotransferase testing to be dictated by clinical criteria. The majority of antiretroviral treatment programmes in sub-Saharan Africa are scaling up antiretroviral treatment (ART) using a fixed dose first-line antiretroviral regimens containing stavudine, lamivudine and nevirapine (NVP) Alcohol and hepatitis C co-infection are well established risk factors for hepatotoxicity but the effects of other host characteristics are not well studied In clinical trials, mostly conducted in resource-rich countries, NVP- associated HT ranges from 8–18% This study included patients from three primary care HIV clinics in Khayelitsha township, South Africa, where antiretroviral treatment (ART) has been provided by Médecins Sans Frontières (MSF) since 2001 in collaboration with the Provincial Department of Health. These clinics follow national South African treatment guidelines which comply with WHO guidelines: all patients were started on either NVP or EFV-based ART. NVP-based regimens were initiated with the recommended lead-in dose of NVP 200 mg daily for 14 days followed by NVP 200 mg twice daily. ALT testing was offered to patients starting NVP-based ART at baseline, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks then every 6 months thereafter. Specimens for laboratory testing were transported the same day to a single regional laboratory run by the National Health Laboratory Services which applied stringent quality control measures. ALT assays were conducted using a spectrophotometer following standard operating procedures.All patients initiating NVP-based ART from November 2002 (when ALT testing became available) until the end of December 2006 and who were on NVP for at least four weeks were considered for analysis. For the analysis of HT, patients were stratified into two groups: those who had ALT monitoring for at least 4 weeks at all time points recommended by national guidelines (complete monitoring) and those with incomplete monitoring. Those who were treatment-experienced and children under 16 years old were excluded. Women who had previously received single or dual NVP-based therapy via the prevention of mother to child transmission (PMTCT) program were also included.Serum ALT was graded according to criteria established by the AIDS Clinical Trial Group Patient time on NVP was calculated from treatment commencement to HT or until the drug was stopped or substituted for other reasons. Mortality was evaluated up to 6 months after initiation of NVP-based ART.Data was exported from a database prospectively maintained for routine monitoring and evaluation. Serum ALT values were obtained from the laboratory and merged using Stata . Baseline characteristics were described using medians and interquartile ranges (IQRs) for continuous variables and counts and percentages for categorical data. Means were compared using the student's t-test and proportions using the chi squared test. Person-time of observation were accrued from the initiation on NVP-based ART until either occurrence of death, loss to follow-up, transfer to another ART programme or censoring of observation in July 2007. Kaplan–Meier analysis was used to estimate cumulative HT. Cox proportional hazards were used to determine associations between baseline characteristics and HT and mortality. Logistic regression was used to determine associations between baseline characteristics and grade 3 or 4 ALT baseline elevation. Variables considered in the analysis for early HT and baseline ALT elevation included age, gender, baseline CD4 cell count (cells/µl), increase in CD4 count >100 cells/µl at 6 months, baseline weight less than 60 kg, concurrent TB treatment, and prior prevention of mother-to-child transmission (PMTCT) program enrolment. Early HT was also considered in the analysis for mortality. All tests and confidence intervals (CI) were considered to be significant at a p≤0.05 (two-sided). An additional chart review was conducted on patients with early HT to obtain information on viral hepatitis testing, associated symptoms, outcome of HT, drug regimen changes, and death attributable to HT.Procedures for selection and inclusion of patients within the ARV program followed the national guidelines for the implementation and roll-out of ART. General measures were provided to ensure patient confidentiality, consent for HIV testing, and counselling and support for those who receive a positive HIV test results. ART and all laboratory testing services (including ALT) were offered free of charge. The University of Cape Town ethics committee gave approval for this study without requiring patient consent because the analysis was based on routine clinical data. No additional data were collected specifically for this study, and the data were fully de-identified prior to analysis.From November 2002 to December 2006, 3728 HIV-infected, ART-naïve individuals were initiated on ART. Of these, 1831 (49%) were started on NVP- based first-line therapy; 1809 (99%) remained on NVP at 4 weeks. Of these 911 (50%) patients had at least a baseline and a 4 week ALT measurement (complete monitoring) and 898 (50%) had either a missing baseline or 4 week ALT measurement (incomplete monitoring). Patient characteristics of both these groups on NVP are shown in Of the 1831 patients on NVP at baseline, 1444 (79%) patients had a baseline ALT measurement. Of the 1809 patients on NVP at 4 weeks, 911(50%) had ALT measured at baseline and 4 weeks. Of the 1705 patients on NVP at 8 weeks, 368 (22%) had complete ALT monitoring , and at 12 weeks, only 127 (8%) had all recommended ALT monitoring . ALT grades for these visits  are shown in Twenty-six patients developed early HT. The median time to early HT was 32 (IQR 28–63) days. Among patients who had complete ALT monitoring, the cumulative proportion of early HT was 2.0% by 102 days on NVP giving an incidence rate of 7.6 (95% CI: 4.8–12.1) per 100 person-years over this period. The cumulative proportion with HT in patients with incomplete ALT monitoring was 1.0% by 102 days on NVP giving an incidence-rate of 3.6 (95% CI: 1.8–7.2) per 100 person-years over this period .Of the 26 patients with early HT, 14 (54%) had grade 3 HT and 12 (46%) had grade 4 HT. Four (15%) were being treated for active tuberculosis. Eight (31%) inadvertently continued NVP-based ART of which 7 (88%) resolved their HT and one was LTFU. Of the 18 who stopped NVP, all switched to efavirenz (EFV) based-ART. Of these, 2 (12%) developed further HT on EFV. Symptoms were known for 17 (65%). Of these 8 (47%) had either a rash or symptoms consistent with hepatitis . Eleven There were no associations found between baseline characteristics and hepatotoxicity including gender , age , baseline CD4 count , and increase in CD4 count >100 cells/µl at 6 months . .Seventy-three (4%) of 1809 patients died within 6 months of starting NVP based ART. In patients with at least baseline and 4 week ALT measurements (complete monitoring), 16/911 (2%) died in the first 6 months. In this group, 18/911 patients had early HT, of whom 2/18 (11%) died. One of these deaths was HT-related. In the patients with incomplete ALT monitoring, 57/898 (6%) died in the first 6 months. In this group, 8/898 had early HT, of whom one (12%) died, of non-HT causes. All three patients who died with early HT had grade 4 ALT elevations. There was no mortality at 6 months in patients who developed late HT.Baseline CD4 count >100 cells/µl was protective against mortality  on multivariate analysis. Variables not associated with mortality included age , gender , HT , ALT elevation at baseline , current TB status , and PMTCT attendance . .The cumulative proportion of early HT was 1.0–2.0% and not associated with increased mortality. This is comparable to low incidence proportions reported by Hahn et al (2.2%) from an ARV program in Uganda and Meyssonnier et al (1.2%) in a program from Niger Programmatic ALT monitoring was enforced in less than one tenth of patients at 3 months. This highlights the difficulty of implementing serial ALT monitoring in high-burden public sector clinics. Even when monitoring schedules were adhered to, the proportion of patients with severe hepatotoxicity was very low. Furthermore, patients with ALT elevations who were also symptomatic could have been identified by their symptoms. Finally, many patients who had HT by ALT elevation were left on NVP due to administrative delays in receiving the laboratory results and subsequently revert to normal levels. This study did not demonstrate that ALT monitoring according to national guidelines adverted severe or fatal hepatotoxicity. The one HT-related death occurred in a patient with complete monitoring.The medium time to early HT was 32 days and was consistent with other studies We did not identify any risk factors for HT. In particular, a higher CD4 was not associated with HT. This contrasts with previous findings that men and women with CD4 counts greater than 400 cells/µl and 250 cells/µl, respectively, are at greatest risk This study had a number of strengths and limitations. First, this is one of the largest cohorts of patients on NVP-based ART in southern Africa. Second, laboratory ALT values were used to determine HT so misclassification was unlikely. Finally, the availability of a group with complete ALT monitoring allowed calculation of ALT grades by weeks on NVP. The main limitation was that there were many patients with incomplete ALT monitoring and transient HT may have been missed which likely accounted for the lower incidence rate of early HT in this group. However, the incidence rate in the group with complete monitoring likely reflects true early HT. Another limitation was that other risk factors for HT such as alcohol use and hepatitis co-infection could not be evaluated We believe our findings have important public health implications regarding ALT monitoring and the risk of nevirapine-based ART regimens. The risk of life-threatening hepatotoxicity has led NVP manufacturers to warn against its use without close serological and clinical monitoring and in the US and Europe serial serum ALT testing is routine. In resource-limited settings such as in sub-Saharan Africa where NVP is the mostly commonly used NNRTI, ALT monitoring is expensive and often technologically not feasible and its use in routine programmes needs to be carefully considered. In our cohort in South Africa, the incidence rate of early HT is low in this population with a low median baseline CD4 count. Routine ALT monitoring was not consistently implemented and not associated with mortality. Taking into account these considerations, focused monitoring in the first month may be a more cost-effective and pragmatic option in settings with limited resources. Correlation with clinical signs and symptoms may allow future ALT testing to be dictated by clinical criteria.Table S1Associations between patient characteristics and hepatotoxicity.(0.06 MB DOC)Click here for additional data file.Table S2Associations between patient characteristics and mortality.(0.06 MB DOC)Click here for additional data file.


Short cycle treatment interruption could reduce toxicity and drug costs and contribute to further expansion of antiretroviral therapy (ART) programs.3 and HIV RNA plasma levels <50 copies/ml) in one of three arms: continuous, 7 days on/7 days off and 5 days on/2 days off treatment. Primary endpoint was ART treatment failure determined by plasma HIV RNA level, CD4+ cell count decrease, death attributed to study participation, or opportunistic infection.A 72 week, non-inferiority trial enrolled one hundred forty six HIV positive persons receiving ART  participants in the 5 days on/2 days off arm failed. Five had virologic failure and one participant had immunologic failure. Eleven of 51 (21.6%) participants in the continuous treatment arm failed. Nine had virologic failure with 1 death (lactic acidosis) and 1 clinical failure (extra-pulmonary TB). The upper 97.5% confidence boundary for the difference between the percent of non-failures in the 5 days on/2 days off arm (88.5% non-failure) compared to continuous treatment (78.4% non failure) was 4.8% which is well within the preset non-inferiority margin of 15%. No significant difference was found in time to failure in the 2 study arms (p = 0.39).Short cycle 5 days on/2 days off intermittent ART was at least as effective as continuous therapy.NCT00339456ClinicalTrials.gov  Combination antiretroviral therapy (ART) has significantly decreased morbidity and mortality for HIV-infected persons who have access to such therapy.Researchers have previously evaluated several approaches of structured treatment interruptions to enhance adherence and reduce toxicity and cost.We here report an evaluation of a randomized, controlled, non-inferiority trial of two regimens of short cycle intermittent compared to continuous ART on virologic, immunologic, toxicity and adherence parameters.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.Ethics Statement: The study protocol was reviewed and approved by the Uganda National Council for Science and Technology (UNCST), and by the Institutional Review Boards (IRBs) of the National AIDS Research Committee of the UNCST, Uganda and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID/NIH).3 of whole blood and plasma HIV RNA below 50 copies per milliliter of plasma were eligible following signed informed consent. A confirmatory HIV test was required prior to enrollment. Participants were required to be receiving at least 3 ARVs including a protease inhibitor or the non-nucleoside reverse transcriptase inhibitor, efavirenz. Patients receiving a nevirapine-based regime were required to switch to a protease inhibitor or efavirenz if they were randomized to the interrupted arm. Nine patients in the 5 days on/2 days off arm and 7 participants in the 7 days on/7 days off arm were on nevirapine and switched to efavirenz for study enrollment. Stavudine was dosed by weight, participants weighing >60 kg received 40 mg, those weighing <60 kg received 30 mg. All participants paid for their ARVs, consistent with standard practice at the JCRC. It should be noted that at the time the study was initiated, large international programs to support the cost of ART had not begun. Such programming was not available until a large number of patients had already been enrolled. It was the decision of the investigators and the review boards that changing the payment practices mid-study could bias the results. Trial oversight was provided by and independent Data Safety and Monitoring Board (DSMB). Clinicaltrials.gov registry number NCT00339456.HIV-infected persons receiving ART at the Joint Clinical Research Center (JCRC) in Kampala, Uganda, with a CD4+ cell count equal to or greater than 125 cells/mmThe study was designed to test non-inferiority of two short cycle intermittent ART regimens (7 days on/7 days off and 5 days on/2 days off) compared to continuous ART with 57 patients  randomized in a 1-1-1 manner to each arm and followed for 72 weeks . The primary endpoint was ART treatment failure determined by a plasma HIV RNA level equal to or greater than 10,000 copies on any one evaluation, a plasma HIV RNA level equal to or greater than 1,000 copies on two consecutive measurements, a plasma HIV RNA level greater than 400 copies/ml at the end of the study, a CD4+ cell count decrease of greater than 30 percent from baseline on 2 consecutive measurements, death attributed to study participation or occurrence of an opportunistic infection. The primary endpoint was changed during the study by the protocol team from <50 copies/ml to <400 copies/ml (end of study) to eliminate the inclusion of low-level viral blips. Patients who experienced treatment failure received standard clinical care in Uganda including switching of ARV regimen, where appropriate. They were followed until 30 days after their plasma viremia and/or CD4+ cell count returned to baseline if they changed therapy or for 30 days from the time of treatment failure if they decided not to change therapy. Laboratory safety monitoring and HIV viral load measurements  were performed on site at the JCRC. HIV genotyping  was performed off-site at SAIC-Frederick, Inc., Frederick, Maryland.Participants were evaluated for standard clinical, virologic, immunologic, and adherence parameters at weeks 2 and 4 following enrollment  and every 6 weeks thereafter including toxicity monitoring . Lipodystropy, peripheral neuropathy and lactic acidosis were determined by the treating physician's assessment, clinical symptoms and laboratory confirmation (serum lactate). All laboratory evaluations were provided to the participants free of charge. Intervening visits were scheduled based on clinical standard of care or relevant failure parameters. All evaluations except the end of study evaluation were performed at the end of the off drug period in the intermittent arm. Adherence to ART was measured using patient maintained study calendars which were recorded at all study visits, adherence rates based on self-reported diaries were then calculated based on the percentage of drugs taken.An independent data and safety monitoring board (DSMB) reviewed all safety data and treatment failures on a bi-annual basis and at least yearly after 50% of participants were enrolled in each treatment arm.A sample size of 57 individuals in each treatment group  was determined for a 95% non-failure rate in each study group with a non-inferiority margin of 15%, an alpha (type I error) of 0.025 and 81% power using the Farrington and Manning method.Paired differences and the median correlations, determined by the Spearman rank method, were tested for significance by the Wilcoxon sign rank test. The Wilcoxon two-sample test was used to compare independent groups, and Fisher's exact test was used to compare frequencies of categorical variables. Comparison of Kaplan-Meier survival curves was done by the log-rank test. The significance of the ratios of rates of occurrences of adverse events was determined by the exact rate ratio test based on the Poisson distribution. Exact methods were used so that p values and confidence intervals are properly defined even when the counts are very small or zero.The study was funded through the Division of Intramural Research, NIAID/NIH. SJR, CWH, MD, RTD, ASF, TCQ & MAD were employees of the NIAID/NIH during the study design and implementation period.A total of 146 HIV-positive participants were enrolled in the trial between 2002 and 2005: 32 in the 7 days on/7 days off arm, 57 in the 5 days on/2 days off arm and 57 in the continuous arm. Five individuals in the 5 days on 2 days off group left the study . Five in the continuous arm were lost-to-follow-up resulting in 52 with data in the interrupted and 51 in the continuous arm at week 72/73 . Fifty-sFollowing the enrollment of 32 participants in the 7 days on and 7 days off arm, the DSMB agreed with the principal investigator to terminate that arm because of a failure rate of 31% found during the interim analysis in the fall of 2004 (3 participants had withdrawn and 9/29 remaining participants had failed).Six of 52 (11.5%) participants who completed the 5 days on/2 days off arm failed. Five of these participants had virologic failure all with a viral load greater than 10,000 copies/ml  14,398 to 50,551 and one participant had immunologic failure. In comparison, eleven of 51 (21.6%) participants who completed the continuous treatment arm failed. Nine of these participants had virologic failure. Six failed with a viral load greater than 10,000 copies/ml, one with a viral load greater than 1,000 and 2 with a viral load greater than 400 at week 72 The remaining 2 failures included 1 death (lactic acidosis) and 1 clinical failure (extra-pulmonary TB). The upper 97.5% confidence boundary for the difference between the percent of non-failures in the 5 days on/2 days off (88.5% non-failure) arm compared to continuous treatment (78.4% non failure) was 4.8% which is well within the preset non-inferiority margin of 15%. In the modified intent-to-treat analysis which designated the 5 in each group who discontinued treatment as failures, the upper 97.5% confidence boundary for the difference between the percent of non-failures in the 5 days on/2 days off, 80.7% (46 of 57), and that in the continuously treated, 71.4% (40 of 56) was 6.6% which is within the preset non-inferiority margin of 15%. In the intent-to-treat analysis with 57 in each group, the upper 97.5% confidence boundary for the difference between the 80.7% (46 of 57) non-failures in the 5 days on/2 days off, and the 70.2% (40 of 57) non-failures in the continuously treated arm was 5.4% which is again well within the preset non-inferiority margin of 15%. No significant difference was found in time to failure in the 2 study arms, p = 0.39 .3, respectively, p = 0.85), at week 24 , at week 48  and marginally different at week 72/73 (p = 0.08) nor at week 48 . The median paired difference in CD4+ T cells was statistically greater in the 5 days on/2 days off arm at week 72/73 (p = 0.01) at week 12 of follow-up. This participant remained virologically suppressed (<50 copies/ml) through week 73 suggesting that this was not a true failure but merely a fluctuation in CD4+ T cell count. CD4+ T cell counts were correlated with weeks on treatment in the continuous treatment arm  and in the 5 days on, 2 days off arm . The correlations in the two groups were not statistically different (p = 0.33). For the 46 individuals in the 5 days on/2 off arm and the 40 in the continuously treated arm who successfully completed the study the median CD4+ T cell counts at baseline were similar in the two arms (255 and 271/mmp = 0.08). The medp = 0.01) None of The rates of lactic acidosis, lipodystrophy and peripheral neuropathy were compared among participants receiving stavudine within the continuous versus 5 days on/2 days off ART groups. The data were normalized for comparison between groups by reporting data as events per 100 person years. Among the 40 participants receiving continuous stavudine, 5 cases of lactic acidosis as determined by clinical findings and arterial blood levels of lactate occurred (resulting in one death), with an incidence of 11.4/100 person years, whereas no occurrences of lactic acidosis occurred among 44 participants receiving intermittent ART that included stavudine (p = 0.04). A reduction in lipodystrophy was also observed with 5 cases in the continuous treatment arm (incidence 11.4/100 person-years) compared with 1 case in the intermittent treatment arm (incidence 1.8/100 person-years) (p = 0.13). The rate of lipodystrophy in the continuous arm was 6.2 times the rate in the 5 days on and 2 days off arm . The rates of peripheral neuropathy were similar in both arms  with theOne patient receiving 5 days on and 2 days off ART died from chronic renal failure that predated enrollment; the event was determined to be unrelated to the study by investigators in Uganda. The patient had a plasma HIV RNA level of less than 50 copies per milliliter of plasma at death and a CD4+ T cell count at baseline and death of 171 and 349, respectively. No other significant hepatotoxicity, renal toxicity or anemia were observed during follow up with similar AST, ALT and creatinine measurements between study arms at 73 weeks . Median Overall adherence to ARVs for the study was greater than 95% in both study arms based on participant diaries. Pharmacy records were consistent with the high level of reported adherence measured during the study (data not shown).Clinical monitoring of mutations associated with antiretroviral drugs resistance was not available in Uganda during the study period. Consistent with standard clinical care at the JCRC and per protocol, and with review and monitoring by the DSMB and the IRB, patients were evaluated for failure by laboratory and clinical evaluations and treated according to standard clinical practice including switching of ARV regimen where appropriate. Genotypic evaluation of mutations associated with ARV resistance was performed by SAIC-Frederick, Inc., Frederick, Maryland and the results were made available to clinicians in Uganda.Genotyping was performed on 4 out of 5 of the participants in the 5 days on 2 days off arm at the first viral load measured >1000 copies/ml. Each participant was receiving an efavirenz-based regimen that included lamivudine and each had mutations consistent with resistance to efavirenz and lamivudine . No partThe present study demonstrates that short cycle intermittent ART defined as 5 days on/2 days off is at least as effective as continuous ART over a 72/73 week follow-up period. The data are consistent with the results of a pilot study that was begun in the United States after this randomized, controlled trial was underway and with a recent report of significantly increased risk of failure with interruptions beyond 2 days.Although the trial was not powered to show superiority, it was interesting that there were nearly twice as many failures in the continuous arm compared to the 5 days on/2 days off arm . The adherence data collected indicated high levels of adherence in each arm. However, 2 of 5 participants with virologic failure in the continuous arm evaluated for drug resistance had wild-type virus, suggesting poor adherence. Those results are compatible with the possibility that scheduled interruptions promote adherence during the on-drug period. All participants in the 5 days on/2 days off arm developed genetic mutations consistent with resistance to both lamivudine and efavirenz while 3 of 5 participants in the continuous arm developed mutations consistent with resistance to efavirenz and only 1 developed resistance to lamivudine with a second participant developing lamivudine resistance at a later follow-up visit. Our study was not powered to specifically look at differences in genotypic resistance patterns and also used self-reported adherence, known to be an imperfect measure, limiting our ability to draw any firm conclusions from these findings.There was evidence of reduced toxicities associated with intermittent ART. This was particularly striking among the subset of participants receiving stavudine containing ART which is commonly used in resource limited settings despite efforts to replace it with other nucleoside reverse transcriptase inhibitors. We observed a marked reduction in the rate of lactic acidosis among participants receiving intermittent stavudine with no cases detected in this group compared to 5 cases in the continuous treatment arm. A similar although not statistically significant reduction in lipodystrophy was also observed among participants receiving intermittent stavudine in our study. This finding is consistent with reports that mitochondrial toxicity associated with stavudine appears to be at least partially dose dependent.Immunologic responses to ART were robust in both intermittent and continuous treatment arms. Although there was a significantly greater increase in CD4+ T cell counts between arms for patients followed more than 52 weeks, the clinical relevance of this finding is unclear. Larger trials would be needed to validate those results and to determine their clinical significance. Consistent with recent publications from other resource limited settings, immunologic failure was not observed despite several episodes of virologic failure.The present study has several limitations. It was not powered to evaluate superiority or long term outcomes including death. The protocol included only subjects on efavirenz in the intermittent arms due to the concern of nevirapine resistance. Because nevirapine is extensively used in low and middle income countries this might limit the applicability of our findings. The continuous treatment arm had a higher than expected failure rate than used in the original statistical design which could impact on the overall conclusion of non-inferiority. The methodology used to measure adherence relied on participant diaries which may not reflect the true adherence levels of the different treatment arms in the study. Finally, the patient population at the JCRC tended to be of higher socioeconomic status during the time of the study and thus, most were able to pay for their ART. A larger trial will be needed to more fully address short cycle intermittent therapy among patients representing a wide range of demographies.Since 5 days on/2 days off intermittent therapy has been shown to be as least as effective as continuous therapy, this strategy could have particular importance for programs of directly observed therapy (DOT) for difficult to treat populations including children and adolescents attending school 5 days a week. It could also be relevant for workplace programs. The possibility of decreased toxicity could be relevant as treatment guidelines shift to initiating ART earlier in the course of disease. Drug treatment costs could also be 29 percent lower with a 5 days on/2 days off treatment regimen. Finally, as guidance shifts to earlier initiation of ART, a less expensive, “user-friendly” approach with reduced toxicity could be significant for scale-up of those strategies, particularly in the developing world.Protocol S1Trial Protocol.(0.08 MB PDF)Click here for additional data file.Checklist S1CONSORT Checklist.(0.19 MB DOC)Click here for additional data file.


The gut-associated lymphoid tissue (GALT) is the largest lymphoid organ infected by human immunodeficiency virus type 1 (HIV-1). It serves as a viral reservoir and host-pathogen interface in infection. This study examined whether different parts of the gut and peripheral blood lymphocytes (PBL) contain different drug-resistant HIV-1 variants.Gut biopsies  and PBL were obtained from 8 HIV-1 infected preHAART  patients at three visits over 18 months. Patients received AZT, ddI or combinations of AZT/ddI. HIV-1 Reverse transcriptase (RT)-coding sequences were amplified from viral DNA obtained from gut tissues and PBL, using nested PCR. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to phylogenetic analyses, and antiretroviral drug mutations were identified.Phylogenetic and drug mutation analyses revealed differential distribution of drug resistant mutations in the gut within patients. The level of drug-resistance conferred by the RT sequences was significantly different between different gut tissues and PBL, and varied with antiretroviral therapy. The sequences conferring the highest level of drug-resistance to AZT were found in the colon.This study confirms that different drug-resistant HIV-1 variants are present in different gut tissues, and it is the first report to document that particular gut tissues may select for drug resistant HIV-1 variants. Science has been confronted with the problem of drug-resistance virtually since the introduction of the first antiretroviral drugs to treat infection by human immunodeficiency virus type 1 (HIV-1)  ,25. The N) pair-wise distances between patients were calculated for the RT-coding sequences obtained from PBL, esophagus, stomach, duodenum, and colon for all patients at all visits  at the last visit for sequences derived from the PBL, esophagus, stomach, and the duodenum   (p < 0.05) towards the last visit between the patients was observed for the RT-coding region for both PBL and duodenum, while in the esophagus and stomach the dN decreased but fluctuated over time. In the colon the dN values increased , and nonsynonymous  Figure . A decred Figure , suggestThe RT sequences were subsequently subjected to phylogenetic analyses. Neighbour-joining trees of all RT sequences and bootstrap analysis revealed clustering of some sequences by tissue and patient . As we previously reported such clustering was not consistent for most of the sequences data not. The repWe analyzed the presence of mutations associated with NRTI resistance using the Stanford HIV-1 Drug-Resistance Database . As showp < 0.05 Chi-square test), and varied over time . Furthermore, the different tissues also differed significantly in distribution of drug-resistance mutations in the viral quasi-species (p < 0.05 Chi-square test). Our analysis revealed no evidence for a preferential presence of any specific drug-resistance mutations for any individual tissue compartment.These observations strongly suggest a differential distribution of antiretroviral drug-resistance in the different gut tissues, with drug-resistance mutations differing from those observed in the blood. These observations were further corroborated by sorting drug-mutations by tissue compartment (summarized in Table p < 0.05 Chi-square test), and similar results were obtained for the other two surviving patients . No significant differences in ddI resistance scores were observed following ddI treatment, although they tended to be higher in the stomach  among the different tissues following AZT or ddI treatment Figure  and 4D. The presence of HIV-1 antiretroviral drug-resistance in different tissues, such as the CNS, has been well document -33. HoweVarying viral diversity was observed for the RT-coding region in gut and PBL over time. Despite the fact that we were unable to obtain sequences for all lower GI tissues for a number of patients at visit 1, we observed a tendency towards a more conserved RT-coding region in the PBL, esophagus, stomach and duodenum between patients at the later visits. This may be a sign of adaptation of the virus to the different tissues, as there are some indications the RT-protein might affect cell tropism ,35. In aFor our current study, we were only able to consistently amplify HIV RT sequences from the integrated and nonintegrated viral DNA found in total tissue DNA. Therefore, our study was restricted to an analysis of HIV drug resistance of the banked viral reservoir and potentially not actively replicating viruses. Despite this limitation our data clearly indicated that antiviral drug resistant mutations are easily detected in the gut viral DNA reservoir. Furthermore, our data revealed that the viral gut reservoir is variable and dynamic. Significant changes together with selection for antiviral drug resistance occurred within a matter of weeks or months under continuous antiviral therapy. These banked viral reservoirs are clinically significant as they could be an important source of drug resistant viruses.Mycobacterium tuberculosis co-infected individuals [et al. [As in our previous study , analyseividuals . Howeverividuals . Moreoveividuals ,46. It i [et al.  postulatBased on our observations, one may conclude that AZT resistant viruses may arise first in the colon, and then start seeding the PBL and other gut tissues. The current data does not allow us to determine this unequivocally and further studies will be necessary. Our data did suggest that the colon selected for highly drug-resistant viruses. This could be due to the different antiretroviral drug-concentrations in the different tissues. Studies in rats have shown that after oral administration the intestinal absorption of zidovudine is lower in the colon compared to other parts of the intestinal tract (i.e. duodenum and jejunum). To our Our analysis focused on the primary drug resistance mutations in the main body of the RT encoding region. The sequencing method used did not analyze either the connection or RNase H domains of RT, both of which are known to contain sites that can affect levels of resistance to AZT -59. It wFinally, the patient samples for this study were collected during the preHAART era (1993-1996). Our analysis of antiretroviral drug-resistance in different parts of the gut in this period of the HIV epidemic is extremely relevant in the current era of HAART. Suboptimal treatment conditions still exist, in part due to patient noncompliance and toxicity of antiretroviral drugs. The data gathered from our studies about preHAART HIV-1 infection of the gut is also of importance for the HIV-1 epidemic in the developing world, where comprehensive HAART regimens may not be consistently available, and the proposed antiviral strategies may not be fully suppressive. Moreover, our observations are also relevant for other HIV-1 subtypes as they also have been shown to replicate differentially in the GALT (reviewed in ). The imIt has been proposed that HIV-1 can "hide" from antiretroviral therapy in the gut, and drug-resistance may be compartmentalized ,16,62. O+ cell counts at each study visit. At approximate 6 month intervals, upper and lower gastrointestinal endoscopies were performed and biopsies were collected from the esophagus, stomach, duodenum, and colon and stored at -70°C [+ cell count of 125 ± 122 cells/μl, and plasma viral load of 4.0 ± 0.8 log10 copies/ml at the last visit (Table Samples for this study had been collected from patients enrolled in a previous cohort study of HIV-1 seropositive men who have sex with men (MSM) followed at the Southern Alberta Clinic (SAC), Calgary, Alberta from 1993-1996 ,25. All at -70°C . Peripheat -70°C ,25. Thisat -70°C . Gut tism = 55°C), and for the second round consisted of RT 2604 5'-CCA AAA GTT AAA CAA TGG CCA TTG ACA-3' and RT 3251 5'-AGT TCA TAA CCC ATC CAA AG-3' (Tm = 55°C). Both primary and nested PCR reactions were performed with a high fidelity Taq polymerase to reduce the incorporation of mutations during amplification. To avoid selective amplification of the most dominant viral sequences at the expense of less frequent viral sequences due to high template concentrations or amplification of single viral DNA copies due to low template concentrations, optimal template concentrations were determined by dilution experiments. We used 2 to 10 fold dilutions of template DNA , and the dilutions that yielded the most abundant PCR products were used for analyses. To prevent contamination with amplicons, DNA isolation, PCR amplifications and subsequent cloning steps, were performed in separated rooms and laboratory areas. Negative  and positive  controls were included during all amplifications. The PCR fragments were separated and isolated from agarose gels, and directly sequenced to ensure genuine HIV-1 viral DNA had been amplified.Total DNA was isolated from tissue using Trizol Reagent , and HIV-1 reverse transcriptase (RT)-coding sequences (at nt. 2604-3251) were amplified from viral DNA by nested PCR as described previously . The ampEF656787 to EF656965, and EU931894 to EU932684).To analyze the HIV-1 quasi-species within each tissue sample, the nested PCR products identified as amplicons of genuine HIV-1 DNA were cloned into pCR2.1 TOPO linearized vector using the TA cloning kit . Plasmids containing relevant inserts were purified from bacteria using a Plasmid Mini-Prep Kit . The inserts were sequenced on an automated ABI sequencer  and a Li-Cor 4300 DNA Analysis sequencing system  according to manufacturers' protocols. For each compartment, 5-10 clones containing HIV-1 RT fragments were sequenced. For a number of samples we repeated both PCR and subsequent sequence analysis, and similar results were obtained, indicating our approach was reproducible. Sequences have been submitted to Genbank (http://www.megasoftware.net) [http://mesquiteproject.org)[N, codon changing substitutions), and synonymous  distances [The inferred amino acid sequence for the cloned DNA fragments was obtained for each sample, and screened for the integrity of coding sequences, i.e. stop codons, deletions, and/or insertions. DNA sequences were subjected to phylogenetic analysis using the Molecular Evolutionary Genetics Analysis (MEGA) version 4.0 software (are.net) . Neighboject.org)-28. Sequistances  for the http://hivdb.stanford.edu/) [All RT sequences were screened for drug-resistance mutations (NRTI as well as NNRTI) using the Stanford HIV-1 Drug-Resistance Database algorithm (rd.edu/) . Mutatiop values < 0.05 were considered significant.All statistical analyses were performed using SPSS version 11.5 , The authors declare that they have no competing interests.GvM, DLC, MAW MJG, and collected and analyzed data, designed the study, recruited patients and were involved in writing the paper. KN and KC were involved in designing the methods for collecting, analyzing and interpreting data, and helped putting the data and parts of the manuscript together for publication.Additional Data Table 1: Drug resistance mutations by tissue source. Drug resistance mutations by tissue source for all patients in the current study.Click here for file