Abstract: BACKGROUND: The prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced. RESULTS: In the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred. CONCLUSION: Subtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.

Abstract: This study was conducted among individuals taking first-line antiretroviral treatment (ART) for at least 12 months under programme conditions in Maputo, Mozambique in order to report on the level of detectable viraemia and the proportion and types of drug resistance mutations among those with detectable viral loads. HIV-1 RNA viral load levels (lower detection limit <50 copies/ml) were measured, and resistance mutations were sequenced. One hundred and forty-nine consecutive patients (69% females, median age 36 years) were included after a mean follow-up time of 23 months. One hundred and seven (72%; 95% CI 64-79) had undetectable viral load, while in 42 (28%, 95% CI 21-36) viral load was detectable (range 50-58884 copies/ml). From 15 patients with viral load >1000 copies/ml, 12 viruses were sequenced: eight were C subtypes and four were circulating recombinant forms (CRF08). Eight (5%; 95% CI 2-9) patients with detectable viral load had one or more major resistance mutations. Nucleoside reverse transcriptase inhibitor (NRTI) and non-NRTI mutations were observed. There were no major mutations for resistance to protease inhibitors. In Maputo, the level of detectable viraemia is reassuringly low. While embarking on ART scale-up, wider surveillance is warranted to monitor programme quality and limit the development of drug resistance, which remains a major potential challenge for the future of ART in Africa.

Abstract: In this study we constructed two phage libraries displaying non-immunized natural human IgM derived HCDR3 repertoires. One library was structurally constrained by a Gly to Cys substitution at position 104 enabling the formation of a disulfide bridge with the Cys at position 92. Panning of these libraries on an anti-human influenza hemagglutinin (HA) antibody resulted in the selection of 16 different HCDR3 loops displaying different degrees of sequence homology with the HA epitope. The specificity of the HCDR3 loops recovered from the structurally constrained library was confirmed by competition assays using the HA epitope. Only one of these HCDR3 peptides contained Cys104. Structural analysis of these sequences revealed that the loss of Cys104 was associated with an increased preference for the formation of the type I beta-turn required for high affinity binding to the antibody. Affinity studies confirmed that the HCDR3 peptides containing the sequence YDVPDY and Gly104 had affinities in the nanomolar range (K(d)=7.6 nM) comparable to the HA epitope. These findings provided evidence that the recovered HCDR3 sequences may bind to their target in a conformation that is unreachable by the parental antibody from which the HCDR3 was derived. Furthermore, the isolation of target-specific and high affinity binders demonstrates the value of HCDR3 libraries as a source of 'biologically randomized' sequences of human origin for the identification of peptidic lead molecules.

Abstract: AIM: To analyze the Hepatitis C virus (HCV) genotype distribution and transmission risk factors in a population of unselected patients in Luxembourg. METHODS: Epidemiological information (gender, age and transmission risks) were collected from 802 patients newly diagnosed for hepatitis C and living in Luxembourg, among whom 228 patients referred from prison. Genotyping using 5'noncoding (5'NC) sequencing was performed. We compared categorical data using the Fisher's exact F-test and odds ratios (OR) were calculated for evaluating association of HCV genotype and risk factors. RESULTS: The sex ratio was predominantly male (2.2) and individuals aged less than 40 years represented 49.6% of the population. Genotype 1 was predominant (53.4%) followed by genotype 3 (33%). Among risk factors, intravenous drug usage (IVDU) was the most frequently reported (71.4%) followed by medical-related transmission (17.6%) including haemophilia, transfusion recipients and other nosocomial reasons. Genotype 3 was significantly associated to IVDU (OR = 4.84, P < 0.0001) whereas genotype 1 was significantly associated with a medical procedure (OR = 2.42, P < 0.001). The HCV genotype distribution from inmate patients differed significantly from the rest of the population (Chi-square test with four degrees of freedom, P < 0.0001) with a higher frequency of genotype 3 (46.5% vs 27.5%) and a lower frequency of genotype 1 and 4 (44.7% vs 56.8% and 5.3% vs 9.6%, respectively). IVDU was nearly exclusively reported as a risk factor in prison. CONCLUSION: We report the first description of the HCV genotype distribution in Luxembourg. The repartition is similar to other European countries, with one of the highest European prevalence rates of genotype 3 (33%). Since serology screening became available in 1991, IVDU remains the most common way of HCV transmission in Luxembourg.

Abstract: BACKGROUND: Guidelines established for the treatment of HIV-1 infection and genotype interpretation do not apply for HIV-2. Data about antiretroviral (ARV) drug efficacy and resistance mutations is scarce. METHODS: Clinical data about HIV-2 infected patients in Belgium and Luxembourg were collected and the effect of ARV therapy on plasma viral load and CD4 counts were analysed. Viral RNA encoding for protease (PR) and reverse transcriptase (RT) from ARV-naïve and treated patients were sequenced. RESULTS: Sixty-five HIV-2 infected patients were included in this cohort. Twenty patients were treated with 25 different ARV combinations in a total of 34 regimens and six months after the start of ARV therapy, only one third achieved viral load suppression. All of these successful regimens bar one contained protease inhibitors (PIs). Mean CD4 gains in the group of viral load suppressors and the group of patients treated with PI-containing regimens were respectively significantly higher than in the group of non-suppressors and the group of PI-sparing regimens. The most frequent mutations selected under therapy (compared to HIV-2 ROD) were V71I, L90M and I89V within PR. Within RT, they were M184V, Q151M, V111I and K65R. All of these mutations, except K65R and M184V, were also found in variable proportions in ARV-naïve patients. CONCLUSION: Despite a high rate of ARV treatment failure, better virological and immunological results were achieved with PI-containing regimens. The analysis of polymorphic positions and HIV-2 specific mutations selected during therapy showed for the first time that transmission of drug resistant viruses has occurred in Belgium and Luxembourg. The high heterogeneity in ARV combinations reflects a lack of guidelines for the treatment of HIV-2 infection.

Abstract: We observed an unusual glycine-to-glutamate substitution at protease (PR) residue position 48 (G48E) in an African patient infected with a subtype A1 HIV-1 strain failing a saquinavir-containing regimen. Phenotypic analysis of protease inhibitor (PI) susceptibility showed that the G48E site-directed mutant, when introduced into an NL4-3 HIV-1 PR backbone, was slightly resistant to SQV (2-fold when compared with the wild-type virus). In addition, the G48E and G48E/V82A site-directed mutants were associated with a decrease in fitness, whereas a reversion to the wild type at position 48 was observed in vitro. Growth competition experiments using a novel growth competition assay based on enhanced green fluorescent protein- or Discosoma spp. red fluorescent protein-expressing viruses showed that the replicative fitness of the G48E virus was reduced to 55% compared with the parental NL4-3 virus. Synthesizing all possible site-directed mutants found in the patient strain is too time-consuming; therefore, a molecular dynamics (MD) simulation approach was used to understand why this mutation survived despite its fitness cost. These simulations documented that the G48E mutant interacted with PI resistance mutations (M46I, I54V, Q58E, and L63P) and with natural polymorphisms specific to subtype A1 (E35D, M36I, and R57K) that were present in the patient's virus. We hypothesize that the polymorphisms contained in the PR flap regions of the patient's virus may compensate for the presence of G48E, possibly by restoring the flexibility of the PR flaps. In summary, our results demonstrate that the G48E substitution, when introduced in the context of an HIV-1 subtype B strain, is highly unstable and gives rise to viruses with a poor replicative fitness in vitro. We also showed that when confronted with too many mutations to evaluate in vitro, MD simulations are helpful to draft hypotheses on how polymorphisms can interact with resistance mutations to stabilize their potential fitness cost.

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Abstract: OBJECTIVE: Drug resistance interpretation systems are used to select the optimal antiretroviral therapy in HIV-infected patients. It is unclear how the systems perform in predicting therapy success and failure and in how far the interpretations are affected by insufficient drug levels. METHODS: The accuracy of nine different interpretation systems in predicting therapy outcomes was evaluated using virological, immunological, pharmacological, and clinical data of 130 patients treated at 13 outpatient centers. Individual susceptibility scores of the interpretation systems were converted into active drug scores (ADS) and correlated with therapy success and failure, defined as viral load reduction of equal to or more (n=66) and less than 1 log10 copies/ml (n=64) at three months after drug resistance testing. RESULTS: Three interpretation systems considered the respective therapies as more active compared to the other interpretation systems (p<0.01). These systems predicted therapy success better than the other systems, while the others performed better in predicting therapy failure. Thus, the overall rate of correctly predicted treatment outcomes was comparable between the different systems (73.1-80.0 %). Univariate and multivariate regression analysis revealed significant correlations between the ADS of all interpretation systems and virological therapy outcomes (p<0.0001). In contrast, only three interpretation systems were significantly correlated with immunological therapy outcomes in univariate and just one in multivariate models (p<0.05). Among 128 determinations of drug levels in 64 patient samples, 19.4 % revealed no detectable drug levels. The consideration of insufficient drug levels significantly improved the prediction accuracy of all interpretation systems (p<0.005). CONCLUSION: Differences between interpretation systems in predicting therapy failures and success need to be considered for future consensus algorithms. The prediction accuracy of interpretation systems can be improved by consideration of plasma drug levels.

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Abstract: The construction is described of a HIV-1 proviral, eGFP-tagged plasmid that allows for the recombination of any selected env gene without the use of restriction enzymes and for the quantitation of the infection by the recombinant virus using flow cytometry. The system was tested showing that an isoleucine to valine substitution at residue position 37 of the HIV-1 gp41 impairs the fitness of the virus but does not lead by itself to T-20 resistance.

Abstract: Our study aimed to complete the published data on ARV therapy in Africa by describing the baseline situation in Rwanda before the launch of a large ARV programme (ESTHER). Prescription habits, frequency and reasons for treatment interruptions but also antiviral efficay, resistance to ARVs and genotypic variability of the viruses present in Rwanda were analysed. Among the 233 patients included in the study, it appeared that a vast majority (91%) were under triple therapy and that half of them had experienced at least one treatment interruption caused mainly by drug shortage or financial difficulties. Among 60 blood samples analysed, 26 were in virological failure with a viral load above 1000 RNA copies/ml and 11 presented major drug resistance mutations. Finally, virological failure could mainly be explained by the high frequency of treatment interruptions but also by the emergence of drug resistance mutations. Consequently the major objective for the ESTHER programme to improve the situation in Rwanda will be to reduce the drug shortage and facilitate the financial accessibility of the treatments.

Abstract: BACKGROUND: The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is an important factor for the development of HIV drug resistance. Because of high variability between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance substitutions. This study compared the genetic barrier between subtypes using some 2000 HIV-1 sequences (>600 of non-B subtype) isolated from anti-retroviral-naive patients in Europe. METHODS: The genetic barrier was calculated as the sum of transitions (scored as 1) and/or transversions (2.5) required for evolution to any major drug resistance substitution. In addition, the number of minor protease substitutions was determined for every subtype. RESULTS: Few dissimilarities were found. An increased genetic barrier was calculated for I82A (subtypes C and G), V108I (subtype G), V118I (subtype G), Q151M (subtypes D and F), L210W (subtypes C, F, G, and CRF02_AG), and P225H (subtype A) (P < 0.001 compared with subtype B). A decreased genetic barrier was found for I82T (subtypes C and G) and V106M (subtype C) (P < 0.001 vs subtype B). Conversely, minor protease substitutions differed extensively between subtypes. CONCLUSIONS: Based on the calculated genetic barrier, the rate of drug resistance development may be similar for different HIV-1 subtypes. Because of differences in minor protease substitutions, protease inhibitor resistance could be enhanced in particular subtypes once the relevant major substitutions are selected.

Abstract: BACKGROUND: Infection with drug-resistant human immunodeficiency virus type 1 (HIV-1) can impair the response to combination therapy. Widespread transmission of drug-resistant variants has the disturbing potential of limiting future therapy options and affecting the efficacy of postexposure prophylaxis. METHODS: We determined the baseline rate of drug resistance in 2208 therapy-naive patients recently and chronically infected with HIV-1 from 19 European countries during 1996-2002. RESULTS: In Europe, 1 of 10 antiretroviral-naive patients carried viruses with > or = 1 drug-resistance mutation. Recently infected patients harbored resistant variants more often than did chronically infected patients (13.5% vs. 8.7%; P=.006). Non-B viruses (30%) less frequently carried resistance mutations than did subtype B viruses (4.8% vs. 12.9%; P<.01). Baseline resistance increased over time in newly diagnosed cases of non-B infection: from 2.0% (1/49) in 1996-1998 to 8.2% (16/194) in 2000-2001. CONCLUSIONS: Drug-resistant variants are frequently present in both recently and chronically infected therapy-naive patients. Drug-resistant variants are most commonly seen in patients infected with subtype B virus, probably because of longer exposure of these viruses to drugs. However, an increase in baseline resistance in non-B viruses is observed. These data argue for testing all drug-naive patients and are of relevance when guidelines for management of postexposure prophylaxis and first-line therapy are updated.

Abstract: HIV-1 drug resistance methodologies are being increasingly utilized to guide treatment decisions; however, information comparing the various assays is limited. Duplicate plasma samples from 70 ART-experienced subjects were analyzed by both the Antivirogram and PhenoSense phenotypic assays and the results compared. HIV genotypes were also obtained and analyzed using seven different resistance algorithms. These results were also compared with the phenotypic assay results. Concordances between the phenotypic tests and between each algorithm, and between the two phenotypic assays were calculated and kappa coefficients (KC) determined. Overall agreement between the two phenotypic assays was good (86.9% concordance; KC 0.621). The highest concordance by drug class was seen for protease inhibitors (93.4%; KC 0.679) and the lowest (79.8%; KC 0.549) for nucleoside reverse transcriptase inhibitors. Concordance between the two phenotypic assays, when evaluating individual drugs, was good for all drugs tested except for abacavir, zalcitabine, and indinavir. Agreement between the seven algorithms and each phenotypic assay was variable, though most had good or excellent agreement. The highest overall level of agreement for an individual drug was observed when comparing lamivudine susceptibility to either assay. Concordance for abacavir, didanosine, zalcitabine, and saquinavir was generally problematic when comparing one or more phenotypic assays to the drug resistance predictive algorithms. In conclusion, results comparing these two phenotypic tests were mostly similar, but comparisons of the predictive resistance algorithms for specific drugs, as well as to specific phenotypic assays, were more inconsistent.

Abstract: Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the pol region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.

Abstract: This study aimed to find out whether genetic polymorphisms were present in positions potentially affecting susceptibility to antiretrovirals in non-B subtypes from HIV-1-infected patients in Rwanda. Viral pol gene diversity was investigated by direct sequencing in 43 treatment-naive women. In addition, 10 DNA sequences from uncultured peripheral blood mononuclear cells were analyzed 6 weeks after a single dose of nevirapine (prevention of mother-to-child transmission program). Phylogenetic analyses have shown 34 subtype A1, 6 subtype C, and 2 subtype D strains. In addition, an A/C recombinant between the protease (PR) (subtype A1) and the reverse transcriptase (RT) (subtype C) was identified. In the PR coding region, high numbers of polymorphisms were found, including substitutions in secondary PR resistance sites. PR 35D, 36I, and 37N were always present within subtype A as were PR 93L in subtype C strains. PR 10I/V, 20R, 33F, and 77V were found in subtype A whereas PR 36I was highly prevalent in subtype C strains. The A/C recombinant displayed substitutions related to resistance (PR 10, 33, 36 and RT 118). One nevirapine resistance mutation (RT 181Y/C) was found in proviral DNA after 6 weeks. In conclusion, subtypes A and C are predominant in this cohort in Rwanda. Substitutions similar to secondary protease inhibitor resistance mutations are common before treatment whereas major resistance mutation may be archived after a single dose of nevirapine. Accordingly, the hypothesis of a genetic background effect in non-B strains has to be further addressed in programs of introduction of antivirals in Africa.

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Abstract: We compared the response to protease inhibitor-containing highly active antiretroviral therapy in 175 HIV-1 treatment-naive patients harbouring subtype B versus non-B. No difference in the proportion of patients with viral loads below 400 copies/ml was observed at month 24. However, there was a significant difference in the median CD4 cell increase at month 24. Whether this is caused by viral or immune factors warrants further investigation.

Abstract: BACKGROUND: Resistance testing is increasingly accepted as a tool in guiding the selection of human immunodeficiency virus type 1 (HIV-1) antiretroviral therapy in HIV-1 infected individuals who fail their current regimen. OBJECTIVES: To descriptively compare the correlation between virologic treatment response and results using three genotypic HIV-1 drug resistance interpretation systems: the VERSANT HIV-1 Resistance Assay (LiPA) system and two sequence-based interpretation systems. STUDY DESIGN: Specimens from 213 HIV-1-infected subjects, either starting (n=104) or switching to (n=109) a regimen of three or four antiretroviral drugs, were collected retrospectively at baseline and after 3 months of uninterrupted therapy. The correlation between viral load change and the number of predicted active drugs in the treatment regimen was assessed. An interpretation algorithm was recently developed to process VERSANT HIV-1 Resistance Assay (LiPA) data. The number of active drugs predicted using this algorithm was rank correlated with the viral load change over a 3-month treatment period. For comparison, a similar calculation was made using two sequence-based algorithms (REGA version 5.5 and VGI GuideLines Rules 4.0), both applied on the same sequences. RESULTS: Statistically significant (p<0.05) correlation coefficients for each of the three HIV-1 drug resistance interpretation systems were observed in the treatment-experienced subjects on a 3-drug regimen (-0.39, -0.38, and -0.42, respectively) as well as on a 4-drug regimen (-0.33, -0.31, and -0.37, respectively). However, no significant correlation was observed in treatment-naive subjects, probably due to the very low frequency of drug resistance in these subjects. CONCLUSION: All three genotypic drug resistance interpretation systems (LiPA version 1, REGA version 5.5, and VGI GuideLines Rules 4.0) were statistically significantly correlated with virologic therapy response as measured by viral load testing.

Abstract: In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Abstract: Enfuvirtide (T-20) is the lead compound of the new class of antiretroviral drugs called fusion inhibitors. T-20 resistance-associated mutations located in the heptad repeat 1 (HR-1) domain of gp41 have been described in vitro and in clinical trials. In this study, the authors investigated the primary genotypic T-20 resistance in subtype B and non-B HIV-1 strains from patients at the beginning of their follow-up in the Luxembourg HIV Cohort as well as the emergence of primary resistance to T-20 in patients who had long-term infection with subtype B HIV-1 strains. HR-1 fragments including the gp41 amino acid 36-45, T-20-sensitive region were screened for amino acid variation. No classic T-20 resistance-associated mutations were identified in subtype B or non-B isolates. However, several uncommon mutations were found at residues 37, 39, and 42 for subtype B isolates and at residue 42 for a subtype non-B isolate. The results indicate that primary genotypic T-20 resistance seems to be rare in HIV-1, regardless of subtype or prior antiretroviral therapy (excluding fusion inhibitors). However, episodic variation within HR-1 can occur and needs further phenotypic evaluation in accurate fusion inhibitor resistance assays.

Abstract: OBJECTIVE: To assess genotypic and phenotypic resistance testing in HIV-1-infected children failing a first protease inhibitor (PI) therapy. METHODS: In a multicenter observational study 21 children, ages 3 to 16 years, were given two reverse transcriptase inhibitors and one PI (mainly ritonavir, n = 18). They were subsequently treated with single or dual PI-based therapy (predominantly nelfinavir, n = 10, or ritonavir-saquinavir, n = 7). Resistance testing was performed at the time of therapy switch via direct sequencing and a recombinant virus susceptibility assay. RESULTS: A total of 21 genotypic and 15 phenotypic resistance profiles were obtained. Most viruses displayed several reverse transcriptase mutations; however, 7 isolates maintained a wild-type protease. Ritonavir targeted the well-known pathway containing 82, 54, 46 and other secondary (nonactive site) mutations including T74A. No in vitro cross-resistance, i.e. > or = 8-fold resistance to saquinavir or amprenavir, was encountered. Secondary mutations enhanced the prediction of ritonavir resistance (i.e. L10I) and in vitro nelfinavir cross-resistance (i.e. K20R/I) conferred by primary (active site) resistance mutations. Either the 82, 54, 46 mutational genotype or the phenotype showing > or = 8-fold nelfinavir cross-resistance predicted a poorer virologic response to nelfinavir salvage therapy. CONCLUSION: In a small cohort of heavily pretreated pediatric patients, resistance testing appears to predict the response to nelfinavir as salvage for a ritonavir-based therapy. This is further supported by the correlation between ritonavir-selected mutations and in vitro nelfinavir cross-resistance. Prospective studies should assess clinical outcome in children undergoing regimen changes based on resistance testing.

Abstract: The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.

Abstract: BACKGROUND: The efficacy of treatment containing nonnucleoside reverse transcriptase inhibitors (NNRTIs) could be compromised in NNRTI-naïve patients already harboring a virus resistant to NNRTIs. On the contrary, hypersusceptibility to NNRTIs in patients having failed nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens has been described and has been associated with improved outcome. METHOD: We assessed the prevalence of NNRTI resistance-associated mutations or polymorphisms in 146 antiretroviral-naïve patients and in 181 HIV-infected patients who were given an NNRTI-based regimen. We phenotypically evaluated the NNRTI susceptibility of 41 strains presenting with amino acid substitutions at positions involved in NNRTI resistance. RESULTS: In the 268 genotypically analyzable samples, the overall prevalence of NNRTI resistance-associated mutations was 2% (6/268 patients). The prevalence of strains with amino acid substitutions at reverse transcriptase (RT) gene positions (A98, K101, K103, V106, V108, V179) involved in NNRTI resistance was 15%. Hypersusceptibility to NNRTI was rare (2%, 1/41) in those samples. RT substitutions at positions involved in NNRTI resistance were not associated with a significantly worse virologic outcome in NNRTI-treated patients. Our understanding of small shifts in IC50 values (higher or lower) toward NNRTI is very limited. The significance of many RT mutations on NNRTI susceptibility is not clear. CONCLUSION: In contrast to resistance mutations, RT substitutions at positions involved in NNRTI resistance are frequent. They are not associated with a worse virologic outcome or with decreased phenotypic susceptibility to NNRTIs. It may be prudent not to rule out the use of NNRTIs in patients with small shifts in IC50 values or poorly understood mutations.

Abstract: An observational study assessed the longitudinal use of a new line probe assay for the detection of protease mutations. Probe assays for detection of reverse transcriptase (Inno-LiPA HIV-1 RT; Innogenetics) and protease (prototype kit Inno-LiPA HIV Protease; Innogenetics) mutations gave results for 177 of 199 sequential samples collected over 2 years from 26 patients failing two nucleoside reverse transcriptase inhibitors and one protease inhibitor (first line: indinavir, n = 6; ritonavir, n = 10; and saquinavir, n = 10). Results were compared to recombinant virus protease inhibitor susceptibility data (n = 87) and to clinical and virological data. Combinations of protease mutations (M46I, G48V, I54V, V82A or -F, I84V, and L90M) predicted phenotypic resistance to the protease inhibitor and to nelfinavir. The sum of protease mutations was associated with virological and clinical outcomes from 6 and 3 months on, respectively. Moreover, a poorer clinical outcome was linked to the sum of reverse transcriptase mutations. In conclusion, despite the limited number of patients studied and the restricted number of codons investigated, probe assay-based genotyping correlates with phenotypic drug resistance and predicts new Centers for Disease Control and Prevention stage B and C clinical events and virological outcome. Line probe assays provide additional prognostic information and should be prospectively investigated for their potential for treatment monitoring.

Abstract: PURPOSE: To describe the prevalence of nine HIV-1 co-receptor polymorphisms in Luxembourg (CCR5-Delta 32, CCR5-58755-A/G, CCR5m303, CCR5-59029-A/G, CCR2-64I, CCR5-59653-C/T, CX(3) CR1-V249I, CX(3) CR1-T280M, and SDF1-3'A) in 288 HIV-1-infected patients and in 158 uninfected, healthy volunteers. METHOD: The presence of mutations was detected using PCR-restriction fragment length polymorphism (RFLP)-based methods. RESULTS: We did not find any significant differences in genotype distributions and allele frequencies between infected and uninfected participants in Luxembourg. The distribution of genotypes agreed in all groups with Hardy-Weinberg predicted proportions. CCR5-Delta 32 was significantly associated with HIV-1-infected slow progressor patients. CONCLUSION: Overall, allele frequencies were comparable to frequencies reported in previous studies in Caucasian populations.

Abstract: The emergence of HIV strains that are resistant to antiretroviral drugs is a major cause of treatment failure. Two sets of mutations: the Q151 M complex and the 69 insert, cause resistance to multiple nucleoside analogues. We report the response to treatment in 12 patients with multiple NRTI-resistant HIV-1 strains. Seven of 12 patients (58%) were able to maintain a viral load below 200 copies/ml at week 48. The patients most likely to obtain therapeutic success were those having no or low-level resistance to non-nucleoside reverse transcriptase inhibitors and/or protease inhibitors. New and more effective drugs are needed for patients with HIV-1 that is resistant to more than one of the current three classes of HIV drugs.

Abstract: OBJECTIVES: To study the prevalence of HIV-1 subtypes in Luxembourg between 1983 and 2000. To compare the drug susceptibility of non-B and B clade viruses and the prevalence of resistance-associated mutations and polymorphisms before antiretroviral treatment. DESIGN: A retrospective study on plasma samples of HIV-infected patients registered at the National Service of Infectious Diseases, Luxembourg, between 1983 and 2000. METHODS: Genotyping was performed by sequencing of the reverse transcriptase (RT) and protease coding region of the pol gene. Drug susceptibility was assessed in a recombinant virus assay. RESULTS: A total of 20.1% of the HIV-positive patients were infected with non-B subtypes, and since 1990 the proportion of non-B viruses has increased ninefold. Eleven out of 14 F1 subtypes occurred in patients native to Luxembourg. Major resistance mutations related to protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) occurred in less than 3% of treatment-naive viruses; however, 87% of the viruses had at least one PI-associated mutation. Natural polymorphism of the protease and RT coding region was observed more frequently among non-B than B viruses. Significantly more B viruses displayed resistance to the tested PI, NRTI and NNRTI (P = 0.044). CONCLUSION: The proportion of non-B viruses has increased dramatically since 1990. Non-B subtypes showed no decreased susceptibility to antiretroviral drugs, but displayed minor mutations and polymorphisms at higher frequency in their protease and RT coding region. In contrast, a significantly higher proportion of B viruses showed resistance to a range of antiretroviral drugs.

Abstract: We identified uncommon amino acid substitutions in the V3 loop regions of HIV-1 strains infecting patients from Rwanda. Their frequency was greater in long-term non-progressors (LTNP) compared with late-stage patients (P = 0.006), particularly in a sequence region that has crucial interactions with the cell surface, and is highly relevant for the host's immune response. These variants might reflect a viral response to a strong immune pressure, or represent attenuated HIV-1 strains infecting LTNP in Rwanda.

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Abstract: An "in-house" recombinant virus protease inhibitor susceptibility assay was carried out (median of 3 per patient) retrospectively in 26 patients failing HIV protease inhibitor based therapy at regular intervals to the initiation of the first protease inhibitor. Patients were treated with either indinavir (N = 6), ritonavir (N = 10), or saquinavir (N = 10) and two nucleoside analogues. Second line therapy was based on single or dual protease inhibitor regimens occasionally containing nelfinavir. Clinically relevant resistance cut-offs associated with a poorer virological outcome from 6 months on and the clinical outcome from 3 months on were determined tentatively as 4- to 8-fold resistance for indinavir and ritonavir and 2.5- to 8-fold to saquinavir. In addition, the degree of cross-resistance at the time of the change of protease inhibitor was associated with the response in viral load at 6 months to the second line therapy (P = 0.018). Cross-resistance (> or = 8-fold) between ritonavir and indinavir was common (78 and 100%). Cross-resistance between indinavir or ritonavir and saquinavir was less frequent (75 and 60% respectively) than the opposite (100%, P = 0.004). Cross-resistance to nelfinavir was encountered more frequently (> 70%) than to amprenavir (9%). The magnitudes of resistance were correlated between each other. In summary, the protease inhibitor susceptibility carried out longitudinally appears to be an earlier prognostic marker than viral load in a context of cross-resistance. The magnitude of resistance, as a marker of cross-resistance, should be useful to guide second line therapy.

Abstract: OBJECTIVES: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS: Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION: Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.

Abstract: BACKGROUND: Azodicarbonamide (ADA), a HIV-1 zinc finger inhibitor, targets a new step in viral replication and cell infectivity. OBJECTIVE: A first phase I/II clinical study of ADA. METHODS: ADA was administered at escalating doses concomitantly with current antiviral therapy during a 3-month open-label period in patients with advanced AIDS and documented virological failure. After 3 months, patients were randomized in a double-blind placebo-controlled withdrawal, ADA being given at the highest tolerated dosage. RESULTS: Fifteen patients with advanced disease failing on combined antiretroviral therapy, 75% of them with proven phenotypic resistance, had a median baseline CD4 cell count of 85 x 10(6) cells/l, CD4/CD8 cell ratio of 0.09 and median plasma RNA viral load of 4.2 log10 copies/ml. Tolerance to ADA was dose dependent and some patients developed nephrolithiasis, glucose intolerance or showed an ADA-related cytotoxicity towards CD4 cells at higher dosages. No patient died during the study period. ADA increased CD4 cell percentage, increased the CD4/CD8 cell ratio and decreased plasma RNA viral load from baseline. At the end of the double-blind period, the ADA group, but not the placebo group, showed a significant response (P < 0.05). No phenotypic resistance to ADA was observed. Overall, 3/11 patients (27%) had consistent viral load reductions > 0.5 log10 copies/ml compared with baseline and 5/ 11 (45%) showed a CD4 cell recovery from baseline > 33%. In responders, ADA induced a median peak increase in CD4 cell percentage change from baseline of 65% (range 47-243%), and viral load decrease of 1.04 log10 copies/ml (range 0.52-1.23). CONCLUSIONS: The maximal tolerated dosage of ADA appears to be 2 g (three times daily). This study provides safety results that will allow larger clinical trials to confirm the preliminary efficacy data.

Abstract: The resistance of human immunodeficiency virus type 1 (HIV-1) to drugs is a major cause of antiretroviral treatment failure. We have compared direct sequencing to a line probe assay (LiPA) for the detection of drug resistance-related mutations in 197 clinical samples, and we have investigated the sequential appearance of mutations under drug pressure. For 26 patients with virological failure despite the use of two nucleoside analogues and one protease inhibitor (indinavir [n = 6], ritonavir [n = 10], and saquinavir [n = 10]), genotypic resistance assays were carried out retrospectively every 3 months for up to 2 years by using direct sequencing (TruGene; Visible Genetics) and a LiPA for detection of mutations in the reverse transcriptase (INNO-LiPA HIV-1 RT; Innogenetics) and the protease (INNO-LiPA HIV Protease, prototype version; Innogenetics) genes. Comparison of the results from both assays found rare major discrepancies (<1% of codons analyzed). INNO-LiPA detected more wild-type-mutant mixtures than sequencing but suffered from a high rate of codon hybridization failures for the reverse transcriptase. LiPA detected earlier and more frequently than sequencing the transient mixed virus population that contained I84V, which appears before V82A in the protease sequence. Mutations M461, G48V, and L90M were often transient and drug pressure related. In conclusion, direct sequencing and LiPAs give concordant results for most clinical isolates. LiPAs are more sensitive for the detection of mixed virus populations. Mutation I84V appears in minor populations in the early steps of the pathways of resistance to indinavir and ritonavir. The fact that some mutations can be found only transiently and in minor virus populations highlights the importance of a low detection limit for resistance assays.

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Abstract: The objective of this observational study was to assess the genetic variability in the human immunodeficiency virus (HIV) protease gene from HIV type 1 (HIV-1)-positive (clade B), protease inhibitor-naïve patients and to evaluate its association with the subsequent effectiveness of a protease inhibitor-containing triple-drug regimen. The protease gene was sequenced from plasma-derived virus from 116 protease inhibitor-naïve patients. The virological response to a triple-drug regimen containing indinavir, ritonavir, or saquinavir was evaluated every 3 months for as long as 2 years (n = 40). A total of 36 different amino acid substitutions compared to the reference sequence (HIV-1 HXB2) were detected. No substitutions at the active site similar to the primary resistance mutations were found. The most frequent substitutions (prevalence, >10%) at baseline were located at codons 15, 13, 12, 62, 36, 64, 41, 35, 3, 93, 77, 63, and 37 (in ascending order of frequency). The mean number of polymorphisms was 4.2. A relatively poorer response to therapy was associated with a high number of baseline polymorphisms and, to a lesser extent, with the presence of I93L at baseline in comparison with the wild-type virus. A71V/T was slightly associated with a poorer response to first-line ritonavir-based therapy. In summary, within clade B viruses, protease gene natural polymorphisms are common. There is evidence suggesting that treatment response is associated with this genetic background, but most of the specific contributors could not be firmly identified. I93L, occurring in about 30% of untreated patients, may play a role, as A71V/T possibly does in ritonavir-treated patients.

Abstract: We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes.

Abstract: The relationship between HIV-1 replication and hematologic parameters was examined in two separate studies. The first study was a cross-sectional evaluation of 207 untreated patients. In this study, the proportion of patients with hematologic disorders increased with disease progression. There was a significant inverse correlation between HIV-1 plasma viral load and all hematologic values (r = -0.266 to -0.331). The second study was a longitudinal evaluation of patients on combination antiretroviral therapy (HAART) with hematologic alterations before treatment ( N = 27 with platelets <150,000/microl, 24 with hemoglobin <12 g/dl, 36 with neutrophils <2000/microl and 29 with leukocytes <3000/microl). Samples were analyzed every 3 months for 2 years. At 2 years, >50% of patients experienced a sustained virologic response, with viral loads <500 RNA copies/ml. Hematologic reconstitution occurred progressively for all blood cell lineages and became statistically significant after the sixth month of therapy ( p <.001). Mean values increased from 110 to 180 x 10(3)/microl for platelets, from 10.7 to 12.3 g/dl for hemoglobin (stabilizing finally at 11.4 g/dl), from 1,260 to 2,240/microl for neutrophils, and from 2,260 to 3,600/microl for leukocytes. In conclusion, hematologic disorders are corrected by combination antiretroviral therapy. This suggests a causative role of HIV-1 in hematologic disorders.

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Abstract: HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Abstract: Direct sequencing of the pol gene was assessed retrospectively with protease inhibitor susceptibility in a longitudinal study. A total of 134 samples from 26 patients were analysed at regular intervals up to 2 years. Patients were included in virological failure despite indinavir, ritonavir or saquinavir based triple-drug therapy. Both the type and number of certain secondary protease mutations modulated the effect of primary mutations on phenotypic resistance. This was notably applicable to L101/V, and to lesser extents to A711V/T. However, combinations of primary mutations, including 154V could predict resistance to the drug used and nelfinavir in more than 80%. In contrast, in vitro cross-resistance to amprenavir was rarely encountered. In addition, there was a relationship between a higher number of key mutations and poorer virological and clinical outcomes, respectively, from 6 and 3 months on. The key mutations were the protease mutations independently conferring phenotypic resistance and/or the reverse transcriptase mutations predicting treatment outcome. This relationship was independent from drug history, viral load and CD4 cell count measurements. In summary, even on a small sample size, sequence-based genotyping seems to be a good prognostic marker when performed longitudinally. In the context of primary resistance mutations, including additional secondary mutations, it may be useful in the prediction of phenotypic and clinical resistance. This should be assessed to optimize treatment monitoring before emergence of broadly cross-resistant virus.

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Abstract: We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

Abstract: The prevalence and the genotypic and phenotypic characteristics of multinucleoside-resistant (MNR) human immunodeficiency virus type 1 (HIV-1) variants in Europe were investigated in a multicenter study that involved centers in nine European countries. Study samples (n = 363) collected between 1991 and 1997 from patients exposed to two or more nucleoside analogue reverse transcriptase inhibitors (NRTIs) and 274 control samples from patients exposed to no or one NRTI were screened for two marker mutations of multinucleoside resistance (the Q151M mutation and a mutation with a 2-amino-acid insertion at codon 69, T69S-XX). Q151M was identified in six of the study samples (1. 6%), and T69S-XX was identified in two of the study samples (0.5%; both of them T69S-SS), but both patterns were absent among control samples. Non-NRTI (NNRTI)-related changes were observed in viral strains from two patients, which displayed the Q151M resistance pattern, although the patients were NNRTI naive. The patients whose isolates displayed multinucleoside resistance had received treatment with zidovudine and either didanosine, zalcitabine, or stavudine. Both resistance patterns conferred broad cross-resistance to NRTIs in vitro and a poor response to treatment in vivo. MNR HIV-1 is found only among multinucleoside-experienced patients. Its prevalence is low in Europe, but it should be closely monitored since it seriously limits treatment options.

Abstract: HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

Abstract: Clinical trials have shown that highly active antiretroviral treatment (HAART) is able to reduce HIV plasma viral loads to undetectable in 70% to 90% of patients and to increase CD4 cell counts. HAART in community settings (i.e., nonclinical trial situations) is reported to be much less effective. STUDY DESIGN: Observational study. PURPOSE: The aim of our study was to evaluate the effectiveness of protease inhibitor (PI)-based HAART in the Luxembourg HIV cohort after 36 months of treatment in previously treated and untreated patients. The secondary aim was to identify surrogate markers associated with long-term virologic and immunologic outcomes. PATIENTS AND METHOD: Seventy-three PI-naive patients, who started on HAART, combining one PI and two nucleoside reverse transcriptase inhibitors (NRTIs),with a follow-up of 3 years, were evaluated with plasma viral load and CD4 cell counts every 3 months and were analyzed retrospectively. Patients who had been treated previously with NRTI (n = 48) were at a more advanced stage of disease. RESULTS: Overall, there was a mean decrease in viral load compared to baseline of -1.89 log RNA copies/mL (SD = 1.40) that persisted at month 36. Sixty-two percent (62%) of patients reached an undetectable viral load (i.e., below 500 copies/mL): 82% and 53% of NRTI-naive and NRTI-experienced patients, respectively (p =.013). CD4 cell counts increased progressively in both groups with a sustained effect (mean increase of 146 cells/mL +/- 241) at month 36. NRTI-naive patients had a mean increase of 257 cells/mL (SD = 305), in contrast to experienced patients who had an increase of 108 cells/mL (SD = 206) at 3 years. Proportions of patients with a CD4 count under 200 cells/mL fell after 3 years for NRTI-naive (from 66% to 43%) and for experienced patients (from 32% to 13%).Predictors of short duration of viral load response were in decreasing order of importance: clinical AIDS, the use of saquinavir hard gel formulation as initial PI, and the number of NRTIs previously used. Viral load response was the only significant predictor of CD4 changes. CONCLUSION: In a community setting, effectiveness of PI-based HAART at 3 years is still achieved for most patients. NRTI-experienced patients have a good long-term response rate even if it is lower than NRTI-naive patients. A poor treatment response is associated with a more advanced stage of disease before HAART is introduced.

Abstract: The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate type's being detected.

Abstract: BACKGROUND: We intended to evaluate the stability of human immunodeficiency virus (HIV) type 1 virions in whole blood and in culture medium. MATERIALS AND METHOD: EDTA whole-blood samples taken from 12 patients were left at room temperature for up to 7 days, and aliquots of a laboratory virus stock spiked in EDTA, in heparinized or in citrated whole blood, with or without the addition of Triton X-100, or spiked in culture medium were left at room temperature for up to 120 days before plasma was separated and frozen at -80 degrees C. Viral load was measured for all frozen plasma samples using different viral load assays. p24 antigen and infectivity were also measured in the spiked samples. RESULTS: The patient whole-blood samples did not show any decrease in viral load during this 7-day period. The spiked samples decayed by not more than 1 log after 120 days (about 4 months), with the fastest decay in medium. Virus infectivity decayed very slowly from 20,000 units mL-1 to undetectable amounts after 56 days. CONCLUSIONS: These results indicate that HIV-1 virions in whole blood possess a long-term stability in terms of viral load, p24 antigen level and infectivity, which is not sufficiently recognized by laboratory and health care workers.

Abstract: We report the development of a new group of nonnucleoside reverse transcriptase inhibitors (NNRTIs). One of the most active congeners of this series of 1,1,3-trioxo-2H,4H-thieno[3,4-e] [1,2,4]thiadiazine (TTD) derivatives, i.e., 2-(3-fluorobenzyl)-4-cyanomethylen-l,1,3-trioxo-2H,4H- thieno [3,4-e] [1,2,4] thiadiazine) (QM96639) was found to inhibit human immunodeficiency virus (HIV) type 1 [HIV-1 (IIIB)] replication in MT-4 cells at a concentration of 0.09 microM. This compound was toxic for the host cells only at a 1,400-fold higher concentration. The TTD derivatives proved effective against a variety of HIV-1 strains, including those that are resistant to 3'-azido-3'-deoxythymidine (AZT), but not against HIV-2 (ROD) or simian immunodeficiency virus (SIV/ MAC251). HIV-1 strains containing the L100I, K103N, V106A, E138K, Y181C, or Y188H mutations in their reverse transcriptase (RT) displayed reduced sensitivity to the compounds. Their cross-resistance patterns correlated with that of nevirapine. 2-Benzyl-4-cyanomethylen-1,1,3-trioxo-2H,4H-thieno[3,4-e] [1,2,4]thiadiazine (QM96521) enhanced the anti-HIV-1 activity of AZT and didanosine in a subsynergistic manner. HIV-1-resistant virus containing the V179D mutation in the RT was selected after approximately six passages of HIV-1 (IIIB) in CEM cells in the presence of different concentrations of QM96521. From structure-activity relationship analysis of a wide variety of TTD derivatives, a number of restrictions appeared as to the chemical modifications that were compatible with anti-HIV activity. Modelling studies suggest that in contrast to most other NNRTIs, but akin to nevirapine, QM96521 does not act as a hydrogen bond donor in the RT-drug complex.

Abstract: The performance to detect drug resistance mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.

Abstract: OBJECTIVE: To study the prevalence of multiple dideoxynucleoside (ddN)-resistant (MddNR) HIV-1 in European patients under treatment with multiple ddN analogues, and to characterize MddNR strains genotypically and phenotypically. DESIGN AND METHODS: Blood samples from patients after > or = 6 months of treatment with multiple ddN were screened for the MddNR mutation Q151M. After confirmation of MddNR in 15 patients from five European countries, genotypic resistance was evaluated by DNA sequencing of the reverse transcriptase (RT) gene. Phenotypic resistance was measured by the recombinant virus assay. Results were compared with the clinical evolution of the patients. RESULTS: The prevalence of MddNR strains in European patients treated with multiple ddN analogues was 3.5%. Viruses typically contained amino acid substitutions V75F, F77L, F116Y and Q151M in the RT gene. A new mutation, S68G, was frequently associated with MddNR. Phenotypically, viruses displayed high-level resistance to zidovudine (ZDV), didanosine (ddl), zalcitabine (ddC), stavudine (d4T) and partial resistance to lamivudine (3TC) once multiple mutations were present. Under in-vivo treatment pressure, some MddNR strains additionally developed resistance to protease inhibitors or non-nucleoside RT inhibitors (NNRTI). Clinically, most patients had advanced HIV disease with low CD4 cell counts, high viral loads and a rapid progression, but two patients harbouring MddNR virus responded well to dual protease inhibitor associations. CONCLUSIONS: MddNR resistant HIV-1 can be found in European patients. MddNR is characterized by a specific set of drug resistance mutations, cross-resistance to most ddN analogues and a fast clinical progression. MddNR can be associated with protease inhibitor or NNRTI resistance.

Abstract: OBJECTIVES: This study was performed to assess the frequency of drug resistance mutations in treatment-naive HIV-1-infected patients. STUDY DESIGN/METHODS: Frozen plasma samples from 135 treatment-naive HIV-infected adults were available from the first time the patients were seen for their infection in our center between 1992 and 1997. A rapid genotypic assay based on reverse DNA hybridization (LiPA HIV-1 RT, Murex, London, U.K.) was used to study substitutions at reverse transcriptase (RT) codons 41, 69, 70, 74, 184, and 215. Additionally, a selective polymerase chain reaction (PCR) for the multiple dideoxynucleoside resistance (MddNR) mutation Q151M was performed. RESULTS: 16 patients (12%) harbored virus with one or more drug resistance mutations. The prevalence of patients with drug-resistant virus was 0% in 1992, 17% in 1993, 0% in 1994 (only 6 samples tested), 18% in 1995, 14% in 1996, and 9% in 1997. Mutation K70R (resistance to zidovudine) was found in 8 patients, M41L (resistance to zidovudine) in 5 patients, M184V/I (resistance to ddI/ddC/3TC) in 2 patients, and T215Y/F (resistance to zidovudine) in 4 patients. All samples were wild type at codons 69 (ddC), 74 (ddI), and 151 (MddNR). CONCLUSIONS: Virus strains containing drug resistance mutations are now found in about 1 of 10 treatment-naive HIV-1-seropositive patients in Luxembourg. We believe that testing for drug-resistant virus before starting treatment should be recommended and will help to improve the selection of the most effective antiretroviral treatment. We also suggest the need for an international surveillance program on HIV drug resistance in treatment-naive patients.

Abstract: Potent new antiretroviral drugs combined with updated treatment strategies have now achieved efficient inhibition of HIV replication in most patients. The major targets for antiretroviral therapy are the reverse transcriptase and the protease of HIV. Up to date, 11 antiretroviral drugs have been licensed in the US. New nucleoside reverse transcriptase inhibitors (NRTI), i.e. abacavir, and adefovir dipivoxil proved to be effective in clinical trials. The antiretroviral activity and impact on clinical outcome of nonnucleoside reverse transcriptase inhibitors (NNRTI) are mostly short lived. Compared to NRTIs, protease inhibitors (PIs) have the highest antiretroviral activity. New PIs that conserve activity against mutant HIV strains or derivatives that have a simpler biosynthesis are being developed. A major drawback of highly active antiretroviral therapy is the selection of resistant mutants under suboptimal dosage, in advanced stages of disease or after pre-treatment with mono- or double-combination regimens. Monitoring of antiretroviral therapy is achieved by measurement of viral load using nucleic acid amplification techniques. The goal of antiretroviral therapy should be to reduce viral load below the detection limit of current assays in order to delay the emergence of resistant mutants and to maximally reduce disease progression. Recommendations for antiretroviral therapy and monitoring are evolving constantly due to the rapid progress in the development of active compounds and new insights into HIV pathogenesis. Resistance determination of HIV patient isolates seems to play a progressively increasing role in monitoring of antiretroviral treatment since treatment-naive patients may already harbour drug-resistant virus. Due to cross-resistance between different compounds, the choice of the treatment regimen should be guided by resistance patterns of HIV in order to warrant maximal and long-lasting efficiency.

Abstract: Sodium valproate (VPA), a simple branched-chain fatty acid that has anticonvulsant activity and is used in the treatment of many forms of epilepsy, has been reported to stimulate human immunodeficiency virus (HIV) type 1 replication in acutely infected CEM and chronically infected U1 cells (Chemico-Biological Interactions 1994;91:111-121). When attempting to reproduce and extend these findings, we confirmed that VPA is able to stimulate HIV-1(IIIB) replication in acutely infected CEM and C8166 T lymphocytic cell lines and chronically infected ACH-2 and U937/IIIB/LAI cells in a concentration-dependent manner. The stimulatory effect of VPA on HIV replication in CEM cells was not increased by pretreatment of the cells with VPA for 24 hr before infection. However, we could not detect any stimulatory effect of VPA on HIV-1(IIIB) replication in acutely infected peripheral blood mononuclear cells (PBMCs), MT-4, MT-2, HUT-78, and MOLT-4 (clone 8) cells and in chronically infected HUT-78/IIIB/LAI cells. The stimulatory effect by VPA under certain conditions (see above) may be ascribed to an enhanced HIV transcription, as VPA was found to enhance the HIV long terminal repeat (LTR)-directed expression of beta-galactosidase in transiently transfected HLtat, P4, and COS7 cells. VPA did not enhance beta-galactoside expression mediated by the cytomegalovirus (CMV) promoter. VPA did not affect HIV-induced syncytium formation. Nor had VPA any direct inactivating effect on HIV.

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Abstract: From a series of macrocyclic diamides possessing the disulfide linkage, only SRR-SB3, a compound that complexes with zinc, was found to inhibit human immunodeficiency virus type 1 (HIV-1; strain IIIB) replication at a concentration of 1.8 to 6.5 micrograms/ml in MT-4, CEM, and peripheral blood mononuclear cells. SRR-SB3 was toxic to MT-4 cells at a concentration of 15.9 micrograms/ml, resulting in a selectivity index of 9 in these cells. This macrolide was also effective against various other HIV-1 strains, including clinical isolates and HIV-1 strains resistant to protease inhibitors and nucleoside and nonnucleoside reverse transcriptase inhibitors. It was also active against various HIV-2 strains, simian immunodeficiency virus (strain MAC251), and Moloney murine sarcoma virus, but not against viruses other than retroviruses. In addition, the compound was found to inhibit chronic HIV-1 infections in vitro. The compound in combination with other antiviral agents, such as zidovudine, zalcitabine, and stavudine, showed an effect that was between additive and synergistic. Time-of-addition experiments indicated that SRR-SB3 acts at a late stage of the HIV-1 replicative cycle.

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Abstract: Bicyclams have recently been identified as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) replication. The prototype of this series, JM3100 exhibits anti-HIV potency at concentrations ranging from 0.001 to 0.01 micrograms/ml. JM3100 proved to be active when tested against HIV strains resistant to the reverse transcriptase (RT) inhibitors 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), 3TC, alpha APA and TIBO, at roughly the same concentrations as for the wild-type strain. The virus was passaged in vitro in the presence of increasing concentrations of either TIBO or alpha APA alone or in combination with JM3100. The combination between TIBO, or alpha APA, and JM3100 delayed the development of TIBO- and alpha APA-resistant strains, without emergence of resistance to JM3100. In separate experiments, it took more than 60 passages (300 days) in MT-4 cells and 20 passages (140 days) in peripheral blood lymphocyte (PBL) cells for the virus to become resistant to JM3100. The JM3100-resistant virus showed cross-resistance to sulfated polysaccharides such as dextran sulfate (DS), pentosan sulfate (PS), heparin and cyclodextrin sulfate (CDS), suggesting that these compounds may share a common mechanism of action. Furthermore, the inhibitory effect of JM3100 on virus-induced syncytium formation was enhanced in the presence of heparin. The results presented here provide further support for the bicyclams as attractive candidate drugs for the chemotherapy of HIV infections.

Abstract: OBJECTIVE: To define genotypic and phenotypic resistance patterns following prolonged therapy with the protease inhibitor ritonavir (ABT-538). DESIGN: Seven HIV-1-infected patients, all but one previously treated with dideoxynucleoside analogues (zidovudine, didanosine, zalcitabine), were treated for 1 year with ritonavir. METHODS: Direct solid-phase sequencing of the protease gene starting from plasma derived viral RNA followed by comparison to phenotypic drug resistance data. RESULTS: The most frequent amino-acid substitutions occurring upon administration of the protease inhibitor were V82A/F (substrate binding site), I54V (flap region), A71V and L10I. Additional mutations found in more than one patient were I15V, M36I, I84V and I93L. Mutation L63P was found both in pre- and post-ritonavir samples. Phenotypic drug resistance assays confirmed resistance to ritonavir in post-treatment samples (approximately 170-fold) and showed cross-resistance to indinavir (approximately 30-fold) and partially to saquinavir (approximately fivefold). At 1 year of treatment, one patient without known resistance-associated mutations in the protease gene still showed a substantial rise in CD4 cell count accompanied by a more than 2.4 log decrease in RNA viral load. However, at week 78, mutations R8Q, E34K, R57K, L63P and I84V were detected and the treatment benefit was partially lost. CONCLUSIONS: Long-term treatment with ritonavir is associated with the emergence of multiple mutations in the HIV-1 protease gene. The mutations L10I, I54V, L63P, A71V, V82A/F and I84V correspond to known drug-resistance mutations for ritonavir and other protease inhibitors. Phenotypic resistance to ritonavir was detected in a majority of ritonavir-treated patients at 1 year of treatment. In addition, long-term ritonavir treatment selects for cross-resistance to the protease inhibitors indinavir and saquinavir. This argues against sequential therapy with several protease inhibitors. Delayed resistance in one patient was accompanied with a prolonged increase in CD4 cell count and decrease in viral load suggesting a temporary benefit of treatment.

Abstract: A human immunodeficiency virus type 1 (HIV-1)-seropositive patient was treated sequentially with the dideoxynucleoside (ddN) analogues zidovudine, didanosine, zalcitabine, stavudine, and lamivudine and the nonnucleoside HIV-1-specific reverse transcriptase inhibitor (NNRTI) loviride (alpha-APA). Accumulation of drug resistance mutations (mainly V75I, F77L, K103N, F116Y, Q151M, and M184V) eventually resulted in a strain that was genotypically and phenotypically resistant to all tested ddNs and the majority of NNRTIs. However, the multidrug-resistant virus retained wild type sensitivities to drugs such as foscarnet, phosphonomethoxyethyl adenine, dextran sulfate, JM3100, saquinavir, and NNRTI TSAO-m3T. Drug-resistant isolates showed replication kinetics and infectivity in an in vitro peripheral blood mononuclear cell system similar to those of the wild type isolate from the same patient. The multi-ddN-resistant isolate was not eliminated in a competition culture with the wild type isolate. Sequential therapy did not prevent the appearance of multidrug-resistant virus with a conserved replication rate.

Abstract: OBJECTIVE: To evaluate the immunological and virological efficacy of triple combination therapy with zidovudine (ZDV) plus zalcitabine (ddC) plus lamivudine (3TC) and a double (ZDV+3TC) combination therapy in patients previously treated with ZDV plus ddC. DESIGN: A 6-month follow-up open-label randomized study was undertaken in 46 HIV-1-infected patients previously treated for at least 6 months with ZDV plus ddC, who were allocated to receive either ZDV/ddC/3TC (n = 15) or ZDV/3TC (n = 15) or to continue with the ZDV/ddC regimen (control group; n = 16). METHODS: Changes in CD4+ cell counts and plasma viral load (VL) were analysed with analysis of variance. Sequencing of the reverse transcriptase gene was performed in a subset of 3TC-treated patients. RESULTS: Mean CD4+ cell counts increased significantly above baseline in both 3TC regimens whereas counts decreased in the control group. Significant plasma VL reduction was achieved in both 3TC combination therapy groups at weeks 4 and 24 compared with the control group. Coexistence of mutations conferring resistance to ZDV and 3TC were found in patients from both 3TC treatment groups. CONCLUSIONS: Both therapy strategies, switching ddC to 3TC or adding 3TC, significantly improved the virological and immunological efficacy compared with continuing ZDV/ddC. Our results support the use of 3TC in patients previously treated with the ZDV/ddC combination.

Abstract: We investigated and compared the reproducibility, accuracy, detection limits, and dynamic ranges of two commercial kits for quantification of RNA viral load using a titrated virus stock (laboratory strain HIV-1 IIIB) and 107 plasma samples of 25 HIV-1-infected patients. The high reproducibility of both methods (SD = 0.2-0.3 log for both methods) allowed reliable detection of a 0.5 log change in RNA viral load. Both methods had a similar detection limit (at least 10(3) RNA copies/ml plasma) and a dynamic range that extended over a 5 log (AMPLICOR) or a 6 log (NASBA) range of HIV-1 input. For HIV-1 IIIB, the viral load was compatible with measurements of virus-associated p24 antigen. For 21 patients (91 samples), the RNA viral load was similar with both methods differing by no more than 0.5 log. For four patients, the difference in viral load between the two methods was > 0.5 log for all 16 samples. For three of these patients, this could be explained by mismatches with primers or probes in the gag sequence: there was no correlation to the viral subtype. The RNA viral load determination was highly sensitive compared with p24 antigen measurement (> 95% of patients had a detectable viral load vs. 40% who had a detectable p24 level), but in the p24-positive samples the correlation between the antigen level and the RNA viral load was of only borderline significance. We also found that the viral RNA in whole blood was stable for at least 48 h during transport at room temperature. These observations show that both the NASBA HIV-1 RNA QT test and the AMPLICOR HIV monitor test are reliable parameters of the viral load, with great promise for their use as potential surrogate markers.

Abstract: The presence of HIV-1 RNA in the plasma and serum of European and African patients was monitored using RNA-polymerase chain reaction (RNA-PCR) and the new isothermal NASBA nucleic acid amplification system encompassing a gel-based detection assay (ELGA). Identical RNA extraction procedures, provided by the NASBA amplification system, were used for both methods. The detection limit for HIV-1 RNA, measured on a 10-fold dilution series of spiked HIVIIIB in negative plasma, was about 0.05 CCID50 per test for both methods. Both NASBA and RNA-PCR were more sensitive than a p24 assay for the detection of circulating HIV-1 virus in blood: 17 of the 34 (50%) p24 antigen-tested seropositives were p24-positive while 32 (94%) were positive by NASBA and 30 (88%) by RNA-PCR. Among the 45 seropositives, 34 of which were tested for p24 antigen, 43 (96%) were positive by NASBA and 41 (91%) by RNA-PCR. Almost all seropositives had a detectable viral load in 100 microliters plasma. Lower viral loads were only encountered in some healthy seropositives with a higher CD4 count. There was no cross-reactivity with HIV-2 or HIV-I with both the RNA-PCR and NASBA. The extraction method used permitted the detection of HIV-1 RNA equally well in serum and in plasma with heparin or EDTA.

Abstract: Penicillium marneffei infection has emerged as a new potential indicator disease for AIDS in Southeast Asia. We report two additional cases of P. marneffei infection in patients infected with HIV who had traveled to endemic areas and review the mucocutaneous features of previously reported cases. Our two patients had many of the typical features associated with invasive infection: fever, weight loss, anemia, lymphadenopathy, hepatomegaly, pulmonary and gastrointestinal symptoms, as well as skin manifestations such as papular molluscum contagiosum-like lesions and oral lesions. Knowledge of cutaneous signs is important for prompt diagnosis of P. marneffei infection and institution of life-saving therapy with antifungal agents.

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