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Human immunodeficiency virus type 1 (HIV-1) was discovered in the early 1980s when the virus had already established a pandemic. For at least three decades the epidemic in the Western World has been dominated by subtype B infections, as part of a sub-epidemic that traveled from Africa through Haiti to United States. However, the pattern of the subsequent spread still remains poorly understood. Here we analyze a large dataset of globally representative HIV-1 subtype B strains to map their spread around the world over the last 50years and describe significant spread patterns. We show that subtype B travelled from North America to Western Europe in different occasions, while Central/Eastern Europe remained isolated for the most part of the early epidemic. Looking with more detail in European countries we see that the United Kingdom, France and Switzerland exchanged viral isolates with non-European countries than with European ones. The observed pattern is likely to mirror geopolitical landmarks in the post-World War II era, namely the rise and the fall of the Iron Curtain and the European colonialism. In conclusion, HIV-1 spread through specific migration routes which are consistent with geopolitical factors that affected human activities during the last 50years, such as migration, tourism and trade. Our findings support the argument that epidemic control policies should be global and incorporate political and socioeconomic factors., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Utilization of dried plasma for HIV-1 viral load testing would significantly decrease sample shipping costs., Objectives: To describe the precision and reproducibility of ViveST(®) (ST) as a transportation method for shipping specimens for HIV-1 viral load (VL) testing., Study Design: Thirty clinical plasma samples were used to generate replicate samples with HIV VL values of 4 log(10), 3 log(10) and 2 log(10) copies/mL for reproducibility testing and an additional 299 samples with HIV VL <50 copies/mL (99); 1.7 log(10) to 3.99 log(10) (100); and 4 log(10) to 5.99 log(10)/mL (100) were used to compare ViveST to frozen plasma samples using the VERSANT(®) HIV-1 RNA 3.0 Assay. Results were compared using Student t-test, Pearson correlation and Bland-Altman analyses., Results: Mean intra-assay variance among frozen and dried plasma triplicates was 0.15 log(10) and 0.09 log(10) copies/mL respectively (n=10, P=NS). Compared to frozen plasma, there was a mean reduction of 0.3 log(10), 0.27 log(10), and 0.35 log(10) copies/mL at the 4 log(10), 3 log(10), and 2 log(10) copy/mL samples respectively (n=30, all comparisons, P<0.01). Overall correlation between 299 frozen and ViveST samples was r=0.97, where 12 of 99 undetectable frozen VL were positive with ST, and 12 of 200 frozen detectable VL were undetectable with ViveST (mean VL 2.1, 1.9 log(10) copies/mL respectively)., Conclusions: HIV-1 viral load results using ViveST were reproducible, correlated well with frozen plasma, though yielding minimally lower values. Our data suggest that dried plasma for HIV-1 VL testing using ViveST has promise for use in HIV clinical practice., (Published by Elsevier B.V.)

Background: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy., Methods: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system., Results: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs., Conclusion: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Purpose: To study the impact of baseline resistance mutations and HIV-1 subtypes on virological response to first-line antiretroviral therapy and to analyse the concordance of the results of two antiretroviral resistance interpretation tools in the INITIO trial., Method: Genotype and virco TYPE resistance analyses were studied at baseline, Year 2, Year 3, and at first therapeutic failure on plasma specimens stored at -80 degrees C. Relations between resistance mutations at baseline, subtype, initial virological response, and virological outcome after Week 24 were studied., Results: 781 participants had genotypic results available at baseline. Therapeutic failure occurred for 112 participants. Initial virological response as well as virological outcome after Week 24 were not associated with HIV subtype. Before Week 24, the proportion of participants remaining under strict initial regimen was lower in patients with resistance mutations at baseline than in those without any resistance mutations. Presenceof resistance mutations at baseline also impacted negatively long-term virological outcome. Few discrepancies were observed between genotypic and virco TYPE for resistance interpretation., Conclusion: These data showed that presence of resistance mutations at baseline was associated with a poorer long-term virological outcome in the INITIO trial.

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.

Objective: To compare virological outcome and genotypic resistance profiles in HIV-1-infected patients starting non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing regimens., Methods: NNRTI-naive patients were included who started treatment with nevirapine (NVP) or efavirenz (EFV) with genotypic resistance test results available at time of initiation (baseline). Virological failure was defined as two consecutive values > 500 copies/ml after starting the regimen. Cox models were used to investigate time to virological failure (the first of two values)., Results: A total of 759 patients were included (13% antiretroviral-naive): 389 initiated NVP and 370 initiated EFV. Baseline IAS-USA NNRTI resistance mutations were detected in 3%. Using the Rega algorithm (version 7.1) to interpret resistance, 460 (64%) patients had resistance (full or intermediate) to at least one drug they were starting (69% NVP, 60% EFV, P = 0.011); 287 (74%) NVP and 168 (45%) EFV patients experienced virological failure after treatment initiation, P < 0.001. After adjustment for previous antiretroviral use, previous AIDS, year started NNRTI, CD4 cell count (baseline, nadir), viral load (baseline, maximum), and baseline drug resistance (measured by Rega), the relative hazards (EFV versus NVP) of virological failure was 0.50, 95% confidence interval: 0.39-0.65, P < 0.001. At time of virological failure, comparable levels of NNRTI resistance were detected. The K103N mutation emerged more in patients failing EFV and Y181C in patients failing NVP., Conclusions: NVP may be associated with an inferior virological outcome compared to EFV in NNRTI-naive patients with extensive resistance to other drug classes. The profile of NNRTI resistance mutations when virologically failing an NNRTI-containing regimen appears to depend on the NNRTI the patients fail.

Escalating drug resistance in treatment-experienced HIV-1-infected patients has made management increasingly difficult. In clinical trials, tipranavir (TPV) has produced potent and durable responses in such patients, although experience in clinical cohorts is limited. A retrospective clinical case review was undertaken of triple-class experienced HIV-1-infected patients receiving optimized boosted TPV-containing regimens and T20 with up to 108 weeks follow-up. Antiretroviral therapy (ART) resistance profiles were characterized using International Aids Society (IAS)-USA scoring and 'TPV resistance score' (TPV-RS) at baseline and failure. Five of 12 patients had undetectable virus (<50 copies/mL) after median 84 weeks (range 60-108), and 1/12 < had 700 copies/mL after 40 weeks. Six of 12 patients failed after 36 (range 12-48) weeks and were more likely to have > or = 3 TPV-RS mutations than non-failures (P = 0.06). Presence of a major IAS-USA mutation at baseline was strongly associated with absence of a 1 log viral load drop at 24 weeks (P = 0.02). TPV-containing regimens showed impressive efficacy and tolerability in this heavily experienced cohort.

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&#95;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.

Background: Combination antiretroviral therapy (cART) may vary in ability to suppress viral load and increase CD4+ T-cell count in people infected with different HIV-1 subtypes, possibly due to differences in resistance development. Antiretroviral drugs have predominantly been developed in Western Europe/North America on the basis of the most prevalent subtype, B. However, non-B subtypes are increasingly spreading worldwide., Objective: To compare virological and immunological response to cART between patients infected with B and non-B subtypes across Europe., Design: EuroSIDA prospective, observational cohort with 11,928 HIV-1-infected patients., Methods: Response to cART was analysed in patients with subtypes determined pre-cART, via multivariable logistic regression on the first measurements 6-12 months after starting cART. A virological response was defined as a viral load <500 copies/mi and immunological response as a CD4+ T-cell count increase of > or =100 cells/mm(3)., Results: Forty-five percent of patients were antiretroviral naive at initiation of cART. Virological suppression was achieved by 58% of 689 subtype-B-infected patients and 66% of 102 non-B-infected patients (P=0.159). After adjustment for potential confounders, there was no significant difference in odds of achieving virological suppression (non-B compared with B; odds ratio [OR]: 1.05, 95% confidence interval [CI]: 0.58-1.93, P=0.866). An immunological response was achieved by 43% of 753 B-infected patients and 48% of 114 non-B-infected patients (P=0.334). After adjustment, there was no significant difference in odds of an immunological response (OR: 1.17, 95% CI: 0.73-1.87, P=0.524)., Conclusions: There was no evidence of significant differences in virological or immunological response to cART between patients infected with HIV-1 B and non-B subtypes.

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.

Objectives: To assess the clinical utility of phenotypic resistance testing in addition to genotypic resistance testing among HIV-1-infected patients experiencing virologic failure and with limited therapeutic options., Design: Multicenter randomized trial., Methods: Patients were eligible if a decision had been made to switch antiretroviral therapy, the most recent HIV-1 RNA plasma viral load (VL) exceeded 2000 copies/mL, and the clinician was unable to select a potent regimen of 3 or more drugs without access to a resistance test. Subjects were randomized to genotypic resistance testing alone (G arm) or to genotypic plus phenotypic testing (G + P arm). Patients had access to resistance testing at any time during follow-up (minimum of 1 year) according to the original allocation. The primary end point was change in plasma VL from baseline at 12 months., Results: Three hundred eleven patients were recruited between February 2000 and July 2001. At baseline, mean VL and CD4 count were 4.23 log10 copies/mL and 275 cells/mm, respectively, and subjects had previous exposure to a mean of 7.7 antiretroviral drugs. There was no appreciable difference between the study arms in the drug regimens prescribed after randomization. Mean reduction in VL load at 12 months was similar in the 2 arms (G: 1.37 log10 reduction, G + P: 1.28 log10 reduction; P = 0.77), as was the proportion of subjects with VL <50 copies/mL (G: 35%, G + P: 27%)., Conclusion: The study did not demonstrate added value of phenotypic resistance testing in conjunction with genotypic resistance testing in patients with limited therapeutic options.

Background: Little is known about the long term risk of development of HIV-1 drug resistance for patients starting antiretroviral therapy (ART) with three or four drug regimens in routine clinical practice., Methods: We analysed a large cohort study of patients seen in one of six large HIV clinics in and around London, UK. The focus of this analysis was on patients who started ART with two nucleosides plus either a single protease inhibitor (PI), a PI with ritonavir, abacavir or a non-nucleoside reverse transcriptase inhibitor (NNRTI)., Results: 4306 patients were followed; 1436 (33%) started with a single PI, 279 (6%) with a PI plus ritonavir, 156 (4%) with triple nucleosides and 2435 (57%) with an NNRTI. The overall cumulative risk of viral load failure was 38% by 6 years. Risk of > or =1 major IAS-USA mutation was 27% by 6 years; risk of mutations from at least two of the three main drug classes was 20% over the same period. These are lower limit estimates as test results were not available for many with viral load failure. Risk of PI mutations being detected in people who started ART with regimens containing a PI with ritonavir was significantly lower than the risk of NNRTI mutations being detected in those starting with NNRTI-containing regimens (relative hazard 0.3195% CI 0.15-0.61; p = 0.0008)., Conclusion: In routine practice, rates of viral load failure and of resistance detection in patients who started ART with three or four drugs are appreciable.

Background: Studies have suggested that HIV-1 may develop thymidine analogue mutations (TAMs) by one of two distinct pathways - the TAM1 pathway (including mutations 41L, 210W and 215Y) or the TAM2 pathway (including mutations 67N, 70R and 219E/Q) - under the pressure of a not fully suppressive thymidine-analogue-containing regimen., Methods: Frozen plasma samples stored in the EuroSIDA repository were selected and sent to two central laboratories for genotypic analysis. We considered 733 patients with at least one genotypic test showing > or =1 TAMs (the first of these tests in chronological order was used). TAM1 and TAM2 genotypic profiles were defined in accordance with previous literature. Statistical modelling involved logistic regression and linear regression analysis for censored data., Results: The observed frequencies of patterns classifiable as TAM1 or TAM2 profiles were markedly higher than the probabilities of falling into these classifications by chance alone. The chance of detecting a TAM2 profile increased by 25% per additional year of exposure to zidovudine. We found that mutations 67N and 184V were not associated with a particular TAM profile. In the presence of TAM2 profiles, the adjusted mean difference in the 6-month viral reduction was 0.96 log10 copies/ml (95% confidence interval: 0.20; 1.73) higher in patients who started stavudine-containing regimens instead of zidovudine-containing regimens., Conclusions: This study provides evidence that the suggested TAM clustering is a real phenomenon and that it may be driven by which thymidine analogue the patients has used. In patients with TAM2-resistant viruses, stavudine appears to retain greater viral activity than zidovudine.

Objectives: To investigate the ability of several HIV-1 drug-resistance interpretation systems, as well as the number of pre-specified combinations of abacavir-related mutations, to predict virological response to abacavir-containing regimens in antiretroviral therapy-experienced, abacavir-naive patients starting an abacavir-containing regimen in the EuroSIDA cohort., Patients and Methods: A total of 100 HIV-infected patients with viral load (VL) >500 copies/ml who had a plasma sample available at the time of starting abacavir (baseline) were included. Resistance to abacavir was interpreted by using eight different commonly used systems that consisted of rules-based algorithms or tables of mutations. Correlation between baseline abacavir-resistance mutations and month 6 virological response was performed on this population using a multivariable linear regression model accounting for censored data., Results: The baseline VL was 4.36 log10 RNA copies/ml [interquartile range (IQR): 3.65-4.99 log10 RNA copies/ml] and the median CD4 cell count was 210 cells/microl (IQR: 67-305 cells/microl). Our patients were pre-exposed to a median of seven antiretrovirals (2-12) before starting abacavir therapy. The median (range) number of abacavir mutations (according to the International AIDS Society-USA) detected at baseline was 3.5 (0-8). Overall, the Kaplan-Meier estimate of the median month 6 VL decline was 0.86 log10 RNA copies/ml [95% confidence intervals (95% CI): 0.45-1.24]. The VL in those patients (n=31) who intensified treatment by adding only abacavir decreased by a median 0.20 log10 RNA copies/ml (95% CI: -0.18; +0.94). The proportion of patients who harboured viruses fully resistant to abacavir among the eight genotypic resistance interpretation algorithms ranged from 12% [Agence Nationale de Recherches sur le SIDA (ANRS)] to 79% [Stanford HIV RT and PR Sequence Database (HIVdb)]. Some interpretation systems showed statistically significant associations between the predicted resistance status and the virological response while others showed no consistent association. The number of active drugs in the regimen was associated with greater virological suppression (additional month 6 VL reduction per additional sensitive drug=0.51, 95% CI: 0.15-0.88, P=0.006); baseline VL was also weakly associated (additional month 6 VL reduction per log10 higher=0.30, 95% CI: -0.02; +0.62, P=0.06). In contrast, the number of drugs previously received was associated with diminished viral reduction (additional month 6 VL reduction per additional drug=-0.14, 95% CI: -0.28; 0.00, P=0.05)., Conclusions: Our results revealed a high degree of variability among several genotypic resistance interpretation algorithms currently in use for abacavir. Therefore, the interpretation of genotypic resistance for predicting response to regimens containing abacavir remains a major challenge.

New information about the benefits and limitations of testing for resistance to human immunodeficiency virus (HIV) type 1 (HIV-1) drugs has emerged. The International AIDS Society-USA convened a panel of physicians and scientists with expertise in antiretroviral drug management, HIV-1 drug resistance, and patient care to provide updated recommendations for HIV-1 resistance testing. Published data and presentations at scientific conferences, as well as strength of the evidence, were considered. Properly used resistance testing can improve virological outcome among HIV-infected individuals. Resistance testing is recommended in cases of acute or recent HIV infection, for certain patients who have been infected as long as 2 years or more prior to initiating therapy, in cases of antiretroviral failure, and during pregnancy. Limitations of resistance testing remain, and more study is needed to refine optimal use and interpretation.

In 1996 the International AIDS Society-USA convened an international panel of experts in HIV drug resistance and clinical management to develop guidelines for the clinical use and limitations of resistance testing. Since then the International AIDS Society-USA Resistance Testing Guidelines Panel has developed and regularly published its recommendations. The latest panel recommendations appear in the July 1 issue of Clinical Infectious Diseases. We periodically pose questions to the panel relating to clinical elements of resistance testing that have been collected from HIV practitioners across the nation. We are happy to feature the latest edition in this issue of Topics in HIV Medicine. It is our hope that addressing these issues will help guide your treatment strategy decisions regarding resistance testing.

Purpose and Methods: To describe the evolution of resistance to zidovudine (ZDV), lamivudine (3TC), abacavir (ABC) and nelfinavir (NFV), 113 previously untreated children in the PENTA 5 trial had resistance assayed at baseline, rebound and/or 24, 48, 72 weeks (VIRCO: phenotyping and genotyping with 'Virtual Phenotype' interpretation)., Results: At baseline, few reverse transcriptase mutations and no primary protease inhibitor mutations were observed. Time to detectable HIV-1 RNA with reduced phenotypic susceptibility to any drug was shortest in the ZDV+3TC arm (overall logrank P=0.02). Through a median follow-up of 55 weeks, at their last assessment 11 (28%), 16 (40%) and 13 (32%) children with detectable HIV-1 RNA and a resistance test available had mutations conferring resistance to none, one, or two or more trial drugs, respectively, according to the virtual phenotype. Reduced phenotypic susceptibility to ABC only occurred in the 3TC+ABC arm and required K65R and/or L74V in addition to M184V. NFV-resistant virus was selected slowly through D30N or L90M pathways, and selection of ZDV-resistant virus was rare., Conclusions: Selection of 3TC-resistant virus was most frequent, followed by NFV and/or ABC; selection of ZDV-resistant virus was rare. Importantly, although in vitro, ABC selects for M184V as the first mutation, ABC did not select for M184V when combined with ZDV without 3TC. The most sustained HIV-1 RNA response was in the 3TC+ABC arm, but mutations conferring reduced susceptibility to 3TC and/or ABC evolved more frequently if virological failure occurred with 3TC+ABC than with ZDV+ABC.

Objective: To use a stochastic model to gain insights into the consequence for resistance development of different drug use patterns., Methods: We consider use of three drugs (A, B and C) where for each drug one and only one viral mutation is associated with ability to replicate (effective reproductive ratio, R > 1) in the presence of that drug as monotherapy. For drug A mutation is a, etc. We define eight populations of short-lived infected cells that live 1 day: Vo with no mutations a, b, c; Va with mutation a only, Vab with mutations a and b, etc. A random number generator was used to determine whether mutations occur in any one round of replication and to sample from a Poisson distribution to determine for each cell the number of cells of the same population created in the next generation, using the R operative at that time. Values of R depended on drug exposure, cost of resistance and availability of target cells., Results: Treatment strategies and the resulting percentage (over 100 runs) developing full "resistance" in 1500 days (Vabc not equal 0) were: (i) ABC 1500 days 0%; (ii) A 300 days, AB 300 days, ABC 900 days 100%; (iii) AB 300 days, ABC 1200 days 33%; (iv) ABC 2/3 1500 days 15%; (v) ABC 1/2 1500 days 100%; (vi) ABC 50 days, no drugs 50 days, for 1500 days 1%, where ABC 2/3 means on-drug for 2 days in every 3, ABC 1/2 represents on-drug for 1 day in every 2, and represents suboptimal adherence., Conclusions: This model helps to develop understanding of key principles concerning development of resistance under different patterns of treatment use.

The relative fitness of HIV-1 viral variants containing a broad range of drug resistance-associated mutations has been little studied in vivo. Understanding the relative fitness associated with viruses containing mutations may aid future therapeutic management. The aim of this study was to investigate the relative fitness of mutant viruses by assessing a cohort of patients who had developed resistance to many drugs and subsequently stopped all therapy. Eleven patients were assessed for drug resistance associated mutations in the protease (PR) and reverse transcriptase (RT) genes before and, at multiple time points, after stopping therapy. Relative fitness was calculated as a function of the rate of disappearance of mutant viruses when therapy was stopped. The least fit viruses were associated with the RT mutation M184I/V (11.6% less fit) and the PR mutations D30N (12.4% less fit) and M46I/L (21% less fit). Mutations at these codons were associated with significant reductions in fitness levels compared to wild-type viruses. Mutations at codons 10, 20, 36, and 63 in the PR gene remained fairly constant when therapy was stopped and may not significantly reduce viral fitness. The rapid re-population of wild-type viruses may allow the recycling of antiretroviral drugs prescribed previously., (Copyright 2001 Wiley-Liss, Inc.)

The aim of this study was to assess the relationships among the detection of p24 antigen, human immunodeficiency virus (HIV) RNA level, CD4 cell count, and disease progression in 111 males with hemophilia who were infected with HIV for < or =20 years. Sixty-four individuals (58%) developed p24 antigenemia a median of 11.6 years after seroconversion. The time to first detection of p24 antigen was shorter among those who were older (P=.04) and those with a high initial HIV RNA level (P=.006). The median HIV RNA level and CD4 cell count at the time of the detection of p24 antigen were 4.95 log(10) copies/mL and 100 cells/mm(3), respectively. In univariate analyses, p24 antigenemia was associated with more-rapid progression to AIDS (relative hazard [RH], 5.50; P=.0001). The effect was reduced (RH, 1.85; P=.06) after adjusting for CD4 cell counts and HIV RNA levels during follow-up, age, and calendar year. A significant relationship between p24 antigenemia and death was nonsignificant after adjusting for CD4 cell count.

Nucleoside reverse transcriptase inhibitors (NRTIs) comprise the first class of drug with proven antiretroviral efficacy against HIV-1, and the first in which drug resistance was reported. Ongoing research in the area of NRTI resistance and cross-resistance contributes much to what we know about the failure of antiretroviral therapy. The genetic mutation patterns responsible for resistance to the available NRTIs have been well documented. This information is being used to plan rational drug therapy. Furthermore, it serves as the standard against which to evaluate response patterns to multiple-drug regimens, ultimately enabling more accurate prediction of outcome with combination therapies. Other features of NRTI resistance, such as the theoretic reversal of zidovudine resistance associated with the M184V mutation or the powerful influence of the Q151M multiple-drug resistance mutation, have revealed the unpredictable nature of HIV resistance and how much we still need to learn. Although NRTIs are the cornerstone of antiretroviral therapy at present and are used to control disease progression for extended periods, it is clear that eventually resistance occurs with all antiretroviral regimens. Future research into NRTI-resistance mutations, mutational interactions, treatment sequencing, and viral fitness and fidelity will continue to refine our understanding of drug resistance and improve our ability to delay or eliminate resistance and advance HIV control.

The correlation of detectable HIV-1 drug resistance mutations in plasma and PBMCs in patients extensively treated with all antiretroviral drug classes has not been fully elucidated. The detection of mutations in PBMCs may reveal resistant HIV-1 associated with past therapies. In addition, these measures in PBMCs may have a practical value when plasma virus is at low levels that are difficult to detect with current assays. The reverse transcriptase (RT) and protease (P) genes were analyzed for drug resistance, using an in-house method carried out on 36 paired samples of plasma and PBMCs from 12 treatment-experienced patients in order to investigate resistance in the two compartments. When viruses in plasma and PBMCs were analyzed by patient, the mean of the Cohen kappa values was 0.728 (substantial agreement). When viruses were analyzed by codon the mean of the Cohen kappa values was 0.715 (substantial agreement). Baseline samples were concordant at 280 of 288 (97%) codons analyzed. This study shows that a minority of mutations associated with previous therapy can be detected in PBMCs and not in plasma. Overall, mutations in plasma and PBMCs showed a substantial correlation in extensively treated patients, suggesting that either compartment is suitable for the detection of mutations as a virological guide for clinical care.

Objective: Assays for drug resistance testing in human immunodeficiency virus type 1 (HIV-1) infection are now available and clinical studies suggest that viral drug resistance is correlated with poor virologic response to new therapy. The International AIDS Society-USA sought to update prior recommendations to provide guidance for clinicians regarding indications for HIV-1 resistance testing., Participants: An International AIDS Society-USA 13-member physician panel with expertise in basic science, clinical research, and patient care involving HIV resistance to antiretroviral drugs was reconvened to provide recommendations for the clinical use of drug resistance testing., Evidence and Consensus Process: The full panel met regularly between January and October 1999. Resistance and resistance testing data appearing in the last decade through April 2000 and presentations at national and international research conferences were reviewed. Recommendations and considerations were developed by 100% group consensus, acknowledging that definitive data to support final recommendations are not yet available., Conclusions: Emerging data indicate that despite limitations, resistance testing should be incorporated into patient management in some settings. Resistance testing is recommended to help guide the choice of new regimens after treatment failure and for guiding therapy for pregnant women. It should be considered in treatment-naive patients with established infection, but cannot be firmly recommended in this setting. Testing also should be considered prior to initiating therapy in patients with acute HIV infection, although therapy should not be delayed pending the results. Expert interpretation is recommended given the complexity of results and assay limitations.

The effect of cytomegalovirus (CMV) seropositivity on the course of human immunodeficiency virus (HIV) type 1 RNA levels and HIV disease progression was assessed in a cohort of 109 hemophilic men infected with HIV-1 for a median of 12.7 years. There was no evidence of higher HIV RNA levels in the first year after HIV seroconversion (P=. 88) or faster rates of increase over infection (P=.20) in the 59 CMV-seropositive individuals than in the CMV-seronegative individuals. In univariate analyses, CMV seropositivity was associated with significantly faster progression to AIDS and death (relative hazards of 1.58 and 2.22, respectively). These effects were unchanged after adjusting for the RNA level, but they were reduced after adjusting for the CD4 cell count, age at seroconversion, and calendar year of follow-up. Thus, the effect of CMV seropositivity on clinical progression remains significant in this cohort but does not appear to be mediated through an increase in HIV RNA levels.

The objective of this study was to investigate the effect of initiating a salvage-therapy regimen on resistant viruses in heavily treated patients infected with human immunodeficiency virus type 1 (HIV-1). Nineteen patients receiving multiple antiretroviral drugs were tested for HIV-1 mutations associated with drug resistance by using an in-house method at baseline and at weeks 2, 4, and 8 of the salvage-therapy regimen. For the majority of mutations analyzed, the mean number of detectable mutations at baseline was significantly higher than the mean at weeks 2, 4, and 8 of salvage therapy. Introducing new and more potent therapy reduces the number of detectable drug resistance-associated mutations within 8 weeks, and no evidence was found that the new therapy promoted the emergence of novel mutations.

HIV-1 RNA levels are routinely monitored as part of patient management. However, little is known about the course of HIV-1 RNA levels over the entire period of infection. The aim of this study was to investigate the course of HIV-1 RNA levels in a cohort of men with hemophilia who were observed for up to 17 years after HIV-1 seroconversion, and to assess the risk of HIV disease progression at any HIV-1 RNA level. Viral loads were measured on annual stored serum samples in 107 men with hemophilia A using the Roche Amplicor Monitor assay with non-B primers. On average, HIV-1 RNA levels increased significantly by 0.11 log10 per year over the course of HIV infection. This rate of increase was significantly faster in those who developed AIDS or died over the subsequent 12 to 17 year period, and in those who were older at HIV- 1 seroconversion. The risk of developing AIDS and death remained low when the HIV-1 RNA level was below 4 log10 copies/ml, but increased rapidly thereafter, supporting current guidelines for the initiation of antiretroviral therapy after the viral load has exceeded this level.

Objective: To investigate the rate of decline of drug resistant viruses after stopping therapy., Design: Twenty-five patients receiving multiple combination therapies (mean five; range three to nine drugs) for more than 3 months were tested for HIV-1 resistance on therapy and off therapy. The sample off therapy was tested 6-175 days after stopping therapy., Methods: Patients were tested for genotypic changes associated with drug resistance using an in-house automated DNA sequencing assay to detect resistance in the protease and reverse transcriptase genes., Results: All samples tested when patients were on therapy showed evidence of drug resistance (range 1-9 mutations). The patients were divided into three groups: <2 weeks after stopping therapy, median 1.1 weeks (n = 8, group A); 2 weeks-2 months, median 6.4 weeks (n = 7, group B) and 2 months-6 months, median 12.9 weeks (n = 10, group C). Of primary mutations (protease: 30N, 461/L, 82A, 90M; reverse transcriptase: 70R, 184I/V, 215 Y/F) detected on therapy 100% remained after stopping therapy in group A; 68% remained in group B and 15% remained in group C. For secondary mutations 98% remained in group A; 99% remained in group B and 57% in group C., Conclusions: This study showed a rapid decline in detectability of the majority of primary mutations within 13 weeks of stopping combination therapy. From this data, HIV-1 resistance testing to direct patients' therapy should only be carried out when on existing therapy, or < 2 weeks off therapy, if reliable decisions are to be made relating to future combinations.

Objective: Previous studies on patients treated with potent antiretroviral therapy have shown that viral clearance rates do not tend to change between early and advanced HIV-1 infection. Our objective was to investigate whether the other major aspect of virus dynamics, viral replicative capacity, does change. In vitro work has indicated that the viral replicative, capacity increases but in vivo evidence has been lacking., Methods: As an in vivo measure of the viral replicative capacity, we studied the rate of rebound of plasma HIV RNA level during a 1-week therapy interruption in previously untreated patients who had received 2 weeks of antiretroviral therapy., Results: Such therapy in five previously drug-naive patients with high CD4 lymphocyte counts (mean, 611 x 10(6)/l) and five patients with low counts (mean, 49 x 10(6)/l) led to a mean 2.2 log10 copies/ml decrease in plasma HIV-1 levels (from 5-6 log10 copies/ml) in 2 weeks. This was similar in the two groups. Interruption of therapy for the ensuing week resulted in a stable HIV-1 level for approximately 2 days followed by a rebound towards pretherapy level, which was much more marked in the patients with low CD4 cell counts (estimated mean rise 2.22 log10 versus 1.06 log10 copies/ml; P < 0.02). After restarting therapy, HIV RNA levels returned to pre-interruption levels., Conclusions: These findings need confirmation, but the ability of HIV-1 to replicate in vivo appears to increase during HIV-1 infection. This increased replicative capacity, for which there are several potential explanations, may be the cause of gradual CD4 lymphocyte depletion.

Objective: To assess the prognostic value of HIV RNA levels measured shortly after HIV seroconversion and whether markers of immune response (CD4+ and CD8+ T-cell counts, IgA and IgG) measured at the same time, continue to provide prognostic information once the HIV RNA level is known., Design and Methods: HIV RNA levels were measured approximately 2.5 years after seroconversion in 97 haemophilic men followed for up to 17 years. Levels of CD4+ and CD8+ T cells, IgA and IgG were measured within 1 year of the HIV RNA level. The relationships between these markers and progression to AIDS and death were studied using Kaplan-Meier plots and proportional hazards regression models., Results: High HIV RNA levels were associated with faster progression to AIDS and shorter survival in univariate Cox regression models. High IgA and IgG levels were also associated with faster disease progression. In multivariate models, high HIV RNA levels remained independently associated with faster disease progression [relative hazard (RH), 1.86; P = 0.01 for AIDS; RH, 1.66; P = 0.05 for death). However, high IgA and IgG levels provided strong independent prognostic information for AIDS and death (IgA: RH, 1.38; P = 0.006 for AIDS; RH, 1.33; P = 0.07 for death; IgG: RH, 1.10; P = 0.02 for AIDS; RH, 1.12; P = 0.01 for death)., Conclusions: Our results confirm the importance of the HIV RNA level in assessing the long-term prognosis in individuals infected with HIV. However, our results suggest that immune activation markers, rather than merely reflecting high HIV RNA levels are important in assessing prognosis in their own right. These findings may improve our understanding of HIV pathogenesis and may aid clinical management of patients.

Objectives: To review current knowledge of the biology and clinical implications of human immunodeficiency virus (HIV) resistance to antiretroviral drugs, describe assays for measuring resistance, and assess their use in clinical practice., Participants: The International AIDS Society-USA assembled a panel of 13 physicians with expertise in basic science, clinical research, and patient care relevant to HIV resistance to antiretroviral drugs., Evidence: We reviewed available data from published reports and presented at national and international research conferences. Basic science research, clinical trial results, and expert opinions were used to form the basis of this report. Data on methods for and characteristics of specific genotypic and phenotypic assays were obtained from manufacturers and service providers., Consensus Process: The panel met regularly between October 1997 and April 1998. Panel subgroups developed and discussed different sections of the report before discussing them with the entire panel. Conclusions and suggested approaches to the use of resistance testing were determined by group consensus., Conclusions: Plasma HIV RNA level and CD4+ cell count are the primary values that should be used to guide the initiation of antiretroviral therapy and subsequent changes in therapy. Possible causes of treatment failure other than development of drug resistance that should be considered are adherence, drug potency, and pharmacokinetic issues. Genotypic and phenotypic testing for HIV resistance to antiretroviral drugs may prove useful for individual patient management. Assays under development need validation, standardization, and a clearer definition of their clinical roles. Possible current roles of resistance testing for choosing an initial regimen or changing antiretroviral therapy, as well as possible implications of the presence or absence of phenotypic resistance and genotypic changes, are discussed.

Replication of HIV introduces errors into the genome which are responsible for conferring a growth advantage over wildtype virus when drugs such as zidovudine (ZDV) exert a selective pressure. The molecular basis for HIV-1 resistance to ZDV has been mapped to codons 41, 67, 70, 215 and 219 of the reverse transcriptase gene both in vitro and in clinical samples of blood. This study has investigated the relationship between the quantitative prevalence of ZDV resistance in multiple organs of the same individual. Proviral HIV-1 load was measured by quantitative-competitive PCR in 90 samples from organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine. The distribution of wildtype and mutant sequences at the positions 41, 67, 70, 215 and 219 of the reverse transcriptase was assessed using a point mutation assay. The results showed that the highest proviral loads were predominately found in lymph node, spleen and lung and there was a significant association between viral load and resistance to ZDV (P=0.008). Inter-organ distribution of wildtype and mutant sequences at codons 41, 67, 70, 215 and 219 was frequently not uniform and in some patients differed markedly between the lymphoreticular system and other organs. These results demonstrate that treatment of HIV-1 infection with zidovudine does not exert uniform selective pressures in multiple organs. These findings have implications for the interpretation of resistance data and design of treatment strategies for HIV, arguing in particular that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in multiple sites within the same individual.

Virological assays for human immunodeficiency virus type 1 load and drug resistance can broadly be divided into culture-based and molecular biology-based methods. Culture-based methods give a direct measure of infectious virus load and phenotypic drug resistance, whereas molecular biology-based methods are indirect, assaying nucleic acid levels to determine virus load and point mutations associated with drug resistance. We have compared culture-based and non-culture-based methods for patients enrolled in a placebo-controlled trial of zidovudine (the Concorde Trial). Virus loads were assayed by culture of peripheral blood mononuclear cells (PBMCs) or quantitative PCR, and drug resistance was assayed in culture or in a quantitative, PCR-based point mutation assay. The rates of detection of viremia and drug resistance were higher by PCR than by culture for this population of subjects. Comparison of the virus loads by the two measures showed a good correlation for virus loads in PBMCs but a poor correlation for virus loads in plasma. The latter result probably reflected the inaccuracies of culture in assaying plasma with the low infectious virus titers seen in the study population. The concordance of phenotypic and genotypic drug resistance methods was high, with all phenotypically resistant isolates having at least one resistance-associated mutation and with no mutations being found in a drug-sensitive isolate. Genomic resistance scores (weighted sums of levels of resistance mutations) showed good correlations with the levels of phenotypic resistance, and both resistance measures were observed to increase as the duration of exposure to drug increased. Overall, non-culture-based methods were shown to correlate well with culture-based methods and offer a low-cost, high-throughput alternative. However, culture-based methods remain the final arbiters of infectious virus load and phenotypic drug resistance and are unlikely to be superseded entirely.

Simple models of CD4 lymphocyte interactions with human immunodeficiency virus (HIV) lead to the hypothesis that progression of HIV infection involves an increase in viral replicative capacity, due either to changes in the virus or in the host environment, or both. In order to consider how changes in plasma virus load after transient, potent antiretroviral therapy can be used to test the above hypothesis--a simple mathematical model that encompasses the processes of (1) arrival of new CD4 lymphocytes, (2) death/removal of these cells by HIV-independent mechanisms, (3) infection of susceptible CD4 lymphocytes by HIV, and (4) death/removal of infected cells was investigated. This showed that the in vivo rate of increase in plasma virus load immediately after transient therapy provides a measure of the viral replicative capacity. Thus, the hypothesis that progression of HIV infection involves an increase in viral replicative capacity can be tested by measuring this viral growth rate in patients with high CD4 counts and in patients with low CD4 counts. Studies should thus investigate dynamics of changes in virus levels after stopping antiretroviral therapy and, in particular, measure rates of increase in virus in patients at high and low CD4 counts. In practice, such data may assist in therapeutic management of patients with HIV infection.

Background: The Delta trial showed that combination therapy (zidovudine plus didanosine and zidovudine plus zalcitabine) substantially lengthened life and reduced disease progression compared with zidovudine monotherapy. We did a nested virological study in three countries (France, the Netherlands, and the UK) to investigate changes in markers for viral load and antiretroviral-drug resistance during therapy., Methods: 240 zidovudine-naive HIV-1-infected patients were randomly assigned zidovudine only (n = 87), zidovudine plus didanosine (n = 80), or zidovudine plus zalcitabine (n = 73). Viral load in peripheral-blood mononuclear cells and plasma was measured by quantitative culture. Plasma HIV-1 RNA was measured by reverse-transcriptase PCR amplification, and serum p24 antigen by ELISA. Resistance to antiretroviral drugs was measured phenotypically by culture and genotypically by detection and quantification of drug-related point mutations in the pol gene. Analyses were done by intention to treat., Findings: The reduction in viral load was greatest 4-12 weeks after the start of therapy and was most pronounced in the combination-therapy study groups (median reductions of RNA at 4 weeks 1.58, 1.28, and 0.49 log10 copies/mL for zidovudine plus didanosine, zidovudine plus zalcitabine, and zidovudine only, respectively). RNA levels at 8 weeks were predictive of disease progression and death after allowance for baseline values. At 48 weeks, the proportion of participants with phenotypic zidovudine resistance was similar in all three groups: didanosine and zalcitabine resistance were rare; zidovudine genomic resistance correlated with phenotypic resistance (r = 0.54, p < 0.0001) and developed earlier in the combined-therapy groups. However, participants in the zidovudine monotherapy group had higher circulating loads of resistant virus than those in the combined-therapy groups., Interpretation: Combined antiretroviral therapy was more efficient at lowering virus load than monotherapy. Although zidovudine resistance was common in monotherapy and combined-therapy groups, circulating concentrations of resistant virus were substantially lower in the combination groups, which is likely to be a result of the continued antiviral activity of didanosine or zalcitabine.

Three procedures for the quantification of human immunodeficiency virus type 1 (HIV-1) RNA from plasma were compared at three laboratories. The comparison involved the Quantiplex branched DNA assay (version 1.0) by Chiron Diagnostics, the NASBA-QT assay by Organon Teknika, and the Amplicor Monitor assay by Roche Molecular Systems. The laboratories performed each of the three assays with the same sets of reconstructed HIV-1-infected human plasma samples, cross-sectionally collected clinical plasma samples and longitudinally collected plasma samples from patients starting zidovudine therapy. Analysis of the reconstruction panel results for interlaboratory variation demonstrated that no laboratory differences in results were detected for any of the assays. A comparison of the reproducibilities of duplicate samples analyzed by batch and in separate assay runs demonstrated that the reproducibilities of the test results were similar within one assay and appeared to be independent of the HIV-1 concentration. The best reproducibility was obtained with the Quantiplex assay, but all three assays demonstrated equal reliability, which was independent of batched or unbatched analysis of replicate samples. Differences in the absolute concentrations calculated were observed for the assays, in particular in the analysis of reconstructed samples. In all assays, similar changes in plasma HIV-1 RNA concentrations were determined for longitudinally collected clinical samples.

Objective: To compare safety and efficacy of lamivudine-zidovudine combination therapy with zidovudine monotherapy in treating human immunodeficiency virus type 1 (HIV-1)-infected, antiretroviral therapy-naive patients., Design: Double-blind, randomized, multicenter, comparative trial of 129 patients throughout 24 weeks followed by 24 weeks of open-label lamivudine in combination with zidovudine., Setting: Outpatients from 14 hospitals in Belgium, France, Germany, Spain, and the United Kingdom were enrolled within 6 months., Patients: HIV-1-positive, antiretroviral-naive ( < or = 4 weeks prior zidovudine use) patients aged atleast 18 years with CD4+ cell counts between 0.10 x 10(9)/L and 0.40 x 10(9)/L (100-400/microL)., Intervention: Patients received either 300 mg of lamivudine every 12 hours in combination with 200 mg of zidovudine every 8 hours for 24 weeks or zidovudine monotherapy for 24 weeks. All patients were then allowed to receive zidovudine in combination with open-label lamivudine (300 mg every 12 hours)., Main Outcome Measures: Efficacy was assessed by changes in CD4+ cell counts beta 2-microglobulin, neopterin, HIV-1 immune-complex dissociated (ICD) p24 antigenemia, and HIV-1 viral load. Safety was assessed by incidence of adverse clinical events and defined laboratory-measured toxic effects., Results: Combination therapy showed superior treatment effects compared with monotherapy during the first 24 weeks as documented by changes in CD4+ cell counts (increase of 0.08 x 10(9)/L vs 0.02 x 10(9)/L; P < .001), ICDp24 (-88% vs -49%; P = .04), cellular viremia (-1.27 vs -0.20 log10 median tissue-culture infected dose [TCID50] per 10(6) peripheral blood mononuclear cells; P = .001), and viral load measured by HIV-1 RNA polymerase chain reaction using a Roche method (-1.33 vs -0.57 log10 copies/mL; P = .001) or an immune-capture method (-0.6 vs -0.14log10 copies/mL; P = .008). Observed changes were sustained to 48 weeks for patients continuing to receive combination therapy. Patients switching to receive combination therapy at week 24 showed improvements in CD4+ cell count and viral load to week 48. Mutation results suggested that mutations associated with zidovudine resistance may have developed more slowly over the first 24 weeks in patients receiving combination therapy. In contrast, mutations associated with lamivudine resistance appeared to develop rapidly, despite sustained antiviral treatment effect. However, the number of patients evaluated for genotypic changes was small, and confirmation of these results is needed in larger studies. No statistically significant differences in incidence or severity of clinically manifested or laboratory-measured toxic effects were noted between treatment groups., Conclusions: The combination of lamivudine and zidovudine results in a potent and sustained antiviral effect in antiretroviral-naive patients that is superior to that observed with zidovudine monotherapy.

Objective: To compare the safety and efficacy of 2 doses of lamivudine given in combination with zidovudine with continued zidovudine monotherapy., Design: Double-blind, randomized, multicenter, comparative trial of 223 patients treated for 24 weeks., Setting: Patients from 32 hospitals in Europe were enrolled throughout a 1-year period., Patients: Adult human immunodeficiency virus type 1 (HIV-1)-positive, zidovudine-experienced ( > or = 24 weeks prior zidovudine) patients with CD4+ cell counts between 0.10 and 0.40 x 10(9)/L (100-400 cells/microL)., Intervention: Patients received either 200 mg of zidovudine every 8 hours, 150 mg of lamivudine every 12 hours plus zidovudine, or 300 mg of lamivudine every 12 hours plus zidovudine for 24 weeks. All patients were then allowed to receive zidovudine and open-label lamivudine combination therapy. Twelve patients withdrew because of adverse events during the 24-week treatment period., Main Outcome Measures: Efficacy was measured by evaluating immunological and viral load changes, and safety was assessed by evaluating clinical manifestations and laboratory indexes of toxic effects., Results: Patients receiving low- or high-dose combination therapy had greater treatment effects compared with patients receiving continued zidovudine monotherapy during the first 24 weeks as documented by changes in CD4+ cell counts (+0.04 vs +0.03 vs -0.02 x 10(9)/L, respectively; P < .001); log10 HIV-1 RNA as measured by the Roche assay (-0.96 vs -0.77 vs +0.07 copies/mL, respectively; P < .001) or log10 HIV-1 RNA measured by the quantitative nucleic acid sequence-based amplification assay (-0.59 vs -1.06 vs -0.02 copies/mL, respectively; P < .011); and immune-complex dissociated (ICD) p24 antigen (-74% vs -68% vs +27%, respectively; P < .001). There were no statistically significant differences in viral measurements, in CD4+ cell counts, or in safety profile between the groups receiving 2 doses of lamivudine in combination with zidovudine. The effects on CD4+ cell counts and ICD p24 antigen were sustained throughout 48 weeks for patients continuing combination therapy. Patients switching to combination therapy at week 24 showed improvement., Conclusion: In zidovudine-experienced HIV-1-infected patients, combination treatment with lamivudine and zidovudine is well tolerated and provides greater and more sustained increases in CD4+ cell counts and decreases in viral load than continued zidovudine monotherapy.

The diagnosis of human immunodeficiency virus (HIV) infection in children born to HIV-infected mothers is complicated by the presence of passively acquired maternal antibodies, and exclusion of infection in these infants remains problematic. The use of genome detection by polymerase chain reaction (PCR) amplification and the quantification of anti-HIV-1 antibodies were examined as methods for early diagnosis. Blood samples were taken from 84 non-breast-fed infants of HIV-infected mothers in five Italian and Spanish centres, a subgroup of children enrolled in the European Collaborative Study (ECS) for whom clinical and immunological information has been documented from birth. Whole blood was added to glycigel cryopreservative, stored, and tested in the United Kingdom by a nested PCR method. Antibody to HIV-1 was detected and quantified by titration using a gelatin particle agglutination test. PCR sensitivity and specificity were assessed. Twenty-one of the 84 children tested were infected. The estimated PCR sensitivity ranged from 0% (95% CI 0-26%) on day 1, 57% (19-85) on day 7, to 63% (33-92) on day 30. The negative predictive value of PCR ranged from 85% (83-88) on day 0 to 98% (94-100) at 3 months of age. On average, the level of maternal antibody halved every 33 days (31-36.5) in uninfected children. Between 6 and 9 months of age, increases in antibody titres in infected children were not more informative than absolute levels. These findings suggest that antibody measurement may supplement genomic diagnosis and that this collection method provides an alternative to the use of dried blood spots.

Point mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type-1 (HIV-1) have been associated with reduced sensitivity of the virus to inhibition by the antiretroviral drug zidovudine (ZDV). During therapy we have previously observed the accumulation of these mutations, at codons 41, 67, 70, 215, and 219 of the RT gene, in proviral DNA sequences from infected peripheral blood mononuclear cells (PBMCs) and in cell-free virus RNA. Using a quantitative point mutation assay (PMA) we have been able to quantify the proportions of mutant and wild-type sequences at these points in mixed populations of viral RNA and DNA derived from clinical samples. Thus we have confirmed that the mutations can be detected earlier in cell-free virus RNA than in PBMC proviral DNA and their accumulation in RNA precedes that in DNA. We have analyzed serial samples from 10 subjects undergoing zidovudine therapy and shown significantly higher levels of mutations in cell-free viral RNA than in PBMC proviral DNA at codons 41, 70, and 215 during the in vivo acquisition of the mutations. In the group of subjects studied the mean time delay between RNA and DNA reaching a given level of mutation was 25 days.

Objective: To derive reliable estimates of the sensitivity of HIV-1 DNA polymerase chain reaction (PCR) in the neonatal period and to quantify the relative contributions of intra-uterine and intra-partum transmission., Methods: After reviewing studies on the early diagnosis of HIV-1 infection, investigators were asked to provide published and unpublished PCR test results on prospectively followed, non-breastfed, vertically infected children. Age-specific estimates of the sensitivity of PCR were derived using distribution-free methods for interval-censored data., Results: Data on 271 infected children were combined for analysis. PCR detected HIV-1 DNA in an estimated 38% [90% confidence interval (CI), 29-46] of HIV-infected children tested on the day of, or day after, birth. Sensitivity was observed to rise rapidly in the second week of life, reaching 93% (90% CI, 76-97) by 14 days of age., Conclusion: The sensitivity of PCR in the neonatal period is higher than previously reported. This affects the clinical interpretation of an early negative test result and encourages the use of PCR as an endpoint for trials to evaluate interventions to reduce vertical transmission in non-breastfed populations. Approximately one-third of vertically acquired HIV-1 infection could be attributable to intra-uterine transmission.

The effect of the appearance of drug-resistant human immunodeficiency virus type 1 (HIV-1) on viral RNA load was studied in patients treated with the reverse transcriptase inhibitor lamivudine. During the first 12 weeks of treatment, HIV-1 RNA concentrations and amino acid changes in codon 184, causing high-level resistance to lamivudine, were determined in longitudinal serum samples from HIV-1 p24 antigen-positive and -negative patients. A marked decline in the amount of HIV-1 RNA (approximately 95% below baseline) and HIV-1 p24 antigen was observed within 2 weeks, followed by a rise that coincided with the appearance of lamivudine-resistant viruses in serum (isoleucine mutants initially, which were subsequently replaced by valine variants). After 12 weeks, a partial antiviral effect was observed despite the presence of a complete codon 184 mutant virus population in serum. This study shows that the rapid appearance of drug-resistant virus in serum is followed by an increase in viral RNA load.

As part of the Medical Research Council prospective study of the neurological complications of HIV infection, neurophysiological tests of spinal cord and peripheral nerve function were recorded in a cohort of homosexual or bisexual men. The studies included motor and sensory nerve conduction studies, vibration perception thresholds, somatosensory evoked potentials and motor evoked potentials elicited by magnetic stimulation. The results were compared with markers of immune function. The findings from 114 volunteers were analysed in a cross-sectional study. Fifty-nine were HIV-seropositive but asymptomatic, 26 had progressed to the symptomatic stages of HIV disease and 29 were persistently HIV-seronegative. There was some evidence of a mild sensory axonopathy in the symptomatic HIV-seropositive group. No differences were detected between the asymptomatic HIV-seropositive group and the HIV-seronegative comparison group. There were no consistently significant correlations between the neurophysiological measurements and CD4 counts and beta 2-microglobulin levels. On repeated testing, there was no evidence of a trend towards deterioration over a mean period of approximately 3 years in 36 HIV-seropositive subjects who remained asymptomatic compared with 22 HIV-seronegatives. These findings have failed to demonstrate neurophysiological evidence of spinal cord or peripheral nerve dysfunction in the asymptomatic stages of HIV infection.

The response of HIV-1 to initial zidovudine (ZDV) treatment was assessed in 11 patients with severe HIV disease. We quantified serum HIV-1 concentrations and mutations associated with ZDV resistance by culture-independent methods. There was a prompt fall in serum HIV-1 RNA within 1-2 days of treatment with maximum suppression by seven days, which was paralleled by changes in serum p24 antigen (p24 Ag). Serum RNA started to return to pretreatment levels within weeks. The HIV reverse transcriptase (RT) gene in most patients developed mutations associated with drug resistance within months and as early as 25 days on therapy in one patient. The codon changes were not sufficient to explain the early return of serum HIV-1 RNA levels and their patterns continued to evolve after patients stopped taking ZDV. The significance of these findings is discussed in relation to the limited long-term efficacy of ZDV. The dynamic time course of viral load and RT responses to ZDV is of particular importance in short-term interventions such as pregnancy.

As part of the Medical Research Council prospective study of the neurological and neuropsychological complications of human immunodeficiency virus (HIV) infection, long-latency event-related potentials were recorded in a cohort of homosexual and bisexual men. The latencies and amplitudes of the potentials, recorded from three scalp sites, were compared with the scores from neuropsychological tests of memory, attention, and concentration and with markers of immune function. The findings from 94 men were analyzed in the cross-sectional study of whom 47 were HIV seropositive without symptoms or signs of HIV type 1 (HIV-1) infection, 24 had progressed to the symptomatic stages of the disease, and 23 were persistently HIV seronegative. There were no consistently significant differences between the three subject groups in mean latencies and amplitudes of the P300 and N200 or in the numbers of abnormal P300 latencies. There were no significant correlations between either the neuropsychological tests scores or the immune measures (CD4 lymphocyte count and beta 2 microglobulin level) and the neurophysiological parameters. On repeated testing an average of 2 years later, there was no evidence of a significant trend towards deterioration in 30 HIV-seropositive subjects who remained asymptomatic compared with 22 HIV seronegatives. These findings indicate that there is no neurophysiological evidence of cognitive dysfunction in the asymptomatic stages of HIV-1 infection.