Operational challenges in delivering CD4 diagnostics in sub-Saharan Africa.

Access to reliable and low cost CD4 T-cell enumeration to stage illness and monitor anti-retroviral therapy remains elusive in resource-limited settings. We report challenges in delivering CD4 testing using the microcapillary Fluorescence-Activated Cell Sorter (FACS) methodology (Guava EasyCD4 instrument Guava Technologies, Hayward) in Burkina Faso and Zimbabwe. Resources, instruments, reagents, and training were provided to local laboratories within the existing infrastructure and data on CD4 were collected from routine laboratory testing. Challenges encountered included frequent instrument breakdown; poor manufacturer maintenance; difficulties in managing reagent stocks; high technician turnover; reliance on antiquated data management systems; redundant service provision; and lack of repeat testing in male HIV+ patients and in patients with higher CD4 counts after initial staging. While adopting newer, less expensive technologies such as fluorescent platforms and point of care tests can facilitate access to lower cost CD4 testing, our experience suggests that supply chain, corporate commitment to implementation, and community factors also require consideration.

Alanine aminotransferase levels are not significantly elevated in patients with HIV/HBV co-infection and lamivudine resistance.

In hepatitis B virus (HBV) monoinfection, alanine aminotransferase (ALT) levels are linearly correlated with HBV DNA levels and lamivudine resistance. In human immunodeficiency virus (HIV)/HBV co-infection, little is known about the association between ALT, HBV DNA, and lamivudine resistance. We assessed HBV DNA, lamivudine resistance and ALT levels in 45 time points in 11 patients with HIV/HBV co-infection during lamivudine-containing antiretroviral therapy. High HBV DNA levels (>10(6) copies/mL) and lamivudine resistance developed in 45% and 91% of patients, respectively. However, ALT levels were not elevated in the setting of high HBV DNA levels (mean ALT, 48 IU/mL) or lamivudine resistance (mean ALT, 44 IU/mL). HBV viraemia and lamivudine resistance during extended lamivudine-containing antiretroviral therapy are common in HIV/HBV co-infection, occurring in the absence of significant ALT elevations. In HIV/HBV co-infection, measurement of HBV DNA and HBV resistance mutations may identify HBV virological failure before biochemical changes and should be routinely used in the management of HIV/HBV co-infection.

Reduction in plasma human immunodeficiency virus ribonucleic acid after dideoxynucleoside therapy as determined by the polymerase chain reaction.

Cell-free HIV RNA in plasma was detected and quantitated after antiviral therapy by the polymerase chain reaction. RNA was extracted from plasma, reverse transcribed to cDNA, amplified by polymerase chain reaction, and quantitated by absorbance based on an enzyme-linked affinity assay. 72 HIV antibody-positive subjects had one plasma sample taken. 39 who were not receiving antiretroviral therapy at the time had a mean plasma HIV RNA copy number of 690 +/- 360 (mean +/- SEM) per 200 microliters of plasma, while 33 subjects who had been receiving zidovudine therapy for a minimum of 3 mo had a mean copy number of 134 +/- 219 (P less than 0.05). 27 additional HIV antibody-positive patients had two plasma samples taken before and 1 mo after initiating dideoxynucleoside therapy. Plasma HIV RNA copy number fell from 540 +/- 175 to 77 +/- 35 (P less than 0.05). Finally, nine of these subjects had two baseline samples obtained before initiating therapy and two posttreatment samples 1 and 2 mo after therapy was begun. Mean plasma RNA copy number declined from 794 +/- 274 to less than 40 (below the lower limit of sensitivity) after 1 mo of therapy, with suppression maintained after 2 mo of therapy. These results suggest that gene amplification can be used to detect and quantitate changes in plasma HIV RNA after dideoxynucleoside therapy. Plasma HIV polymerase chain reaction may be a more sensitive marker to monitor antiviral therapy, particularly in asymptomatic patients where measurement of p24 antigen or quantitative plasma cultures are negative.

A 6-basepair insert in the reverse transcriptase gene of human immunodeficiency virus type 1 confers resistance to multiple nucleoside inhibitors.

While many point mutations in the HIV-1 reverse transcriptase (RT) confer resistance to antiretroviral drugs, inserts or deletions in this gene have not been previously characterized. In this report, 14 RT inhibitor-treated patients were found to have HIV-1 strains possessing a 6-basepair insert between codons 69 and 70 of the RT gene. Known drug resistance mutations were also observed in these strains, with T215Y appearing in all strains. Genotypic analysis indicated that the inserts had substantial nucleotide variability that resulted in relatively restricted sets of amino acid sequences. Linkage of patients' treatment histories with longitudinal sequencing data showed that insert strains appeared during drug regimens containing ddI or ddC, with prior or concurrent AZT treatment. Drug susceptibility tests of recombinant patient isolates showed reduced susceptibility to nearly all nucleoside RT inhibitors. Site- directed mutagenesis studies confirmed the role of the inserts alone in conferring reduced susceptibility to most RT inhibitors. The addition of AZT-associated drug resistance mutations further increased the range and magnitude of resistance. These results establish that inserts, like point mutations, are selected in vivo during antiretroviral therapy and provide resistance to multiple nucleoside analogs.

Bayesian network analysis of resistance pathways against HIV-1 protease inhibitors.

Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.

A pilot study to assess the immunologic and virologic efficacy of generic nevirapine, zidovudine and lamivudine in the treatment of HIV-1 infected women with pre-exposure to single dose nevirapine or short course zidovudine and their spouses in Chitungwiza, Zimbabwe.

OBJECTIVE: A pilot study to assess effectiveness of generic Nevirapine (NVP)+Zidovudine (AZT)+Lamivudine (3TC) as potent antiretroviral therapy (ART) in women exposed to either SD NVP or short course (SC) AZT through participation in prevention of mother-to-child transmission of HIV-1 (pMTCT) interventions, and their spouses. DESIGN: A pilot study of antiretroviral treatment of adults with AIDS. SETTING: Primary health care clinics; Seke North and St Mary's in Chitungwiza, Zimbabwe. SUBJECTS: Women with pre-exposure to SD NVP or SC AZT and their spouses with CD4 count < 200 cells/ INTERVENTIONS: Generic AZT/3TC twice daily plus NVP daily for the first 14 days and then twice a day thereafter, administered to the cohort. MAIN OUTCOME MEASURES: The baseline median CD4 count for women and men was 128.5 and 119.0 cells/ microL respectively. The geomean virus load was similar for the women and men. At weeks 16, 24 and 48, 82.8%, 85.1% and 73.8% had < 400 copies/ml of HIV RNA respectively. Only at 16 weeks, was the proportion of women (75.9%) with undetectable virus significantly lower than that for men (93.9%), p = 0.031. Median CD4 count for both men and women increased significantly, p < 0.001. There were no significant differences in virologic responses between the women with pre-exposure to SD NVP and SC AZT. The mean adherence for women and men was similar, > 98%. CONCLUSION: Women showed a significantly reduced response top ART relative to men only at 16. However, prior exposure to SD NVP for PMTCT was no more likely to negatively influence responses to ART than use of SC AZT.

Risk factors for prevalent and incident HIV infection in a cohort of volunteer blood donors in Harare, Zimbabwe: implications for blood safety.

OBJECTIVES: To compare risk factors for HIV seropositivity with risk factors for HIV seroconversion in a population of volunteer blood donors in Harare, Zimbabwe, and to assess the impact of risk factor exclusion strategies on the safety of the blood supply. DESIGN: A secondary analysis of a longitudinal cohort study was performed. SUBJECTS AND METHODS: The subjects were volunteer blood donors who were also taking part in a prospective cohort study in Harare, Zimbabwe. They were tested for HIV antibodies upon enrollment and at 6-month intervals. Their donation history, age, marital status and the presence or absence of multiple sex partners and paying for sex were assessed as predictors of HIV seropositivity and HIV seroconversion. The impact of exclusion strategies on blood safety was modeled by estimating the number of HIV-infected units that would escape detection by antibody screening if blood donors with these risk factors were excluded. RESULTS: The HIV seroprevalence among persons accepted for blood donation was 8.8%; the HIV seroincidence was 2.1 per 100 person-years. Significant risk factors for HIV seropositivity were being a new donor (odds ratio 7.3, 95% confidence interval 4.4-1 2.1), age over 25 years (odds ratio 1.6, confidence interval 1.1-2.4), being married (odds ratio 1.7, confidence interval 1.2-2.6), paying for sex (odds ratio 2.6, confidence interval 1.7-3.9) and multiple sex partners (odds ratio 2.1, confidence interval 1.4-2.9). Significant risk factors for HIV seroconversion were age under 25 years (hazard ratio 2.5, confidence interval 1.4-5.0) and being unmarried (hazard ratio 2.5, confidence interval 1.4-5.0). Of note, age and marital status reversed their direction of association with respect to HIV seropositivity and HIV seroconversion. Exclusion strategies based on strong predictors of HIV seroconversion were the most effective in improving the safety of the blood supply. CONCLUSIONS: A distinction between risk factors for HIV seropositivity and HIV seroconversion is necessary in order to develop strategies to reduce the residual risk of transfusion-associated HIV transmission. Because window-period donations are the most important source of residual HIV contamination and arise from incident infections, research to develop risk factor exclusion strategies must focus on predictors of HIV seroconversion.

PIP: Secondary analysis of data from a longitudinal cohort study of voluntary blood donors in Harare, Zimbabwe, confirmed a residual risk of HIV contamination of blood due to laboratory false-negatives and donations made during the window period. It further identified different risk factors for prevalent HIV infection (HIV seropositivity) compared with incident HIV (HIV seroconversion). The HIV prevalence rate among the 1515 blood donors enrolled in the study during 1993-95 was 8.8% (2.1 per 100 person-years). HIV seroprevalence was highest among first-time donors, those 21-45 years of age, married persons, those with more than 1 sexual partner in the preceding year, and those who had paid for sex in the past year. Among the 1142 initially HIV-negative donors who had at least 1 6-month follow-up test, there were 40 seroconversions (2.1 per 100 person-years). Significant risk factors for seroconversion were age under 25 years and single marital status. Since window-period donations account for substantially more HIV contamination of the blood supply than laboratory false-negatives, donor exclusion criteria based on risk factors for incident HIV infection are more likely to reduce the residual risk of HIV contamination than exclusion criteria based on risk factors for prevalent HIV infection.

Estimating HIV incidence from age-specific prevalence data: comparison with concurrent cohort estimates in a study of male factory workers, Harare, Zimbabwe.

OBJECTIVE: To compare HIV incidence estimates from cross-sectional age-specific prevalence data with concurrent cohort estimates and to examine the sensitivity of the estimates to changes in age-categorization and survivorship assumptions. METHODS: Two previously described methods of estimating HIV incidence from cross-sectional prevalence data - the cumulative incidence and survival (CIS) and constant prevalence (CP) methods - are applied using data from a study of male factory workers in Harare, Zimbabwe. The methods are applied under two alternative groupings of the HIV prevalence data and under alternative survivorship assumptions: (a) Weibull distribution providing the best fit to the HIV prevalence data using the CIS method; (b) Weibull distribution matching data from an HIV natural history cohort study in Uganda; and (c) survivorship pattern as in (b) with survival periods reducing with increasing age at infection. Age-specific, age-standardized and cumulative HIV incidence estimates are calculated. The results are compared with concurrent longitudinal estimates from 3 years of follow-up of the Harare cohort (1993-1995). RESULTS: Age-standardized HIV incidence was estimated at 2.02 per 100 man years (95% CI, 1.57-2.47) in the cohort study. There was evidence of recent variability in HIV incidence in these data. Estimates from the cross-sectional methods ranged from 1.98 to 2.74 per 100 man years and were sensitive to changes in age-categorization of the HIV prevalence data and changes in survivorship assumptions. The cross-sectional estimates were higher at central ages and lower at older ages than the cohort estimates. The age-specific estimates from the CIS method were less sensitive to changes in age grouping than those from the CP method. CONCLUSIONS: HIV incidence remains high in Harare. Incidence estimates broadly consistent with cohort estimates can be obtained from single-round cross-sectional HIV prevalence data in established epidemics - even when the underlying assumption of stable endemic prevalence is not fully met. Estimates based on cross-sectional surveys should therefore be explored when reliable longitudinal estimates cannot be obtained. More data on post-HIV infection survivorship distributions in sub-Saharan Africa would facilitate the improvement of estimates of incidence based on cross-sectional surveys.

Characterizing patterns of drug-taking behavior with a multiple drug regimen in an AIDS clinical trial.

OBJECTIVE: To characterize drug-taking behavior using continuous electronic monitoring in an AIDS clinical trial. SETTING: This was a substudy of AIDS Clinical Trials Group (ACTG) protocol 175, a phase II/III study of dideoxynucleoside monotherapy versus combination therapy in asymptomatic HIV-positive subjects. Participants were required to comply with regimens containing zidovudine, zalcitabine and didanosine, or matching placebos; the total daily pill count was 16. DESIGN: For participants at two ACTG 175 sites, electronic devices were used to monitor drug-taking behavior of all study medications over a period of approximately 90 days. MEASUREMENTS: Four indices of drug-taking behavior were calculated and their distributions and relationship to the prescribed regimen were examined. RESULTS: Data from 41 subjects were analyzed. Of the prescribed doses of zidovudine, zalcitabine and didanosine, 88, 84 and 82%, respectively, were taken. Of these, 55, 66 and 79%, respectively, were taken at the prescribed dosing frequency. The median percentage of days on which participants failed to take any of the doses was 2-5%. There was a trend towards lower adherence in the combination therapy arms compared with those assigned to receive monotherapy. In this analysis, older patients demonstrated better adherence, although patient characteristics, in general, were poorly predictive of adherence. CONCLUSION: Drug-taking behavior for all three active study medications differed from that prescribed. One result of this erratic adherence was that study participants sustained little antiretroviral effect during more than 25% of the monitoring period.

The extent of non-adherence in a large AIDS clinical trial using plasma dideoxynucleoside concentrations as a marker.

OBJECTIVES: To assess adherence to study medications in an AIDS clinical trial, to evaluate whether study participants adhered to only one component of a multidrug regimen ('differential adherence'), and to determine whether there was evidence of non-uniform adherence to study medications among treatment groups. SETTING: This was a substudy of AIDS Clinical Trials Group protocol 175, a large, double-blind, randomized study of monotherapy versus combination dideoxynucleoside therapy. Participants were required to adhere to a complex regimen of zidovudine, zalcitabine and didanosine, or their matching placebos. DESIGN: Between October 1992 and January 1994, study sites were selected at random, and a 1-week period was designated during which study participants attending routine clinic visits provided a blood sample and dosing history. Participants were not informed of the purpose of the substudy. MEASUREMENTS: Adherence was assessed using plasma drug concentrations and defined by the presence of detectable drug in a plasma sample obtained within a specified analysis window. RESULTS: Of 722 plasma samples analyzed, approximately 75% contained detectable concentrations of the assigned drugs and 5-14.5% contained no detectable drugs. Approximately 7 and 13% of samples from participants assigned to monotherapy arms contained non-prescribed dideoxynucleosides, and 14 and 19% assigned to combination therapies contained only one drug. CONCLUSIONS: Various non-adherence behaviors were observed, including patterns of underdosing and taking non-prescribed drugs. Non-adherence was moderate but uniform amongst the treatment groups and may have contributed to a marginal reduction in the power of the primary intent-to-treat analysis to detect differences in efficacy amongst the assigned treatments.

HIV seroincidence and correlates of seroconversion in a cohort of male factory workers in Harare, Zimbabwe.

OBJECTIVES: Given that health promotion messages on transmission of HIV and other sexually transmitted diseases (STD) have been widely publicized in Zimbabwe and elsewhere in the late 1980s, it is vital to analyse which risk events still expose individuals to infection. A cohort was established with the objectives of estimating HIV seroincidence, behavioural and biological determinants of infection, and ultimately, evaluating the impact of AIDS prevention interventions in the workplace. METHODS: HIV seroincidence was estimated in a prospectively followed cohort of male factory workers recruited in Harare, Zimbabwe during the period prior to a workplace AIDS prevention intervention. Correlates of HIV seroconversion were identified using Cox regression analysis. RESULTS: There were 51 seroconversions among 1607 HIV-negative participants following 1738 person-years (PY) of observation [seroincidence, 2.93 per 100 PY; 95% confidence interval (CI), 2.18-3.86]. The prevalence of HIV in the cohort was 19.1%. HIV seroincidence was significantly increased among men who were younger [hazard ratio (HR) per year, 0.96; 95% CI, 0.93-0.99], were single (HR, 3.29; 95% CI, 1.56-6.96), were married but resided separately from their wives (HR, 2.18; 95% CI, 0.99-4.80), reported having any STD (HR, 3.00; 95% CI, 1.53-5.86), reported having a genital ulcer (HR, 4.87; 95% CI, 2.18-10.91), and reported paying for sex (HR, 2.01; 95% CI, 1.06-3.77). Seroincidence also increased with the number of sex partners reported in the year preceding enrolment (HR per partner, 1.10; 95% CI, 1.01-1.21). In multiple Cox regression analysis, three independent associations with HIV seroconversion were reporting a genital ulcer (adjusted HR, 3.55; 95% CI, 1.52-8.29), number of sex partners (adjusted HR, 1.10; 95% CI, 1.01-1.21), and being married but residing separately from one's wife (adjusted HR, 2.21; 95% CI, 1.00-4.89). CONCLUSIONS: Innovative and sustained workplace-based interventions are needed to address the high risk of HIV infection in this economically productive population. The predictors of HIV seroconversion described in this study underscore the need for public health efforts to simultaneously address the biological, socioeconomic and behavioural factors that continue to place individuals at risk of HIV in general populations of Africa.

PIP: Results of a prospective HIV testing and questionnaire response study of 1607 male factory workers in Harare, Zimbabwe, were used to identify demographic and behavioral characteristics associated with HIV seroconversion. Specifically, the objectives of this study were to estimate HIV seroincidence, identify behavioral and biological determinants of infection, and evaluate the impact of current AIDS prevention intervention programs presented in the workplace. This male cohort was followed from a pre-intervention program phase through the establishment of a workplace/post-intervention program. Results were compared. Correlates of HIV seroconversion were identified using Cox regression analysis. There were 51 (3.2%) seroconversions among the 1607 HIV-negative participants following 1738 person-years (PY) of observation (seroincidence, 2.93/100 PY; 95% confidence interval (CI), 2.18-3.86). The HIV prevalence in the cohort was 19.1%. HIV seroincidence was significantly increased among men who were younger (hazard ratio (HR) per year, 0.96; 95% CI, 0.93-0.99), were single (HR, 3.29; 95% CI, 1.56-6.96), were married but resided separately from their wives (HR, 2.18; 95% CI, 0.99-4.80), reported having an STD (HR, 3.00; 95% CI, 1.53-5.86), reported having a genital ulcer (HR, 4.87; 95% CI, 2.18-10.91), and reported paying for sex (HR, 2.01; 95% CI, 1.06-3.77). Seroincidence also increased with the number of sex partners reported from the preceding calendar year (HR per partner, 1.10; 95% CI, 1.01-1.21). The authors conclude that innovative and sustained workplace-based interventions are needed to address the high risk of HIV infection in this economically productive segment of the Zimbabwe population. The risk factors and predictors of HIV seroconversion described in this study underscore the great need for public health efforts designed to address the biological, socioeconomic, cultural, and behavioral factors surrounding the spread of HIV and STDs.

Genital ulcers and transmission of HIV among couples in Zimbabwe.

Seventy-five married men found to be positive for HIV-1 in Harare, Zimbabwe, were interviewed in order to define behaviours associated with acquisition of infection and to determine factors associated with transmission of infection to their wives. The majority of infected men reported sexual intercourse with multiple heterosexual partners and female prostitutes, and gave a history of sexually transmitted diseases (STDs). All subjects denied homosexual activity and parenteral drug abuse. Serological testing of the wives of seropositive men showed that 45 (60%) were HIV-antibody-positive. Wives of men with AIDS and AIDS-related complex (ARC) and wives of men who gave a history of genital ulcer disease were more likely to be seropositive. The study demonstrates that HIV-1 infection in Zimbabwe occurs through heterosexual intercourse and is associated with other STDs. In addition, the study shows that male to female transmission of HIV-1 is facilitated by the presence of genital ulcers in infected men.

PIP: As part of a prospective cohort study of Zimbabweans seropositive for human immunodeficiency virus (HIV) infection, the factors associated with HIV transmission from husband to wife were analyzed in 75 couples. The mean age of the infected men was 32.1 years; all had been married for at least 1 year and 53 had at least 1 child. None of the men acknowledged a history of homosexual practices or intravenous drug use. All 75 had received injections, but only at reputable health care facilities, and none had been blood transfusion recipients. 4 men (5%) were asymptomatic, 40 (53%) had persistent generalized lymphadenopathy, 24 (32%) had acquired immunodeficiency syndrome (AIDS)-related complex (ARC), and 7 (9%) had full-blown AIDS. Of the 75 wives tested, 45 (60%) were seropositive for HIV-1 infection. Seropositivity was significantly more common among women married to men with ARC (71% infection rate) and AIDS (86% infection rate). Comparison of concordant (both seropositive) and discordant (husband only seropositive) couples indicated no significant differences in terms of age, duration of marriage, number of children, oral contraceptive use by the wife, husband's contact with prostitutes, or sexual activity on the part of the husband with multiple partners in the preceding 3 years. In addition, there was no significant difference between groups in terms of history of sexually transmitted diseases. However, men who reported a history of genital ulcer disease were significantly more likely to have a wife who was seropositive (relative risk, 1.94; 95% confidence interval, 1.62-15.13). This difference persisted even when the male's stage of disease was controlled. Thus, it appears that HIV-1 infected men with genital ulcers are likely to transmit the infection through vaginal intercourse.

Neuromuscular function in HIV infection: analysis of a placebo-controlled combination antiretroviral trial. AIDS Clinical Group 175/801 Study Team.

OBJECTIVE: To determine the frequency of peripheral neuropathy and myopathy in HIV-infected subjects enrolled in a combination antiretroviral treatment trial. DESIGN AND METHODS: AIDS Clinical Trial Group (ACTG) protocol 175 was a multicenter, double-blind, placebo-controlled, clinical trial. A total of 2467 subjects were randomized to one of four single or combination regimens, containing zidovudine (ZDV), didanosine (ddl), zalcitabine (ddC), and their respective placebos. Site investigators reported peripheral neuropathy, and the diagnosis of distal symmetrical neuropathy (DSP) was established by the study authors. Myalgia, muscle weakness and creatine phosphokinase (CPK) were prospectively assessed in a subset of the antiretroviral-naive cohort (n = 1067). RESULTS: Of 222 site diagnoses of neuropathy, 109 (49%) were DSP. There was a significant difference between treatment arms for rate of DSP and time to first grade 2 or higher DSP (ZDV-ddC, 6%; ZDV, 4%; ZDV-ddl, 4%; ddl, 3%; P = 0.029). Age and Karnofsky score were significant predictors of DSP. Fifty-six (54%) out of 104 patients with DSP remained on study medication at full (n = 29) or reduced (n = 27) dose within 6 months of developing neuropathy. There was no significant difference between treatment arms in the rate of myalgia or muscle weakness. The median CPK of subjects on ZDV-ddC was significantly higher than other study treatments, although CPK levels did not correlate with symptoms of myopathy. Only six subjects were diagnosed with myopathy during the study (one ZDV-ddl, one ZDV-ddC, and four ddl). CONCLUSIONS: DSP and myopathy may occur with current dosing regimens of combination antiretroviral therapy, and should be diagnosed using stringent criteria. ZDV-ddC was associated with the highest rate of DSP, although features of myopathy were not significantly different between treatment regimens.

Phenotypic hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in treatment-experienced HIV-infected patients: impact on virological response to efavirenz-based therapy.

BACKGROUND: Enhanced susceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) was recently described in association with increased resistance to nucleoside analogs (nucleoside reverse transcriptase inhibitors; NRTI). OBJECTIVES: To determine the prevalence of NNRTI hypersusceptibility, the genotypic correlates, and its impact on virologic response to efavirenz-based salvage therapy. METHODS: Genotype and phenotype testing was performed retrospectively on baseline isolates from 30 patients who received salvage therapy containing efavirenz. NNRTI hypersusceptibility was defined as a 50% inhibitory concentration (IC(50)) of < 0.5 that of the wild-type control. RESULTS: Eight isolates had major NNRTI mutations. Among the 22 isolates with no major NNRTI mutations, 11 (50%) were hypersusceptible to efavirenz, 10 (45%) to delavirdine, and eight (36%) to nevirapine. Among eight isolates with NNRTI mutations, NNRTI resistance was present, but at lower than expected levels. The number of NRTI mutations was correlated inversely with the fold decrease in susceptibility to efavirenz (Spearman's rho, -0.57; P = 0.005), delavirdine (rho, -0.43; P = 0.04), and nevirapine (rho, -0.69; P < 0.001). Excluding subjects with NNRTI mutations, subjects with efavirenz hypersusceptibility at baseline had significantly better virologic suppression over 24 weeks than those without efavirenz hypersusceptibility (P < 0.001). CONCLUSION: NNRTI hypersusceptibility is common in heavily treated but NNRTI naive patients and is related directly to NRTI resistance mutations. Among patients receiving efavirenz-containing regimens, NNRTI hypersusceptibility was associated with an improved virologic outcome after 24 weeks of therapy. A reversal of phenotypic resistance was seen in patients with NNRTI mutations in the presence of multiple NRTI mutations, but no obvious virologic benefit of this phenomenon was seen in this study.

The contribution of assay variation and biological variation to the total variability of plasma HIV-1 RNA measurements. The Women Infant Transmission Study Clinics. Virology Quality Assurance Program.

OBJECTIVES: To assess the specific contributions of assay variation and biological variation to the total variation of plasma HIV-1 RNA measured by the Roche Monitor assay and the extent to which batch assays reduced both assay variability and total variability compared with real-time determinations. DESIGN: A retrospective analysis of data obtained from three trials conducted by the Adult and Pediatric AIDS Clinical Trials Groups (ATCG), the Women and Infants Transmission Study (WITS) and the NIAID-sponsored Virology Quality Assurance Program. METHODS: Within-subject variation was assessed from stored, serially collected plasma samples from 663 subjects enrolled in the ACTG and WITS studies. Interassay and intra-assay variation were estimated from two of the clinical trials and 22 laboratories that participated in a quality assurance program and were used to estimate the effect of real-time testing on total variation. RESULTS: The total variation (standard deviation) from a random effects model was 0.26 log10 RNA copies/ml. The estimated interassay variation was 0.08 log10 and intra-assay variation was 0.12 log10 RNA copies/ml. Biological variation accounted for 56-80% of total variation. The effect of real-time testing compared with batch testing was minimal. CONCLUSION: Our estimates of total within-subject HIV-1 RNA variation support the current recommendation to obtain at least two specimens, preferably obtained less than 2 weeks apart, for viral RNA measurement before starting therapy. The major contribution of biological variation to the total variation supports the use of real-time HIV-1 RNA assays, provided that consistent specimen collection procedures are followed and acceptable assay proficiency is maintained.

Competing drug-drug interactions among multidrug antiretroviral regimens used in the treatment of HIV- infected subjects: ACTG 884.

OBJECTIVE: To evaluate the steady state concentrations of saquinavir, ritonavir, nelfinavir, delavirdine, and adefovir in six different three- and four-drug combination regimens. DESIGN: Randomized, partially double-blinded, multicenter study in a population of indinavir-experienced subjects with virologic failure. The first seven subjects enrolled in each of the six treatment arms from 10 participating sites were entered into this pharmacokinetic evaluation. SETTING: Multicenter study of the AIDS Clinical Trials Group (ACTG). PATIENTS: HIV-infected subjects. INTERVENTIONS: A 12-hour pharmacokinetic study was conducted after 2 weeks of drug administration. MAIN OUTCOME MEASURES: Area under the concentration-time curve with statistical comparisons to evaluate the effect of the second protease inhibitor and the effect of the non-protease inhibitors. RESULTS: There was no difference in saquinavir concentrations according to whether the second protease inhibitor was ritonavir or nelfinavir. Saquinavir concentrations in the groups receiving the combination of delavirdine plus adefovir dipivoxil were reduced by approximately 50% compared with those receiving delavirdine. Delavirdine concentrations were reduced by approximately 50%, in the delavirdine plus adefovir dipivoxil arms compared with the delavirdine arms. CONCLUSIONS: Saquinavir concentrations were significantly lower in the arms containing the combination of delavirdine and adefovir dipivoxil compared with the arms containing delavirdine. Delavirdine concentrations were significantly lower when coadministered with adefovir dipivoxil. These drug-drug interactions were not expected, the mechanism(s) is (are) not clear, and additional studies are warranted. This study illustrates the need to understand more fully the pharmacokinetic characteristics of complex combination antiretroviral regimens prior to use in patient management.

Quantification and comparison of HIV-1 proviral load in peripheral blood mononuclear cells and isolated CD4+ T cells.

HIV proviral load was determined by quantitative DNA polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMC) and lymphocyte subsets isolated by cell sorter. Provirus measured in PBMC, when expressed as HIV copy number per million CD4+ cells, resulted in values which approximated those obtained from sorted CD4+ T lymphocytes. A cross sectional analysis of HIV proviral load in CD4+ T cells from 25 previously untreated and 30 zidovudine-treated seropositive patients with CD4+ T-cell counts between 25 and 802/mm3 demonstrated HIV copy numbers ranging from 1 copy per 10,000 cells in early disease to 1 copy per 10 cells in advanced disease. HIV proviral load can be rapidly assayed by PCR to give a reproducible value which varies over a 1,000-fold range and is positively correlated with cell infectivity as measured by a quantitative micrococulture assay. A less technically demanding assay using PBMC as substrate can give similar results to those obtained with sorted CD4+ T cells.

Decrease in HIV provirus in peripheral blood mononuclear cells during zidovudine and human rIL-2 administration.

Quantification of human immunodeficiency virus (HIV) proviral DNA in peripheral blood mononuclear cells (PBMC) was performed in 13 HIV-seropositive asymptomatic individuals during 10-24 months by polymerase chain reaction amplification of multiple half-log dilutions of cellular DNA. At enrollment, subjects had a geometric mean titer of 100 copies of HIV provirus per 10(6) PBMC (mean +/- SD, 2 +/- 0.9 log10). In four untreated individuals there was no significant change in provirus levels during a mean period of 13.3 months. In eight patients treated with zidovudine (ZDV) and human recombinant interleukin 2 (rIL-2), HIV provirus copies declined to 13 per 10(6) cells (1.1 +/- 0.8 log10) at the end of the first course of ZDV and rIL-2 at week 20 (p less than 0.01), and to 40 per 10(6) cells (1.6 +/- 0.9 log10) after 12 months of treatment (p less than 0.04). Subsequent courses, which included 12 weeks of ZDV alone or 4 weeks of IL-2 alone, did not significantly change the already depressed provirus copy numbers. Proviral copy number also remained depressed during drug-free "washout periods" between courses. Finally, we observed a return to a geometric mean of 400 copies per 10(6) cells (2.6 +/- 0.3 log10) a mean of 7.9 months after discontinuation of therapy. Measurement of changes in HIV provirus should provide a direct marker for defining antiviral activity of drugs, biologics, and combination therapy.

Sexual behavior and risk factors for HIV infection in a group of male factory workers who donated blood in Harare, Zimbabwe.

Zimbabwe has experienced a rapid rise in HIV seroprevalence in recent years. As many as 1 million people (10% of the population) are predicted to become seropositive by 2000. We examined social and behavioral factors associated with HIV infection in a case-control study among male factory workers who donated blood before the launching of the AIDS Awareness Campaign. There were 188 subjects: 69 were HIV-positive and 119 were HIV-negative. High-risk behavior was common in both groups. Among seronegative men, 73.1% reported a sexually transmitted disease (STD), 55.5% reported cash payment for sex, and 73.1% had outside girlfriends. Nonetheless, HIV-positive men were more likely to report a history of STD (OR = 3.9; 95% CI = 1.5-11.9), particularly genital ulcers (OR = 2.4; 95% CI = 1.2-4.8), and extramarital partners (OR = 2.8; 95% CI = 1.1-7.1). HIV-positive men reported more lifetime partners (16.5 vs. 12; p less than 0.05) and were less likely to live with their wives (OR = 0.51; 95% CI = 0.23-1.15). Our findings support the importance of genital ulcer as a risk factor and suggest widespread high-risk sexual behavior among urban working-class men.

HIV-1 syncytium-inducing phenotype, virus burden, codon 215 reverse transcriptase mutation and CD4 cell decline in zidovudine-treated patients.

The variable rate of disease progression in HIV-1-infected patients treated with zidovudine may be related to certain viral characteristics, such as, antiviral drug resistance, virus burden, and viral syncytium-inducing (SI) capacity. Thirty-two HIV-1-infected patients treated with zidovudine (mean of 34 months) were studied to determine the relationship of SI phenotype and the codon 215 pol gene mutation (a marker of zidovudine resistance) to virus burden and CD4 cell decline. Patients with SI strains and the codon 215 mutation in their proviral DNA had a 54% decline in CD4 cells and a virus burden of 21,480 proviral DNA copies/10(6) CD4 cells. In contrast, patients with non-SI (NSI) strains and wild-type at codon 215 had a 10% increase in CD4 cells and had a viral burden 1/46 that of patients with SI and the 215 mutation. Among patients with NSI strains, changes in CD4 cells depended on the presence of the codon 215 mutation (-160 CD4 cells/microliters), compared with those wild-type at codon 215 (+28 CD4 cells/microliters) (p < 0.01). There was a concordant rise in virus burden between proviral DNA and plasma HIV RNA depending on HIV phenotype and genotype. Using multiple linear regression, SI phenotype and the codon 215 mutation were found to independently predict CD4 cell decline and increased virus burden in zidovudine-treated patients.