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Background: A substantial proportion of patients with HIV infection will not respond to antiretroviral therapy. Early predictors of response to treatment are needed to identify patients who are at risk for treatment failure.Objective: To determine predictors of virologic and clinical response to indinavir, zidovudine, and lamivudine therapy.Design: Observational analysis of one treatment group in a phase III trial.Setting: 40 AIDS Clinical Trials units.Patients: 489 patients receiving indinavir, zidovudine, and lamivudine who had 1) a CD4 count of 0.200 x 10(9) cells/L or less after 8 or more weeks of study therapy and 2) plasma HIV-1 RNA measurements obtained at baseline and week 8.Measurements: HIV-1 RNA level and CD4 cell count at weeks 0, 4, 8, 24, and 40. Clinical progression was defined as a new AIDS-defining illness or death.Results: Patients' levels of HIV-1 RNA at the 8th study week of therapy predicted whether patients would achieve virologic suppression to below 500 (or 50) copies/mL at study week 24. An HIV-1 RNA level less than 500 copies/mL at week 24 was achieved in 71% of patients whose level at week 8 had been less than 500 copies/mL, 53% of those with a level of 500 copies/mL or more and at least 2-log(10) copies/mL reduction since baseline, 29% of those with a level of 500 copies/mL or more with a 1- to 1.99-log(10) copies/mL reduction, and 9% of those with a level of 500 copies/mL or greater and less than 1-log(10) copies/mL reduction since baseline (P < 0.001). HIV-1 RNA level at week 8 also predicted clinical progression. HIV-1 disease progressed in 2.2% of the patients with a week-8 HIV-1 RNA level less than 500 copies/mL, 2.3% of patients with 500 copies/mL or greater and at least 2-log(10) copies/mL reduction since baseline, 4.9% of patients with 500 copies/mL or greater and 1- to 1.99-log(10) copies/mL reduction since baseline, and 10.6% of patients with 500 copies/mL or greater and less than 1-log(10) copies/mL decrease since baseline (P = 0.009). After adjustment for HIV-1 RNA level, patients with a higher week-8 CD4 cell count were more likely to have a week-24 HIV-1 RNA level less than 500 copies/mL (relative risk for patients with a week-8 CD4 count >/= 0.10 x 10(9) cells/L, 1.47 [95% CI, 1.00 to 2.16] compared with <0.050 x 10(9) cells/L; relative risk for patients with a week-8 CD4 count of 0.05 to 0.099 x 10(9) cells/L, 0.98 [CI, 0.61 to 1.57] compared with <0.050 x 10(9) cells/L). After adjustment for HIV-1 RNA level, patients with a week-8 CD4 count of 0.05 x 10(9) cells/L or greater (compared with <0.05 x 10(9) cells/L) had a decreased hazard for clinical progression (hazard ratio, 0.25 [CI, 0.09 to 0.67]).Conclusions: The HIV-1 RNA level and CD4 cell count achieved at 8 weeks of treatment are important predictors of subsequent virologic and clinical outcomes. [ABSTRACT FROM AUTHOR]

Anatomical compartments (e.g., the reproductive tract) are reservoirs of human immunodeficiency virus type-I (HIV-1) and potential sites of residual infection in patients receiving anti-retroviral therapy (ART). Viral hyper-excretion relative to blood is a hallmark of reservoirs. To determine whether hyper-excretion can occur in the oral cavity, we compared viral loads in blood plasma and saliva of 67 adults. Salivary viral hyper-excretion was defined as a four-fold or higher viral load in saliva than in plasma. HIV-1 RNA was detected in 79% of plasma samples, in 44% of unfiltered saliva samples, in 16% of filtered saliva samples, and in 59% of saliva-derived cell pellets. Compared with non-hyper-excretors (n =62), hyper-excretors (n =5) had elevated levels of viral RNA in unfiltered saliva and saliva-derived cells, HIV-associated periodontal disease, gingival inflammation, and no combination ART. Morphological characterization of cell pellets identified lymphocytes as a likely HIV-1 source. These collective findings are consistent with an oral HIV-1 reservoir in selected individuals. [ABSTRACT FROM AUTHOR]

Context: Although protease inhibitors are used routinely in adults with human immunodeficiency virus (HIV) infection, the role of these drugs in the treatment of clinically stable HIV-infected children is not clear.Objective: To evaluate the safety, tolerance, and virologic response produced by a change in antiretroviral therapy in HIV-infected children who were clinically and immunologically stable while receiving previous therapy.Design: The Pediatric AIDS Clinical Trials Group 338, a multicenter, phase 2, randomized, open-label controlled trial conducted from February 6 to April 30, 1997 (patient entry period); patients were followed up for 48 weeks.Setting: Pediatric HIV research clinics in the United States and Puerto Rico.Patients: Two hundred ninety-seven antiretroviral-experienced, protease inhibitor-naive, clinically stable HIV-infected children aged 2 to 17 years.Interventions: Children were randomized to receive zidovudine, 160 mg/m2 3 times per day, plus lamivudine, 4 mg/kg 2 times per day (n = 100); the same regimen plus ritonavir, 350 mg/m2 2 times per day (n = 100); or ritonavir, 350 mg/m2 2 times per day, and stavudine, 4 mg/kg 2 times per day (n = 97).Main Outcome Measure: Plasma HIV-1 RNA levels at study weeks 12 and 48, compared among the 3 treatment groups.Results: At study week 12, 12% of patients in the zidovudine-lamivudine group had undetectable plasma HIV RNA levels (<400 copies/mL) compared with 52% and 54% of patients in the 2- and 3-drug ritonavir-containing groups, respectively (P<.001). Through study week 48, 70% of children continued receiving their ritonavir-containing regimen. At study week 48, 42% of children receiving ritonavir plus 2 nucleosides compared with 27% of those receiving ritonavir and a single nucleoside had undetectable HIV RNA levels (P = .04); however, similar proportions in each group continuing initial therapy had HIV RNA levels of less than 10000 copies/mL (58% vs 48%, respectively; P = .19).Conclusions: In our study, change in antiretroviral therapy to a ritonavir-containing regimen was associated with superior virologic response at study week 12 compared with change to a dual nucleoside analog regimen. More children receiving ritonavir in combination with 2 compared with 1 nucleoside analog had undetectable HIV RNA levels at study week 48. [ABSTRACT FROM AUTHOR]

Context: Pediatric human immunodeficiency virus (HIV) infection has unique viral pathogenetic features that preclude routine extrapolation from adult studies and require specific analysis.Objectives: To evaluate the prognostic value of 2 key laboratory markers-plasma RNA and CD4+ lymphocyte count-for HIV disease progression in infants and children and to establish targeted values for optimal outcome.Design: Data from a cohort of 566 infants and children who participated in a randomized, placebo-controlled trial of nucleoside reverse transcriptase inhibitors (ACTG 152) were analyzed. The trial was conducted between 1991 and 1995 and enrolled a heterogeneous cohort of antiretroviral therapy-naive children (age, 3 months to 18 years); patients had a median follow-up of 32 months.Main Outcome Measures: The trial clinical end points consisted of time to first HIV disease progression (growth failure, decline in neurologic or neurodevelopmental function, opportunistic infections) or death.Results: Baseline plasma RNA levels were high (age group medians, 5 x 10(4) to >10(6) copies/mL), and both baseline RNA and CD4+ lymphocyte count were independently predictive of subsequent clinical course. Risk reduction for disease progression between 49% and 64% was observed for each log10 reduction in baseline RNA and was linear without suggestion of a threshold or age effect. Disease progression predictive power was enhanced by the combined use of plasma RNA and CD4+ cell count. Marker values of less than 10000 copies/mL for plasma RNA and greater than 500 x 10(6)/L (<6.5 years of age) or greater than 200 x 10(6)/L (>6.5 years) for CD4+ cell count were associated with a 2-year disease progression rate of less than 5%.Conclusions: Two key laboratory markers--plasma RNA and CD4+ lymphocyte count-are independent predictors of clinical course among HIV-infected infants and children. The linear, age-independent relationship between log10 plasma RNA and relative risk of disease progression strongly supports therapeutic efforts to achieve plasma virus levels as low as possible. [ABSTRACT FROM AUTHOR]

Objectives: To assess health care providers' identification of human immunodeficiency virus (HIV)-exposed infants, to ascertain the prevalence of transplacental or oral zidovudine treatment among infants exposed to HIV, and to estimate the impact of zidovudine use on perinatal transmission in rural and urban North Carolina.Design: Survey of North Carolina newborns tested for HIV infection in 1993 and 1994 compared with the number of anonymous HIV-positive childbearing women.Setting: North Carolina hospitals, public health clinics, and private physicians' offices.Main Outcome Measures: Rates of identification of HIV-exposed infants and of perinatal HIV-1 transmission, determined by HIV culture and polymerase chain reaction testing in the infants.Results: The proportion of HIV-exposed children in North Carolina who were identified and tested increased from 60% in 1993 to 82% for all of 1994, and to more than 90% for the last quarter of 1994. The HIV-exposed infants born in rural counties were more likely to be recognized than those born in urban counties (P<.001). In 1994, most infants were evaluated relatively early in life: 39% by 1 week of age, 63% by 6 weeks, and 76% by 3 months. Among infants with recognized HIV exposure, transmission decreased significantly between 1993 and 1994, from 21% to 8.5%, respectively (P=.009). After the announcement of the results of the AIDS Clinical Trials Group Protocol 076, zidovudine was given to 75% of HIV-positive women who delivered infants in North Carolina. Only 5.7% of infants who received any zidovudine became infected, compared with 18.9% of infants who received no zidovudine (P=.007).Conclusions: Health care providers in North Carolina are identifying most of the state's HIV-seropositive pregnant women, treating them with zidovudine, and testing their infants soon after birth for HIV infection. The use of zidovudine in pregnant women and their infants has reduced perinatal HIV transmission in the state. [ABSTRACT FROM AUTHOR]

CD4 cell counts and blood plasma and seminal plasma human immunodeficiency virus type 1 (HIV-1) concentrations were compared in HIV-1 RNA-seropositive men with urethritis and with or without genital ulcer disease (GUD). GUD was associated with lower CD4 cell counts (median, 258 vs. 348/μL) and increased blood plasma HIV-1 RNA (median, 240 × 103 vs. 79.4 × 103 copies/ mL). Men with nongonococcal urethritis and GUD shed significantly greater quantities of HIV-1 in semen (median, 195 × 103 vs. 4.0 × 103 copies/mL) than men with nongonococcal urethritis without GUD. These levels decreased ∽4-fold following antibiotic therapy. The results indicate an association between GUD and increased blood HIV-1 RNA levels. Increased HIV-1 in semen was demonstrated in some men with GUD; such an increase could lead to increased transmission, thus complicating interpretation of the role of the genital ulcer itself in the infectiousness of HIV. Reasons for increased HIV RNA in semen in men with GUD remain to be determined. [ABSTRACT FROM PUBLISHER]

Dried blood spots that had been stored ambiently for 3 to 6 years lost approximately 1 log10 of human immunodeficiency virus type 1 (HIV-1) RNA, but the majority could still be genotyped for resistance. Nevirapine resistance was found in 7/16 (43.5%) HIV-1-positive HIVNET 024 infants at 4 to 6 weeks, but no resistance was found at other time points.

Background: Randomized-trial data on the risks and benefits of antiretroviral therapy (ART) as compared with zidovudine and single-dose nevirapine to prevent transmission of the human immunodeficiency virus (HIV) in HIV-infected pregnant women with high CD4 counts are lacking.Methods: We randomly assigned HIV-infected women at 14 or more weeks of gestation with CD4 counts of at least 350 cells per cubic millimeter to zidovudine and single-dose nevirapine plus a 1-to-2-week postpartum "tail" of tenofovir and emtricitabine (zidovudine alone); zidovudine, lamivudine, and lopinavir-ritonavir (zidovudine-based ART); or tenofovir, emtricitabine, and lopinavir-ritonavir (tenofovir-based ART). The primary outcomes were HIV transmission at 1 week of age in the infant and maternal and infant safety.Results: The median CD4 count was 530 cells per cubic millimeter among 3490 primarily black African HIV-infected women enrolled at a median of 26 weeks of gestation (interquartile range, 21 to 30). The rate of transmission was significantly lower with ART than with zidovudine alone (0.5% in the combined ART groups vs. 1.8%; difference, -1.3 percentage points; repeated confidence interval, -2.1 to -0.4). However, the rate of maternal grade 2 to 4 adverse events was significantly higher with zidovudine-based ART than with zidovudine alone (21.1% vs. 17.3%, P=0.008), and the rate of grade 2 to 4 abnormal blood chemical values was higher with tenofovir-based ART than with zidovudine alone (2.9% vs. 0.8%, P=0.03). Adverse events did not differ significantly between the ART groups (P>0.99). A birth weight of less than 2500 g was more frequent with zidovudine-based ART than with zidovudine alone (23.0% vs. 12.0%, P<0.001) and was more frequent with tenofovir-based ART than with zidovudine alone (16.9% vs. 8.9%, P=0.004); preterm delivery before 37 weeks was more frequent with zidovudine-based ART than with zidovudine alone (20.5% vs. 13.1%, P<0.001). Tenofovir-based ART was associated with higher rates than zidovudine-based ART of very preterm delivery before 34 weeks (6.0% vs. 2.6%, P=0.04) and early infant death (4.4% vs. 0.6%, P=0.001), but there were no significant differences between tenofovir-based ART and zidovudine alone (P=0.10 and P=0.43). The rate of HIV-free survival was highest among infants whose mothers received zidovudine-based ART.Conclusions: Antenatal ART resulted in significantly lower rates of early HIV transmission than zidovudine alone but a higher risk of adverse maternal and neonatal outcomes. (Funded by the National Institutes of Health; PROMISE ClinicalTrials.gov numbers, NCT01061151 and NCT01253538 .). [ABSTRACT FROM AUTHOR]

Identifying the specific genetic characteristics of successfully transmitted variants may prove central to the development of effective vaccine and microbicide interventions. Although human immunodeficiency virus transmission is associated with a population bottleneck, the extent to which different factors influence the diversity of transmitted viruses is unclear. We estimate here the number of transmitted variants in 69 heterosexual men and women with primary subtype C infections. From 1,505 env sequences obtained using a single genome amplification approach we show that 78% of infections involved single variant transmission and 22% involved multiple variant transmissions (median of 3). We found evidence for mutations selected for cytotoxic-T-lymphocyte or antibody escape and a high prevalence of recombination in individuals infected with multiple variants representing another potential escape pathway in these individuals. In a combined analysis of 171 subtype B and C transmission events, we found that infection with more than one variant does not follow a Poisson distribution, indicating that transmission of individual virions cannot be seen as independent events, each occurring with low probability. While most transmissions resulted from a single infectious unit, multiple variant transmissions represent a significant fraction of transmission events, suggesting that there may be important mechanistic differences between these groups that are not yet understood. [ABSTRACT FROM AUTHOR]

The protease (PR) and reverse transcriptase (RT) regions of HIV-1 isolates from 21 antiretroviral (ARV)-naive Malawian adults were sequenced and analyzed to determine the prevalence of drug resistance-associated mutations in this population. Phylogenetic analysis confirmed that all isolates grouped with HIV-1 subtype C, the predominant subtype in Malawi. No major mutations associated with resistance to PR inhibitors (PIs), nucleoside RT inhibitors (NRTIs), or nonnucleoside RT inhibitors (NNRTIs) were found. In contrast, accessory mutations were found in the protease region at positions 10, 20, 36, 63, 77, and 93, and in the RT region at positions 118, 211, and 214. Further studies will be needed to determine the clinical impact of these polymorphisms on viral susceptibility to existing antiretroviral drugs.

Fifty-eight HIV-infected children with acute rotavirus diarrhea were tested for plasma HIV RNA. There was no difference between acute and convalescent mean viral loads, and little change in CD4 cell counts. Compared with the 16 children who died within 4 weeks, 31 survivors had slightly lower viral loads at presentation and significantly higher CD4 cell counts. Low CD4 cell counts, but not HIV-1-RNA concentrations, were predictive of Death. Local, enteric rotavirus infection did not appear to affect blood HIV viral load or CD4 cell counts in this small group of children.

Objectives: The objectives of this study were 1) to evaluate testing regimens of human immunodeficiency virus (HIV)-exposed infants and 2) to determine optimal methods of follow-up by enzyme-linked immunosorbent assay (ELISA) testing., Methods: We reviewed the results from 742 HIV-exposed infants in the state of North Carolina; 2474 samples were tested for HIV by DNA polymerase chain reaction (PCR) at the University of North Carolina Retrovirology Core Laboratory. We then reviewed the utility and costs of ELISA testing of all HIV-exposed infants who were seen at the Duke University Pediatric Infectious Disease Clinic between January 1, 1993, and May 5, 1998. We used likelihood ratios to model probability of HIV infection given 3 negative DNA (PCR) tests and to provide recommendations on the use of ELISA follow-up., Results: The overall sensitivity of the DNA PCR was 87.1%, and its specificity was 99.9%. We evaluated 224 HIV-exposed infants who were seen at Duke University and who had at least 3 negative diagnostic tests using either DNA PCR tests or HIV blood cultures. All 178 infants who subsequently underwent ELISA testing ultimately demonstrated seroreversion. The Duke University Pediatric Infectious Disease Clinic transferred the care of 65 patients to primary care physicians before ELISA testing and retained the care of the remaining 159 patients. Children who remained in Duke's care were more likely to have documentation of seroreversion (158 of 159 vs 20 of 65). We reviewed costs of travel, physician appointment, and HIV antibody testing in a tertiary care setting. Given 3 negative PCR tests, the expected cost per case of HIV detected by a positive ELISA assay is $23.8 million., Conclusions: Documentation of seroreversion in this cohort was nearly complete in the multidisciplinary subspecialty clinic but not when such responsibility was left to the primary care physician. Given the low probability of disease in patients who have had 3 negative PCR tests, documentation of a negative ELISA may not be an appropriate use of medical resources.

To confirm the vertical transmission of multidrug-resistant (MDR) human immunodeficiency virus type 1 (HIV-1) and to assess its impact on further evolution of drug-resistant virus in an infant, proviral DNA amplified from infected peripheral blood mononuclear cell cultures was sequenced to identify reverse transcriptase (RT) and protease (PR) mutations. The infant had proviral DNA with evidence of RT mutations (M41L, L74V, and T215Y) and 3 PR substitutions (K20R, M36I, and V82A). After delivery, the mother's proviral DNA had the same substitutions. Phylogenetic analyses of these HIV-1 RT and PR sequences indicated epidemiological linkage. Plasma drug susceptibility was determined by using a recombinant virus assay. Plasma HIV-1 obtained after the infant's birth demonstrated reduced susceptibility to zidovudine and ritonavir. Thus, vertical transmission of MDR HIV-1 was demonstrated in the setting of detectable maternal plasma viremia. Further accumulation of broad MDR in the infant's virus to 3 antiretroviral classes occurred, despite postnatal therapy.

Objective: To describe initial viral dissemination to peripheral tissues and infectious body fluids during human primary HIV infection., Design: Observational cohort study., Methods: Blood plasma, cerebrospinal fluid (CSF), seminal plasma, cervicovaginal lavage fluid and/or saliva were sampled from 17 individuals with primary HIV infection (range of time from symptoms onset to sampling, 8--70 days) and one individual with early infection (168 days). Subjects' HIV-1 RNA levels in each fluid were compared with levels from antiretroviral-naive controls with established HIV infection. For study subjects, correlations were assessed between HIV-1 RNA levels and time from symptoms onset. Responses to antiretroviral therapy with didanosine + stavudine + nevirapine +/- hydroxyurea were assessed in each compartment., Results: HIV-1 RNA levels were highest closest to symptoms onset in blood plasma (18 patients) and saliva (11 patients). CSF HIV-1 RNA levels (five patients) appeared lower closer to symptoms onset, although they were higher overall in primary versus established infection. Shedding into seminal plasma (eight patients) and cervicovaginal fluid (two patients) was established at levels observed in chronic infection within 3--5 weeks of symptoms onset. High-level seminal plasma shedding was associated with coinfection with other sexually transmitted pathogens. Virus replication was suppressed in all compartments by antiretroviral therapy., Conclusions: Peak level HIV replication is established in blood, oropharyngeal tissues and genital tract, but potentially not in CSF, by the time patients are commonly diagnosed with primary HIV infection. Antiretroviral therapy is unlikely to limit initial virus spread to most tissue compartments, but may control genital tract shedding and central nervous system expansion in primary infection.

The gp120 V3-encoding region of human immunodeficiency virus type 1 (HIV-1) RNA derived from the saliva and blood plasma of 11 individuals was characterized by heteroduplex tracking assay and sequence analyses. R5-like viral variants were identified in both fluids of all subjects. X4-like variants were detected in the plasma and/or saliva of three subjects, indicating that X4-like variants are not excluded from the saliva compartment. Viral subpopulations were similar in both fluids of most subjects, suggesting that HIV-1 in oral fluids and blood may stem from a common source. These findings raise the possibility of using saliva as a noninvasive fluid for evaluating and monitoring viral evolution in infected persons.

We compared Roche MONITOR and Organon Teknika NucliSens assays for human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF). Results of 282 assays were highly correlated (r = 0.826), with MONITOR values being 0.29 +/- 0.4 log(10) copies/ml (mean +/- standard deviation) values. Both assays can reliably quantify HIV-1 RNA in CSF.

Objective: To develop a model to predict transmission of HIV-1 from men to women., Design: HIV-1 in seminal plasma, and endocervical CCR5 receptors were correlated with epidemiological studies of HIV-1 transmission to develop a probabilistic model., Settings: Semen samples were collected from patient subjects in Seattle Washington, Chapel Hill, North Carolina, and St. Gallen, Switzerland. Endocervical biopsy specimens were obtained from women in Chicago, Illinois., Participants: Eighty-six men (not receiving antiretroviral therapy) in whom CD4 cell count and semen volume were available, and 24 women in whom the number of endocervical CCR5 receptors were determined., Main Outcome Measures: Prediction of transmission of HIV-1 from men to women per episode of vaginal intercourse based on the absolute burden of HIV (volume x HIV RNA copies/ml seminal plasma)., Results: The model suggests efficient heterosexual transmission of HIV-1 when semen viral burden is high. When semen contains 100 000 copies of non-syncytium-inducing (NSI) HIV RNA the probability of HIV-1 transmission is 1 per 100 episodes of intercourse; conversely, with 1000 copies NSI HIV RNA in semen, transmission probability is 3 per 10 000 episodes of intercourse., Conclusions: This model links biological and epidemiological data related to heterosexual HIV-1 transmission. The model can be used to estimate transmission of HIV from men with high semen viral burden from inflammation, or reduced burden after antiretroviral therapy. The results offer a biological explanation for the magnitude of the HIV epidemic in places where earlier studies have shown men have high semen viral burden, such as in sub-Saharan Africa. The model can be used to develop and test HIV-1 prevention strategies.

This study compared antiretroviral activity among 6 "salvage" therapy regimens. The study was a prospective, randomized, 2x3 factorial, multicenter study of the AIDS Clinical Trials Group. The study enrolled 277 human immunodeficiency virus (HIV)-infected patients naive to nonnucleoside analogues who had taken indinavir >6 months. The patients had 2000-200,000 HIV RNA copies/mL. Patients received saquinavir with ritonavir or nelfinavir together with delavirdine and/or adefovir and were followed for safety and antiretroviral response between baseline and week 16. At week 16, 30% (77/254) of patients had

We have examined cell-free viral populations in the blood plasma and seminal plasma compartments of men infected with subtype C human immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD) clinic for genital tract inflammation in the form of urethritis (n = 43) or a dermatology clinic (controls, n = 14) in Malawi. We have previously shown that the presence of urethritis is associated with an eightfold increase in virus load in the seminal plasma compartment (M. S. Cohen et al., Lancet 349:1868-1873, 1997). The purpose of this study was to determine whether genital tract inflammation and its treatment caused genetic instability in cell-free HIV-1 populations. In a cross-sectional analysis at study entry, three-fourths of the STD and control subjects had multiple V3 populations in their blood while 60% of the STD subjects and 79% of the control subjects had multiple V3 populations in their semen. Overall, one-fourth of all of the subjects showed discordance between results with blood and semen specimens when samples were compared for the presence and absence of subpopulations. When differences in the relative levels of abundance of bands were also taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between STD and control cohorts for these comparisons of the compartments in this cross-sectional analysis at study entry. Longitudinal analysis of the viral populations from two separate clinic visits over 1 to 4 weeks showed that the complexity of each V3 population as measured by Shannon entropy was different in blood and semen at the two time points, indicating that the blood and semen constitute different compartments for HIV-1. The seminal plasma compartment was more dynamic than the blood plasma compartment for the STD subjects who were treated for urethritis, with changes being noted in the presence or absence of V3-HTA bands in the semen of 29% of these subjects but in the blood of only 9% of these subjects. However, the changes were generally small. Overall, our results suggest that 40% of male subjects show discordance between seminal and blood viral populations and that the complexity of each V3 population was different between the two compartments. Both of these results point to the partial independence of the seminal compartment as a viral niche within the body.

We have evaluated two commercially available kits (AMPLICOR MONITOR [Roche] and NASBA HIV-1 QT or NucliSens HIV-1 QT [Organon Teknika]) and two noncommercial methods for the accurate quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in seminal plasma. The same panels of coded specimens were tested on four separate occasions. Laboratories using the commercial assays employed silica beads to isolate HIV-1 RNA, which removed inhibitory factors sometimes found in seminal plasma. Sensitivities and specificities, respectively, for each assay were as follows: AMPLICOR MONITOR, 100 and 73%; NASBA HIV-1 QT, 84 and 100%; NucliSens HIV-1 QT, 99 and 98%; and noncommercial assays, 91 and 73%. When results from the laboratory that was inexperienced with the silica bead extraction method were excluded from the analysis, specificity for the Roche assay increased to 100%. The commercial assays demonstrated highly reproducible results, with intra-assay standard deviations (measured in log(10) RNA copies/milliliter of seminal plasma) ranging from 0.11 to 0.32; those of the noncommercial assays ranged from 0.12 to 0.75. Differences in mean estimated HIV-1 RNA concentrations were

Antiretroviral therapy may lead to decreased shedding of human immunodeficiency virus type 1 (HIV-1) in genital secretions. Thirty men, 19 receiving amprenavir and 11 receiving amprenavir, zidovudine, and lamivudine, donated blood and semen while undergoing treatment, to evaluate the effects of these medications on HIV-1 shedding in semen. Before therapy, 4 men had HIV-1 RNA levels in seminal plasma >6.0 log10 (1 million) copies/mL, markedly higher than levels in blood plasma. Most men (77%) had HIV-1 RNA levels in seminal plasma below the limit of quantification during therapy. Amprenavir alone suppressed HIV-1 RNA levels to <400 copies/mL in seminal plasma in the majority of patients, the first direct demonstration of the antiretroviral effects of a protease inhibitor in the male genital tract. However, 8 men (27%) had measurable HIV-1 in seminal plasma at their last study visit, 4 with increasing levels. Persistent replication of HIV in the genital tract may have implications for the selection of resistant virus and sexual transmission of HIV-1.

Little information is available describing viral loads in body fluids other than blood. In addition, the suitability of commercially available assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation has not been evaluated in most nonblood fluids. We compared Organon Teknika's nucleic acid sequence-based amplification method (NASBA) and Roche's Amplicor HIV-1 Monitor (reverse transcriptase PCR [RT-PCR]) for quantitating HIV-1 RNA in cerebrospinal fluid (CSF), saliva, breast milk, seminal plasma, and cervical-vaginal lavage fluid (CVL). Saliva and breast milk frequently demonstrated some inhibition in the RT-PCR assay, similar to the inhibition previously described in seminal plasma. Inhibition of the RT-PCR assay was not observed with CSF or CVL, nor in any of the NASBA assays. When fluids from HIV-infected individuals were tested by RT-PCR and NASBA, 73 and 27% of CSF samples and 60 and 40% of breast milk specimens had detectable RNA, respectively. These differences were not statistically significant. In cross-sectional studies using RT-PCR to measure viral RNA in paired blood plasma and CSF samples, 71% of blood plasma samples and 42% of CSF samples were positive. A similar analysis using NASBA with paired blood plasma and CVL, saliva, or seminal plasma samples revealed 91% were blood plasma positive and 55% were CVL positive, 76% were blood plasma positive and 46% were saliva positive, and 83% were blood plasma positive and 63% were seminal plasma positive. NASBA worked fairly well to quantitate HIV-1 RNA from all fluids without apparent inhibition. RT-PCR performed well on CVL and CSF, frequently with greater sensitivity, although its use in other fluids appears limited due to the presence of inhibitors. These studies demonstrate that viral loads in nonblood fluids were generally lower than in blood.

Objective: The purpose of this investigation was to quantify human immunodeficiency virus type-1 (HIV-1) RNA in saliva and plasma and identify factors associated with increased salivary viral load., Study Design: Forty HIV-1-seropositive adults underwent oral examinations to assess mucosal and periodontal health. Whole saliva was evaluated for HIV-1 RNA titer and occult blood. Plasma viral load, CD4 cell count, HIV-1 staging, and antiretroviral therapy data were obtained from medical records. Associations between salivary titers and oral/systemic parameters were analyzed by means of t tests, Wilcoxon signed rank tests, Pearson's correlation coefficient, and analysis of covariance., Results: Forty-two percent of the subjects had detectable salivary HIV-1 RNA. Oral titers were highly correlated with plasma viral levels (r = 0.51, P <.01). HIV-associated periodontal disease (in particular, linear gingival erythema), severe gingival inflammation, and absence of antiretroviral therapy were associated with high salivary titers (P <.01)., Conclusions: Substantial quantities of HIV-1 can be shed in the oral cavity, particularly when inflammatory conditions are present. Salivary titer may be a useful indicator of systemic viral burden.

Paired blood samples collected in acid-citrate-dextrose and EDTA were compared for human immunodeficiency virus (HIV) infectivity on the day of collection or after 1 day of storage at room temperature. No significant differences between the anticoagulants were observed. Culture positivity was significantly associated with HIV RNA viral loads for both anticoagulants.

Human immunodeficiency virus (HIV) can be transmitted in semen from a man to his sexual partners. Antiretroviral drugs are likely to affect the amount of HIV-1 in semen and perhaps transmission of the virus. The concentrations of zidovudine, lamivudine, and HIV-1 RNA in blood and seminal plasma were measured in 9 HIV-positive men over

We have examined the nature of V3 sequence variability among subtype C human immunodeficiency virus type 1 (HIV-1) sequences from plasma-derived viral RNA present in infected men from Malawi. Sequence variability was assessed by direct sequence analysis of the V3 reverse transcription-PCR products, examination of virus populations by a subtype C V3-specific heteroduplex tracking assay (V3-HTA), and selected sequence analysis of molecular clones derived from the PCR products. Sequence variability in V3 among the subtype C viruses was not associated with the presence of basic amino acid substitutions. This observation is in contrast to that for subtype B HIV-1, where sequence variability is associated with such substitutions, and these substitutions are determinants of altered coreceptor usage. Evolutionary variants in subtype C V3 sequences, as defined by the V3-HTA, were not correlated with the CD4 level in the infected person, while such a correlation was found with subtype B V3 sequences. Viruses were isolated from a subset of the subjects; all isolates used CCR5 and not CXCR4 as a coreceptor, and none was able to grow in MT-2 cells, a hallmark of the syncytium-inducing phenotype that is correlated with CXCR4 usage. The overall sequence variability of the subtype C V3 region was no greater than that of the conserved regions of gp120. This limited sequence variability was also a feature of subtype B V3 sequences that do not carry the basic amino acid substitutions associated with altered coreceptor usage. Our results indicate that altered coreceptor usage is rare in subtype C HIV-1 isolates in sub-Saharan Africa and that sequence variability is not a feature of the V3 region of env in the absence of altered coreceptor usage.

Since 1993, trends in perinatal human immunodeficiency virus (HIV) transmission have been monitored by use of chart review of patients identified at a central diagnostic laboratory. In the population studied, either pre- or postnatal antiretroviral therapy to the infant increased from 21% in 1993 to 95% in 1997. Concurrently, the number of HIV-infected infants declined from 25 in 1993 to 4 in 1997. The complete Pediatric AIDS Clinical Trials Group Protocol 076 regimen was the most effective in reducing transmission (3.1%). Twenty-two of 35 infants who became infected in 1995-1997 had mothers who did not receive antiretroviral therapy, although counseling practices improved with time. In 1995, 87% of the mothers of HIV-seropositive infants were counseled, whereas in 1997, 96% were counseled (P<.005). None of 59 infants tested had high-level phenotypic zidovudine resistance, although 5 (8.8%) of 57 infants had virus isolates with at least one mutation in the reverse transcriptase gene associated with reduced phenotypic susceptibility to zidovudine.

Before December 1997, in Espírito Santo, Brazil, combination antiretroviral therapy was used without routine virologic or immunologic monitoring. To examine consequences of therapy in this setting, clinical information, human immunodeficiency virus type 1 (HIV-1) RNA levels, CD4 cell counts, and protease and reverse transcriptase sequences were determined for consecutive HIV-1-infected outpatients. Of 48 treatment-naive individuals, 11 were started on therapy for HIV-related symptoms; however, 44 (92%) had an RNA level >20,000 copies/mL, a CD4 cell count <500/mm3, or symptoms. Eighteen (51%) of 35 patients on therapy had an RNA level >20,000 copies/mL. Nucleoside-resistance mutations were observed in 21 (68%) of 31 nucleoside-experienced subjects. Protease mutations necessary for high-level protease inhibitor (PI) resistance were present together with permissive mutations in 3 of 10 PI-experienced patients. Inability to identify high-risk individuals and to detect virologic failure may limit the effectiveness of antiretroviral drug programs and may promote the spread of drug resistance where virologic and immunologic monitoring are not available.

Objectives: This study was undertaken to determine the relative effect of malaria infection on HIV concentration in blood plasma, and prospectively to monitor viral concentrations after antimalarial therapy., Design: A prospective, double cohort study was designed to compare the blood HIV-1 RNA concentrations of HIV-positive individuals with and without acute malaria illness. Subjects were followed for 4 weeks after successful malaria therapy, or for 4 weeks from enrollment (controls)., Methods: Malawian adults with symptomatic Plasmodium falciparum parasitemia (malaria group) and asymptomatic, aparasitemic blood donors (control group) were tested for HIV-1 antibodies to identify appropriate study groups. The malaria group received antimalarial chemotherapy only and were followed with sequential blood films. In both groups, blood plasma HIV-1 RNA viral concentrations were determined at enrollment and again at 1, 2 and 4 weeks., Results: Forty-seven malaria patients and 42 blood donors were enrolled. At enrollment blood plasma HIV-1 RNA concentrations were approximately sevenfold higher in patients with malaria than in blood donors (medians 15.1 x 10(4) and 2.24 x 10(4) copies/ml, respectively, P = 0.0001). No significant changes in median HIV-1 concentrations occurred in the 21 blood donors followed to week 4 (P = 0.68). In the 27 subjects successfully treated for malaria who were followed to week 4, a reduction in plasma HIV-1 RNA was observed from a median of 19.1 x 10(4) RNA copies/ml at enrollment, to 12.0 x 10(4) copies/ml at week 4, (P = 0.02). Plasma HIV-1 concentrations remained higher in malaria patients than controls (median 12.0 x 10(4) compared with 4.17 x 10(4) copies/ml, P = 0.086)., Conclusions: HIV-1 blood viral burden is higher in patients with P. falciparum malaria than in controls and this viral burden can, in some patients, be partly reduced with antimalarial therapy.

We compared the performance of Organon Teknika's NucliSens and Roche Diagnostic Systems' Monitor quantitative human immunodeficiency type 1 RNA assays. Both had similar linearity and sensitivity over most of the dynamic range of the assays, although the Monitor assay was superior at the low range of RNA values while the NucliSens assay was more consistent at higher RNA values. NucliSens generally showed less interassay variability.

Semen is the major vehicle for the sexual transmission of HIV-1. The ability to isolate infectious HIV from the semen and to quantitate viral burden in the form of cell-free or cell-associated HIV-1 RNA in semen are important for epidemiologic and public health aspects of the epidemic. Earlier studies used viral culture to detect HIV in semen. Cell associated culturable virus recovery rates ranged from 8 to 55% (1-8). Much lower recovery rates (3-15%) were reported by these investigators for cell-free seminal plasma. In general, subjects with lower CD4 counts, higher seminal plasma viral load (>3.5-4 log(10)), and an AIDS diagnosis were more apt to have positive seminal cell HIV cultures.

Most individuals can be diagnosed as being infected with HIV-1 with an enzyme immunoassay (EIA), which detects antibodies to the virus. Repeatedly positive results must be confirmed, usually by Western blot. These assays have been improved since their introduction in 1985. When used together, the EIA and Western blot are now extremely reliable, with sensitivities of 99.5% and specificities of 99.8% for the diagnosis of HIV-1 (1). These tests are fairly rapid, reproducible across laboratories, well quality-controlled, relatively inexpensive, and form the basis of the protection of the blood supply.

Objectives: To evaluate blood and genital secretions from HIV-infected men for HIV-1 resistant to antiretroviral agents., Design: A longitudinal study of 11 men with HIV infection and persistent detectable HIV RNA levels in blood and semen on antiretroviral therapy., Methods: HIV-1 from the blood and seminal plasma, obtained before the initiation of a new therapeutic regimen and on therapy, were evaluated by population-based sequencing of reverse transcriptase (RT) and protease RNA for the development of resistance to antiretroviral therapy. The genetic relatedness of sequences over time was compared., Results: RT genotypic resistance markers were present in seminal plasma at baseline in three out of six individuals with previous RT inhibitor experience. Eight out of 10 men, from whom the viral sequence was available on new therapy, demonstrated the evolution of new resistance mutations in the blood or seminal plasma, or both. The evolution of resistance mutations in blood and semen were frequently discordant, although over time similar patterns were seen. In two individuals, protease inhibitor resistance mutations evolved in the blood but not in the major variant in seminal plasma. Comparisons of the viral sequences between blood and seminal plasma from six men revealed two patterns. Three men showed a clustering of sequences from blood and semen. Three had sequences that appeared to evolve separately in the two compartments., Conclusions: HIV-1 variants with genotypic resistance markers are present in the male genital tract and evolve over time on incompletely suppressive antiretroviral therapy. The absence of genotypic changes consistent with protease inhibitor resistance in the semen, despite their presence in blood plasma, suggests the possibility of limited penetration of these agents into the male genital tract. Sexual transmission of resistant variants may have a negative impact on treatment outcome in newly infected individuals and on the spread of the diseases within a population. Therapeutic strategies that fully suppress HIV-1 in the genital tract should be a public health priority.

High levels of human immunodeficiency virus type 1 (HIV-1) replication, as reflected in HIV-1 RNA concentrations in blood and semen, probably contribute to both rapid disease progression and enhanced sexual transmission. Semen and blood were collected from 49 Malawian and 61 US and Swiss (US/Swiss) HIV-1-seropositive men with similar CD4 cell counts and no urethritis or exposure to antiretroviral drugs. Median seminal plasma and blood plasma HIV-1 RNA concentrations were >3-fold (P = .034) and 5-fold (P = .0003) higher, respectively, in the Malawian men. Similar differences were observed in subsets of the Malawian and US/Swiss study groups matched individually for CD4 cell count (P = .035 and P < .002, respectively). These observations may help explain the high rates of HIV-1 sexual transmission and accelerated HIV-1 disease progression in sub-Saharan Africa.

The efficiency and duration of transmissibilty of HIV seems to be highly variable and dependent on a number of factors related to both the donor and the recipient as well as characteristics intrinsic to the virus itself. Some of the factors likely to be important in sexual transmission of HIV include stage of disease, antiretroviral therapy, and concomitant systemic or mucosal infections, including sexually transmitted diseases. This paper describes recent work from our group in each of these areas.

The associations of CD4 cell count, plasma human immunodeficiency virus (HIV) type 1 RNA, infectious HIV titer in peripheral blood mononuclear cells, immune complex-disrupted (ICD) p24 antigen, and MT-2 assays with measures of disease progression after drug treatment were assessed in a subset of patients enrolled in AIDS Clinical Trials Group Study 175. Baseline plasma RNA levels and changes in RNA values at weeks 8 or 56 were more important predictors of disease progression than were baseline or changes in CD4 cell counts. Each 10-fold lower HIV RNA concentration at baseline and each 10-fold decrease in HIV RNA between baseline and week 8 was associated with increases of 49-61 CD4 cells/mm3 at weeks 56 and 104. In multivariate analyses, neither baseline values nor changes in infectious HIV titer nor ICD p24 antigen concentrations were associated with long-term changes in CD4 cell count. Plasma HIV-1 RNA appears to be the best predictor of long-term CD4 cell count responses and disease progression.

Virologic measurements are increasingly used to evaluate prognosis and treatment responses in human immunodeficiency virus (HIV) type 1 infection. Markers of HIV-1 replication, including infectious HIV-1 titer from peripheral blood mononuclear cells, serum HIV-1 p24 antigen, plasma HIV-1 RNA, CD4 cell numbers, and viral syncytium-inducing (SI) phenotype, were determined in 391 virology substudy participants in AIDS Clinical Trials Group study 175. The subjects had 200-500 CD4 cells/mm3. All markers of viral replication significantly correlated with one another and were inversely related to CD4 cell number. Disease progression to an AIDS-defining event or death or loss of >50% of CD4 cells was associated with infectious HIV-1 titer (P < .001), HIV-1 RNA (P < .001), and HIV-1 p24 antigen (P = .007). In multivariate proportional hazards models, p24 antigen was never significant when HIV-1 RNA level was included. In a model containing infectious HIV-1 titer (P = .038), HIV-1 RNA (P < .001), SI phenotype (P < .001), and CD4 cell number (P = .18), only the virologic parameters remained significantly associated with progression.