Determinants of HIV-1 shedding in the genital tract of women.

BACKGROUND: Plasma HIV-1 RNA concentration has been the best predictor for risk of heterosexual and perinatal transmission. However, direct contact with HIV-1 present locally in the genital tract might be necessary for transmission. We aimed to assess the relation between HIV-1 shedding (RNA or culturable virus) in female genital secretions and other factors that might affect HIV-1 shedding. METHODS: This was a cross-sectional study within the Women's Interagency HIV Study (WIHS), a prospective longitudinal cohort study of HIV-infected women. We enrolled 311 HIV positive women from Jan 30, 1997 to July 1, 1998. We did clinical assessments, cultured HIV-1, and measured RNA in peripheral blood mononuclear cells (PBMC) and genital secretions. We compared the results with univariate and multivariate analyses. Presence of HIV-1 RNA or culturable virus in genital secretions was defined as HIV-1 shedding. FINDINGS: HIV-1 RNA was present in genital secretions of 57% (152/268) of women whereas infectious virus was detected only in 6% (17/271). Genital tract HIV-1 shedding was found in 80% (130/163) of women with detectable plasma RNA and 78% (116/148) of women with positive PBMC cultures. 33% (27/83) of women with less than 500 copies/mL plasma RNA and 39% (35/90) of those with negative PBMC cultures also had genital tract shedding. INTERPRETATION: Plasma RNA concentration, both qualitatively and quantitatively, was the most important factor in predicting genital HIV-1 shedding, even among women receiving potent antiretroviral therapy. However, HIV-1 shedding did occur in women with less than 500 copies/mL plasma HIV-1 RNA. This finding suggests that a separate reservoir of HIV-1 replication may exist in some women.

Variation of human immunodeficiency virus type 1 viral RNA levels in the female genital tract: implications for applying measurements to individual women.

The short-term detection and variability of human immunodeficiency virus type 1 (HIV-1) RNA level was assessed in the blood plasma and genital tracts of 55 HIV-1-infected women. Specimens were collected weekly for 8 weeks from the endocervical canal with wicks and cytobrushes and from the ectocervix and vagina with cervicovaginal lavage. In all, 48 women (87.3%) had detectable genital tract HIV-1 RNA at > or =1 collection times. HIV-1 RNA levels varied least in specimens from endocervical canal wick and most in cervicovaginal lavage samples. The within-subject variation for genital-tract virus level was greater than that for blood. Overall, the odds for viral RNA detection in the genital tract approximately tripled for each 10-fold increase in plasma viral RNA concentration (P<.001) or with concomitant genital tract infection (P=.003). Endocervical canal wicks should be considered as an adjunct to cervicovaginal lavage, to improve the sensitivity and precision of HIV-1 RNA detection.

A menstrual cycle pattern for cytokine levels exists in HIV-positive women: implication for HIV vaginal and plasma shedding.

OBJECTIVES: To evaluate the effect of the menstrual cycle in HIV-positive women on plasma and genital cytokine levels, interrelationships between vaginal and plasma cytokines, CD4 and CD8 T cell fluctuations, and genital and plasma viral loads. METHODS: Plasma and cervicovaginal lavage specimens were collected from 55 HIV-positive women with CD4 cell counts < 350 cells/microl during phases of the menstrual cycle. Samples were assayed for IL-1beta, IL-6, IL-4, IL-8, IL-10, TGFbeta, TNFalpha, INFgamma, MIP1alpha, MIP1beta, RANTES, and TNFR-II using enzyme-linked immunosorbent assays. CD4 and CD8 T cell expression was evaluated by flow cytometry. Repeated measures regression models were used to assess the effect of the menstrual cycle on cytokines and viral load. Multivariate repeated regression models were used to assess the correlation among selected cytokines and between selected cytokines and HIV viral load. RESULTS: Vaginal IL-1beta, IL-4, IL-6, IL-8, IL-10, MIP1beta, RANTES, TGFbeta, and TNFR-II were significantly elevated during menses but were not altered during other phases. Plasma cytokine levels were not altered during the menstrual cycle. A positive Candida culture increased vaginal IL-8 during menses, whereas vaginal discharge was associated with a reduction in vaginal IL-4, IL-10, and RANTES. CD4 and CD8 cell numbers did not vary with the menstrual cycle. Vaginal cytokine levels correlated only with vaginal viral load, in a sampling method-dependent manner. CONCLUSION: We provide evidence of elevated vaginal cytokine levels during menses, which appear to regulate vaginal and not plasma HIV shedding, suggesting that a menstrual cycle pattern exists for cytokine production in HIV-positive women impacting vaginal shedding of HIV.

Herpes simplex virus shedding and plasma human immunodeficiency virus RNA levels in coinfected women.

Asymptomatic herpes simplex virus (HSV) shedding was described in a cohort of human immunodeficiency virus (HIV)-infected women, and the association of HSV shedding with changes in plasma HIV RNA load was investigated. Genital, rectal, and oral swabs were obtained daily during a 4-week period for polymerase chain reaction and culture, and concomitant plasma specimens were drawn 3 times weekly for determination of HIV RNA load. During the study, 70% and 79% of subjects shed HSV from the oral cavity and genital area, respectively. Shedding of HSV occurred for a mean of 3.2 days for oral shedding and 5.4 days for genital shedding. Mean plasma HIV RNA loads during periods of HSV shedding and nonshedding and for periods 3 days after the cessation of shedding were compared; no significant differences were found (P=.74). In women who shed HSV, as evaluated by detection of virus, plasma HIV RNA load did not fluctuate with HSV shedding.

Hormonal levels among HIV-1-seropositive women compared with high-risk HIV-seronegative women during the menstrual cycle. Women's Health Study (WHS) 001 and WHS 001a Study Team.

There is a paucity of normative data on hormonal levels among HIV-infected women. Hormonal levels may influence fertility and HIV-related immunological and virological factors. The objective of this study was to determine progesterone and estradiol levels during the menstrual cycle in HIV-seropositive women compared with high-risk seronegative women. The study enrolled 55 HIV-infected and 10 high-risk uninfected women with self-reported regular menstrual cycles (25-30-day cycles). Progesterone and estradiol levels were determined on a weekly basis for 8 weeks. The analysis included evaluations from the first complete menstrual cycle for the 54 HIV-infected and 9 uninfected women who had at least one complete cycle. The median age was 35 years for HIV-infected women and 36 years for uninfected women. The median CD4+ count for HIV-seropositive women was 210 cells/mm3. The median menstrual cycle length was 28 days (range 22-49 days) for HIV-infected women and 25 days (range 24-44 days) for uninfected women. The maximum progesterone level during the luteal phase was normal (>3.0 ng/ml) for 52 (96%) of 54 HIV-seropositive women and 7 (78%) of 9 HIV-seronegative women (p = 0.09, Fisher's exact test). The median maximum progesterone level was 12.2 ng/ml in HIV-seropositive women and 7.2 ng/ml in HIV-seronegative women (p = 0.07, Wilcoxon test). The median maximum estradiol value during the follicular phase was 148 pg/ml for HIV-seropositive women and 111 pg/ml for HIV-seronegative women (p = 0.04, Wilcoxon test). Among HIV-infected women, there were no significant differences in progesterone and estradiol levels by antiretroviral therapy, baseline plasma viral load, or median CD4+ cell count. We conclude that HIV-infected women with self-reported normal menstrual cycles have normal levels of progesterone and estradiol during the menstrual cycle.

Effect of menstrual cycle on HIV-1 levels in the peripheral blood and genital tract. WHS 001 Study Team.

OBJECTIVE: To assess the variation in HIV-1 over the menstrual cycle, including RNA levels in the female genital tract, plasma HIV-1-RNA levels, CD4 cell counts, and culturable virus. DESIGN: A prospective analysis of 55 HIV-1-infected women. METHODS: Blood and genital tract specimens were collected weekly over 8 weeks, spanning two complete menstrual cycles. Applying repeated-measures models that used menses as the reference level, the variation in viral RNA levels was compared in endocervical canal fluid and cells (collected by Sno-strips and cytobrush, respectively) and ectocervicovaginal lavage (CVL) fluid. Repeated-measures models were also used to assess the variation in plasma CD4 cell counts and viral load. RESULTS: Shedding patterns differed among the three sampling methods, independent of genital tract co-infections. Genital tract HIV-1-RNA levels from CVL fluid and endocervical canal cytobrush specimens were highest during menses and lowest immediately thereafter (P = 0.001 and P = 0.04). The HIV-1-RNA level in endocervical canal fluid was highest in the week preceding menses (P = 0.003). The menstrual cycle had no effect on blood levels of RNA (P = 0.62), culturable virus (P = 0.34), or CD4 cell counts (P = 0.55). HIV-1-RNA levels were higher in endocervical canal fluid than in peripheral blood plasma during the late luteal phase (P = 0.03). CONCLUSION: HIV-1-RNA levels vary with the menstrual cycle in the female genital tract but not the blood compartment. HIV-1-RNA levels are higher in endocervical canal fluid than in blood plasma. These findings may have important implications for sex-specific pathogenesis, heterosexual transmission, and contraceptive hormone interventions in HIV-1-infected women.

Detection and quantitation of human immunodeficiency virus type 1 in the female genital tract. The Division of AIDS Treatment Research Initiative 009 Study Group.

Human immunodeficiency virus type 1 (HIV-1) was detected in the genital tracts of 59% of 225 women by RNA PCR and in 7% of the women by culture. In a comparison of two sampling methods, endocervical swabs were more sensitive than cervicovaginal lavage for HIV-1 RNA detection by PCR but not by culture and their sensitivity was independent of the concentration of HIV-1 RNA.

Co-receptor usage was more predictive than NSI/SI phenotype for HIV replication in macrophages: is NSI/SI phenotyping sufficient?

A monocyte-derived macrophage (MDM) culture assay was used to define the replication kinetics of HIV isolates. Ten-day-old MDMs were infected with HIV. Supernatants were collected and assayed for HIV p24 on days 3, 7, 10, and 14 post-infection (PI). In this assay, SF162 (macrophage tropic, NSI) produced increasing amounts of HIV p24 antigen with increasing time in culture. BRU (nonmacrophage tropic, SI) infection resulted in low levels of HIV p24 antigen with no increase in production during the culture period. A panel of 12 clinical isolates was evaluated. All isolates produced detectable levels of HIV p24 antigen in MDMs. However, the NSI viruses had significantly higher log10 HIV p24 antigen values at all times PI (P < 0.01). Co-receptor usage was determined for all 12 isolates (8 NSI and 4 SI). All SI isolates used CXCR4 for entry; two used CXCR4 only, one used CXCR4, CCR5, and CCR3, and one was a mixture of two isolates using CXCR4 and CCR5. None of the NSI viruses used CXCR4 for entry. All used CCR5 as their predominant co-receptor. Of the eight NSI isolates, three used CCR5 only, two used CCR5 and CCR2b, one used CCR5 and CCR3, and one used CCR5, CCR3, and CCR2b. Log10 HIV p24 antigen production on day 14 PI for viruses that used CCR5+CCR3 (3.79 + 1.40) was greater than for viruses that used CCR5+CCR2b (3.22 + 1.55) or CCR5 (3.32 + 1.49), and all were greater than those that used CXCR4 only (1.69 + 0.28), regardless of SI phenotype (P < 0.05). Thus, in these primary isolates, macrophage tropism and replication kinetics were closely linked to CCR5 utilization, whereas SI capacity was closely linked to CXCR4 utilization. Furthermore, viruses, which could use CCR5 and CCR3 for entry, had a replication advantage in macrophages, regardless of SI phenotype.

The impact of the ovulatory cycle on cytokine production: evaluation of systemic, cervicovaginal, and salivary compartments.

To understand the impact of the menstrual cycle on immunologic parameters, we measured the level of cytokines and chemokines from plasma, cervicovaginal lavage (CVL), and saliva samples of 6 premenopausal women during the follicular and luteal phases of the ovulatory cycle. We demonstrate that the level of plasma interleukin-8 (IL-8) was 4-fold higher during the follicular phase than the luteal phase (p = 0.004), whereas plasma IL-1beta, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and TNF receptor II (TNFR II) were not altered during the ovulatory cycle. In the vaginal compartment, as measured from CVL samples, the levels of IL-6 and IL-1beta were both 5-fold higher in the follicular than the luteal phase (p = 0.0002 and 0.03, respectively). Salivary cytokine and chemokine samples were similar when measured during the luteal and the follicular phases. Additional analysis of lymphocyte subsets for phenotypic and functional markers indicated that they were not influenced by the ovulatory cycle. Collectively, these data suggest that IL-6, IL-8, and IL-1beta are differentially regulated during the ovulatory cycle.

Comparison of two amplification technologies for detection and quantitation of human immunodeficiency virus type 1 RNA in the female genital tract. Division of AIDS Treatment Research Initiative 009 Study Team.

Human immunodeficiency virus type 1 (HIV-1) RNA levels in female genital tract and peripheral blood samples were compared using two commercial amplification technologies: the Roche AMPLICOR HIV-1 MONITOR test and either the Organon Teknika nucleic acid sequence-based amplification (NASBA-QT) assay or the NucliSens assay. Estimates of HIV-1 RNA copy number were derived from internal kit standards and analyzed unadjusted and adjusted to a common set of external standards. We found a discordance rate of approximately 18% between the two technologies for the detection of HIV-1 in either the genital tract or peripheral blood samples. Detection discordance was not consistent among specimens or among women. There were no significant differences in adjusted or unadjusted estimates of HIV-1 RNA copy number in the genital tract samples using the AMPLICOR HIV-1 MONITOR test and either the NASBA-QT assay or the NucliSens assay. In addition, the estimated HIV-1 RNA copy number in peripheral blood samples did not differ when tested with the NucliSens assay and the AMPLICOR HIV-1 MONITOR test using kit standards. However, there was a significant difference in estimated RNA copy number between the NASBA-QT assay and the AMPLICOR HIV-1 MONITOR test for internal kit standards, which, as we have previously shown, was eliminated after adjustment with the external standards. Our results suggest that the Roche and Organon Teknika assays are equivalent for quantifying HIV-1 RNA in female genital tract specimens, although variation in detection does exist.

Multicenter evaluation of methods to quantitate human immunodeficiency virus type 1 RNA in seminal plasma.

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

A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates.

Twelve laboratories collaborated in formulating and testing a standardized plaque reduction assay for cytomegalovirus (CMV) cell-associated clinical isolates. Four characterized and plaque-purified CMV strains, as well as six coded clinical isolates obtained after antiviral therapy, were distributed and tested. Good agreement was obtained for four of the clinical isolates, but a broad distribution of results was obtained for two isolates. Analysis of these results indicates the problems associated with clinical isolates, including the large genetic variability and the highly cell-associated phenotype. This collaborative effort, by addressing these problems, represents a significant step toward the development of a standardized assay.

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.

Risk factors for perinatal transmission of human immunodeficiency virus type 1 in women treated with zidovudine. Pediatric AIDS Clinical Trials Group Study 185 Team.

BACKGROUND: Maternal, obstetrical, and infant-related factors associated with the risk of perinatal transmission of human immunodeficiency virus type 1 (HIV-1) were identified before the widespread use of zidovudine therapy in pregnant women. The risk factors for transmission when women and infants receive zidovudine are not well characterized. METHODS: We examined the effects of maternal, obstetrical, and infant-related characteristics and maternal virologic and immunologic variables on the risk of perinatal transmission of HIV-1 among 480 women and their infants, all of whom received zidovudine. The women and infants were participating in a phase 3 trial of passive immunoprophylaxis for the prevention of perinatal transmission. RESULTS: In univariate analyses, the risk of perinatal transmission was associated with each of the following: decreased maternal CD4+ lymphocyte counts at base line; decreased maternal HIV p24 antibody levels at base line and delivery; increased maternal HIV-1 titer at base line and delivery; increased maternal HIV-1 RNA levels at base line and delivery; and the presence of chorioamnionitis at delivery. In multivariate analyses, the only independent risk factor was the maternal HIV-1 RNA level at base line (odds ratio for transmission, 2.4 per log increase in the number of copies; 95 percent confidence interval, 1.2 to 4.7; P=0.02) and at delivery (odds ratio, 3.4; 95 percent confidence interval, 1.7 to 6.8; P=0.001). There was no perinatal transmission of HIV-1 among the 84 women who had HIV-1 levels below the limit of detection (500 copies per milliliter) at base line or the 107 women who had undetectable levels at delivery. CONCLUSIONS: Among pregnant women and their infants, all treated with zidovudine, the maternal plasma HIV-1 RNA level was the best predictor of the risk of perinatal transmission of HIV-1. Antiretroviral therapy that reduces the HIV-1 RNA level to below 500 copies per milliliter appears to minimize the risk of perinatal transmission as well as improve the health of the women.

Worldwide evaluation of DNA sequencing approaches for identification of drug resistance mutations in the human immunodeficiency virus type 1 reverse transcriptase.

A panel (ENVA-1) of well-defined blinded samples containing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was analyzed by automated DNA sequencing in 23 laboratories worldwide. Drug resistance mutations at codons 41, 215, and 184 were present in the panel samples at different ratios to the wild type. The presence of mutant genotypes was determined qualitatively and quantitatively. All laboratories reported the presence of sequence heterogeneities at codons 41, 215, and 184 in one or more of the panel samples, though not all reported the correct codon genotypes. Two laboratories reported a mutant genotype in samples containing only the wild type, whereas two and three laboratories failed to detect the mutant genotypes at codons 41 and 215, respectively, in a completely mutant DNA population. Mutations present at relative concentrations of 25% of the total DNA population were successfully identified by 13 of 23, 10 of 23, and 16 of 23 labs for codons 41, 215, and 184Val, respectively. For more than 80% of those laboratories that qualitatively detected the presence of a mutation correctly, the estimated wild type/mutant ratio was less than 25% different from the input ratio in those samples containing 25 to 50% or 75% mutant input. This first multicenter study on the quality of DNA sequencing approaches for identifying HIV-1 drug resistance mutations revealed large interlaboratory differences in the quality of the results. The application of these procedures in their current state would in several cases lead to inaccurate or even incorrect diagnostic results. Therefore, proper quality control and standardization are urgently needed.

Efficacy of zidovudine and human immunodeficiency virus (HIV) hyperimmune immunoglobulin for reducing perinatal HIV transmission from HIV-infected women with advanced disease: results of Pediatric AIDS Clinical Trials Group protocol 185.

Pediatric AIDS Clinical Trials Group protocol 185 evaluated whether zidovudine combined with human immunodeficiency virus (HIV) hyperimmune immunoglobulin (HIVIG) infusions administered monthly during pregnancy and to the neonate at birth would significantly lower perinatal HIV transmission compared with treatment with zidovudine and intravenous immunoglobulin (IVIG) without HIV antibody. Subjects had baseline CD4 cell counts /=200/microL) but not with time of zidovudine initiation (5.6% vs. 4.8% if started before vs. during pregnancy; P=. 75). The Kaplan-Meier transmission rate for HIVIG recipients was 4. 1% (95% confidence interval, 1.5%-6.7%) and for IVIG recipients was 6.0% (2.8%-9.1%) (P=.36). The unexpectedly low transmission confirmed that zidovudine prophylaxis is highly effective, even for women with advanced HIV disease and prior zidovudine therapy, although it limited the study's ability to address whether passive immunization diminishes perinatal transmission.

Statistical issues for HIV surrogate endpoints: point/counterpoint. An NIAID workshop.

This paper summarizes the proceedings of an NIAID-sponsored workshop on statistical issues for HIV surrogate endpoints. The workshop brought together statisticians and clinicians in an attempt to shed light on some unresolved issues in the use of HIV laboratory markers (such as HIV RNA and CD4+ cell counts) in the design and analysis of clinical studies and in patient management. Utilizing a debate format, the workshop explored a series of specific questions dealing with the relationship between markers and clinical endpoints, and the choice of endpoints and methods of analysis in clinical studies. This paper provides the position statements from the two debaters on each issue. Consensus conclusions, based on the presentations and discussion, are outlined. While not providing final answers, we hope that these discussions have helped clarify a number of issues, and will stimulate further consideration of some of the highlighted problems. These issues will be critical in the proper assessment and use of future therapies for HIV disease.

Acupuncture and amitriptyline for pain due to HIV-related peripheral neuropathy: a randomized controlled trial. Terry Beirn Community Programs for Clinical Research on AIDS.

CONTEXT: Peripheral neuropathy is common in persons infected with the human immunodeficiency virus (HIV) but few data on symptomatic treatment are available. OBJECTIVE: To evaluate the efficacy of a standardized acupuncture regimen (SAR) and amitriptyline hydrochloride for the relief of pain due to HIV-related peripheral neuropathy in HIV-infected patients. DESIGN: Randomized, placebo-controlled, multicenter clinical trial. Each site enrolled patients into 1 of the following 3 options: (1) a modified double-blind 2 x 2 factorial design of SAR, amitriptyline, or the combination compared with placebo, (2) a modified double-blind design of an SAR vs control points, or (3) a double-blind design of amitriptyline vs placebo. SETTING: Terry Beirn Community Programs for Clinical Research on AIDS (HIV primary care providers) in 10 US cities. PATIENTS: Patients with HIV-associated, symptomatic, lower-extremity peripheral neuropathy. Of 250 patients enrolled, 239 were in the acupuncture comparison (125 in the factorial option and 114 in the SAR option vs control points option), and 136 patients were in the amitriptyline comparison (125 in the factorial option and 11 in amitriptyline option vs placebo option). INTERVENTIONS: Standardized acupuncture regimen vs control points, amitriptyline (75 mg/d) vs placebo, or both for 14 weeks. MAIN OUTCOME MEASURE: Changes in mean pain scores at 6 and 14 weeks, using a pain scale ranging from 0.0 (no pain) to 1.75 (extremely intense), recorded daily. RESULTS: Patients in all 4 groups showed reduction in mean pain scores at 6 and 14 weeks compared with baseline values. For both the acupuncture and amitriptyline comparisons, changes in pain score were not significantly different between the 2 groups. At 6 weeks, the estimated difference in pain reduction for patients in the SAR group compared with those in the control points group (a negative value indicates a greater reduction for the "active" treatment) was 0.01 (95% confidence interval [CI], -0.11 to 0.12; P=.88) and for patients in the amitriptyline group vs those in the placebo group was -0.07 (95% CI, -0.22 to 0.08; P=.38). At 14 weeks, the difference for those in the SAR group compared with those in the control points group was -0.08 (95% CI, -0.21 to 0.06; P=.26) and for amitriptyline compared with placebo was 0.00 (95% CI, -0.18 to 0.19; P=.99). CONCLUSIONS: In this study, neither acupuncture nor amitriptyline was more effective than placebo in relieving pain caused by HIV-related peripheral neuropathy.

Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

Cartesian coordinate analysis of viral burden and CD4+ T-cell count in human immunodeficiency virus type-1 infection.

We have used a Cartesian coordinate plot to analyze the inverse relationship between viral burden (x-axis) and peripheral blood CD4+ cell count (y-axis) to extend our understanding of the mechanisms of antiviral drugs and differences in outcome resulting from variability in virus and host responses. Each of 186 subjects studied were assigned to one of four response quadrants. Quadrants A (x-, y+) and D (x+, y-) defined the effect of a change in virus load on the inverse change in CD4+ cell count expected from the natural history of HIV infection or antiretroviral therapy. Quadrants B (x+, y+) and C (x-, y-) defined the dissociation of the inverse relationship between the relative changes in CD4+ cell count and viral load that resulted from the hypothesized effect of putative virologic or immunologic response modifiers. Of the response modifiers studied, only the syncytium-inducing phenotype resulted in a complete dissociation of this inverse relationship. The analysis provided an integrated virological and immunological approach to better understand therapeutic responses and potential dissociation between changes in viral RNA and CD4+ cell count. This type of analysis may be helpful for individualizing patient management as well as designing and analyzing studies of HIV-1 antiretroviral drugs and disease pathogenesis.