Phalguni Gupta

Prognosis in HIV-1 Infection Predicted by the Quantity of Virus in Plasma

The relation between viremia and clinical outcome in individuals infected with human immunodeficiency virus-type 1 (HIV-1) has important implications for therapeutic research and clinical care. HIV-1 RNA in plasma was quantified with a branched-DNA signal amplification assay as a measure of viral load in a cohort of 180 seropositive men studied for more than 10 years. The risk of acquired immunodeficiency syndrome (AIDS) and death in study subjects, including those with normal numbers of CD4+ T cells, was directly related to plasma viral load at study entry. Plasma viral load was a better predictor of progression to AIDS and death than was the number of CD4+ T cells.

Quantitation of HIV-1 RNA in Plasma Predicts Outcome after Seroconversion

The course of infection with human immunodeficiency virus type 1 (HIV-1) varies considerably. Although the median interval between HIV-1 infection and the development of the acquired immunodeficiency syndrome (AIDS) in adults is 10 to 11 years [1], some infected persons rapidly progress to AIDS in less than 5 years [2]. Still others remain asymptomatic without evidence of immunologic decline for more than 6 years [3]. The biological basis of this variability is unknown, but differences in viral strains, host immune responses [4], and exposure to microbial [5] or environmental cofactors probably contribute. The variable course of HIV-1 infection causes uncertainty for the infected person and complicates the design and interpretation of therapeutic trials because of unrecognized differences in prognosis. Many clinical and laboratory markers have been used to estimate prognosis in patients with HIV-1 infection [6]. Markers of AIDS development include HIV-related symptoms [7, 8], depletion of CD4+ T cells [9], cutaneous anergy [7, 10], elevated serum 2-microglobulin and neopterin levels [9], HIV-1 p24 (core) antigenemia [11, 12], and syncytium-inducing HIV-1 phenotype [13]. None of these markers is ideal; all have limitations in sensitivity, specificity, or predictive power. The single best predictor of AIDS onset identified thus far is the percentage or absolute number of circulating CD4+ T cells [9], but less variable and earlier markers of risk for AIDS are needed. Several new methods have been developed to directly measure HIV-1 nucleic acid in body fluids. One of these technologies is the branched-DNA (bDNA) signal amplification method for quantitating HIV-1 RNA in plasma [14]. Although less sensitive than RNA detection by the polymerase chain reaction (PCR), the bDNA method has the advantage of large sample capacity, speed, reproducibility, and a format similar to an enzyme-linked immunosorbent assay. The ability of the bDNA assay or other HIV-1 RNA detection methods to predict clinical outcome in HIV-1 infection has not been clearly defined in appropriate cohorts or been compared with the ability of other predictive markers. Previous studies have shown a strong correlation between disease stage and the amount of circulating HIV-1, whether measured as cell-free infectious virus [15, 16], viral proteins [11, 12], or RNA [17, 18]. Recent studies have shown that an increase in HIV-1 expression in peripheral blood mononuclear cells can precede immunologic deterioration by 1 to 2 years [17, 18]. Our objective, therefore, was to compare plasma HIV-1 RNA with determinations of serum p24 antigen, neopterin, and 2-microglobulin levels and CD4+ T-cell counts as predictors of outcome in a cohort of homosexual men with documented HIV-1 seroconversion. Methods Study Populations The initial pilot study population consisted of 10 seroprevalent men (unknown date of seroconversion) enrolled in the Pittsburgh portion of the Multicenter AIDS Cohort Study (MACS). Five of these men developed AIDS (Centers for Disease Control and Prevention [CDC] 1987 definition) after 35 to 74 months of follow-up (median, 59 months), and five remained asymptomatic with stable CD4+ T-cell counts after a similar follow-up interval (median, 56 months). The second study population consisted of 62 homosexual men enrolled in the MACS who had documented seroconversion (change from negativity for HIV-1 antibody to positivity). Eighteen of these men progressed to AIDS (CDC 1987 definition) by a median of 3.8 years after seroconversion (maximum, 6.5 years), and 44 did not develop AIDS after a median follow-up of 5.4 years (maximum, 8.3 years). Details about the recruitment and characteristics of the MACS cohort have been described previously [19]. All participants gave written informed consent, and the MACS protocol was approved by the Internal Review Board of the University of Pittsburgh. Study Samples The study samples were selected from stored ( 70C) longitudinal plasma and serum samples obtained from enrollees at 6-month intervals as part of the MACS protocol. In patients who developed AIDS, the samples tested were obtained from the seroconversion visit (first visit at which the patient was positive for the HIV-1 antibody), the most recent visit before AIDS diagnosis, and equally spaced visits in between. In patients without AIDS, the samples tested were obtained from the seroconversion visit; visits 1, 2, and 3 years after seroconversion; and the last available visit, which occurred as long as 8.3 years after seroconversion. Definition of Outcomes Study patients were classified into one of three outcome groups: 1) AIDS; 2) decline in the CD4 count; and 3) stable CD4 count. Patients in the AIDS outcome group (n = 18) met the CDC 1987 case definition for AIDS. For each patient who had seroconversion and did not develop AIDS, we used linear regression to fit a line through prospective CD4+ T-cell measurements (minimum of three measurements per patient). We calculated the slope of each line and determined the statistical significance of the negative slopes. Patients with declining CD4 counts (n = 21) had statistically significant (P < 0.05) negative slopes but did not develop AIDS during follow-up. Patients with stable CD4 counts (n = 23) had no significant decline in the CD4+ T-cell count during follow-up, and 6 of 23 patients (26.1%) had a positive slope, that is, an increasing linear trend in the number of CD4+ T cells. Measurement of T-Lymphocyte Subsets We measured T-lymphocyte subsets in whole blood by staining them with fluorescent dye-conjugated monoclonal antibodies specific for CD3, CD4, and CD8 (Becton Dickinson, Mountain View, California) as previously described [20]. The total number of CD4+ T cells was determined by multiplying the percentage of lymphocytes that were CD4+ T cells by the total lymphocyte count. Serum 2-Microglobulin and Neopterin Assays We measured serum 2-microglobulin (Kabi Pharmacia, Uppsala, Sweden) and serum neopterin levels (Henning, Berlin, Germany) with commercial radioimmunoassays and standards provided by the manufacturers. Four replicates of normal control serum were included in each assay to assess variability. The coefficient of variation for control samples was 15% or less. Serum Immune Complex Dissociated p24 Assay Immune complex dissociated (ICD) p24 antigen levels were measured with a commercial enzyme immunoassay (Dupont, NEN Products, Wilmington, Delaware). The ICD p24 antigen levels in serum were interpolated from a standard curve provided by the manufacturer. The assay has a sensitivity of 12 pg of p24 antigen/mL. The interassay coefficient of variation for the p24 standards was less than 10%. Plasma and Cellular HIV-1 RNA Assays Levels of HIV-1 RNA in plasma samples were quantitated with the Quantiplex HIV-1 RNA assay, which is based on bDNA signal amplification technology (Chiron Corp., Emeryville, California). This assay measures HIV-1 RNA associated with viral particles that are pelleted from 1.0-mL plasma samples (23 500 g for 1 hour at 4 C). The assay has a quantitation limit of 1 104 HIV-1 genome equivalents per mL of plasma (Eq/mL) and is linear at levels as high as 1.6 106 Eq/mL. For this study, the interassay coefficient of variation for the positive control samples run with each assay was 11.2%. Additional details about the assay procedure and its performance characteristics have been described previously [14]. We categorized longitudinal plasma HIV-1 RNA results from individual patients into one of four groups: 1) detection of HIV-1 RNA (>1 104 Eq/mL) in all samples tested [n = 9]; 2) detection in most ( 50%) samples [n = 24; mean percentage of positive samples, 67.3%]; 3) detection in fewer than 50% of samples [n = 16; mean percentage of positive samples, 29.3%]; and 4) detection in none of the samples tested (n = 13). We identified an additional subgroup (n = 6) that showed evidence for clearance of detectable HIV-1 RNA from plasma, that is, two or more consecutive negative samples and no further positive samples after one or two initial positive samples. Assays for neopterin, 2-microglobulin, ICD p24, and HIV-1 RNA were done in duplicate on coded serum or plasma samples. Samples from a given patient were batch-tested to minimize the potential effect of interassay variability. Semi-quantitative PCR-based assays for cellular HIV-1 gag RNA were done on stored peripheral blood mononuclear cell samples as described previously [17]. Statistical Analyses The pilot study data are shown in the tables and figures to familiarize the reader with the raw data obtained from the bDNA assay. All cellular PCR results were adjusted per million CD4+ T cells. Analyses of the data set from patients with HIV-1 seroconversion were similarly stratified by outcome group. The Fisher exact test, chi-square test, and Wilcoxon rank-sum test were done where noted in the text. We estimated the association between progression to AIDS and laboratory covariates at seroconversion by multiple logistic regression analysis using BMDP statistical software (BMDP Statistical Software, Inc., Los Angeles, California). Results HIV-1 Quantitation by Branched DNA and Polymerase Chain Reaction in Seroprevalent Patients An initial pilot study of plasma HIV-1 RNA quantitation was done in 10 seroprevalent men enrolled in the MACS. Five of the men developed AIDS after 35 to 64 months of follow-up (progressors), and 5 remained asymptomatic with stable CD4+ T-cell counts (nonprogressors) after 38 to 74 months of follow-up. The median duration of follow-up for progressors and nonprogressors was similar (59 and 56 months, respectively). The bDNA assay was done on stored longitudinal plasma samples from 4 to 6 time points for each patient. Figure 1 shows the plasma HIV-1 RNA levels in the nonprogressors and progressors. Levels of HIV-1 RNA in all five nonprogressors were less than the limit of quantitation (<1 104 Eq/mL) at each time point dur

Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma

Objectives:Kaposis sarcoma-associated herpesvirus (KSHV), a newly discovered human gammaherpesvirus, is found in the majority of KS lesions from patients with and without AIDS. Peripheral blood mononuclear cells (PBMC) were examined for KSHV DNA to determine whether viral infection precedes onset of this neoplasm. Design:Randomized and blinded case–control study of prospectively collected PBMC samples from ongoing cohort studies. Methods:Paired PBMC drawn before and after KS onset from 21 AIDS-KS patients were compared to paired PBMC from 23 high-risk HIV-infected homo-/bisexual patients who did not develop KS and to a single PBMC sample from 19 low-risk, HIV-infected hemophiliac patients. Extracted DNA samples were amplified by polymerase chain reaction (PCR) using two non-overlapping nested primer sets to control for potential PCR contamination. Results:In all comparisons, patients who went on to develop KS were significantly more likely to show evidence of KSHV infection prior to onset of KS than either control group. Of PBMC samples from AIDS-KS patients drawn prior to KS, 52% were positive for KSHV DNA whereas both high- and low-risk control groups had lower rates of PBMC infection (9–13%). KSHV infection can precede KS onset by up to 21 months among AIDS-KS patients. Conclusions:AIDS-KS patients are significantly more likely to show evidence of KSHV infection in PBMC prior to KS onset than control HIV-infected patients. Because identical PBMC samples from cases and controls were examined blindly, these results are not caused by a bias in tissue sampling. Homo-/bisexual and hemophiliac AIDS patients who do not develop KS appear to have a low prevalence of infection. These findings indicate that KSHV infection is specifically associated with the subsequent development of KS in AIDS patients.

Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract

Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract

Memory CD4+ T Cells Are the Earliest Detectable Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells in the Female Genital Mucosal Tissue during HIV-1 Transmission in an Organ Culture System

ABSTRACT The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4+ T cells and not Langerhans cells were the first HIV-1 RNA-positive cells detected at the epithelial-submucosal junction 6 h after virus exposure. Multicolor laser confocal microscopy demonstrated a globular distribution of HIV-1 gag-pol mRNA in the cytoplasm, and the distribution of CD4 and the CD45RO isoform was irregular on the cellular membrane. At 96 h postinoculation, in addition to memory CD4+ T cells, HIV-1 RNA-positive Langerhans cells and macrophages were also detected. The identification of CD4+ T cells in the tissue at 6 h was confirmed by flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay on immune cells isolated from disaggregated tissue. Furthermore, PMPA {9-[2-(phosphonomethoxy)propyl] adenine}, an antiretroviral compound, and UC781, a microbicide, inhibited HIV-1 transmission across the mucosa, indicating the utility of the organ culture to screen topical microbicides for their ability to block sexual transmission of HIV-1.

Human Immunodeficiency Virus Type 1 Shedding Pattern in Semen Correlates with the Compartmentalization of Viral Quasi Species between Blood and Semen

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study, viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects. Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period. Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

ABSTRACT In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model.

Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log‐phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log‐phase vegetative cultures of wild‐type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron® technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre‐incubation of wild‐type CN3685 with a CPB‐neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild‐type CN3685 and that damage was prevented by pre‐incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685‐induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.

Human Immunodeficiency Virus Type 1 Env Sequences from Calcutta in Eastern India: Identification of Features That Distinguish Subtype C Sequences in India from Other Subtype C Sequences

ABSTRACT India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viralenv from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype Cenv, and one had viruses with a subtype Aenv. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate CIN. Overall, CIN lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for CIN sequences (K340E, K350A, and G429E), 56% of the CIN sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-CINsequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in India.

Effect of antiretroviral therapy on HIV shedding in semen.

Sexual activity is the most common method of HIV transmission (1). Strong evidence supports a correlation between blood plasma HIV viral load and transmissibility (2). In addition, small short-term studies have demonstrated that antiretroviral therapy reduces levels of HIV RNA in semen and may thereby reduce HIV transmission (3-5). Recent improvements in antiretroviral regimens have been shown to have dramatic effects on HIV-related morbidity and mortality (6). However, these advances have led to the existence of a large pool of potentially infectious HIV-seropositive persons. The effect of antiretroviral therapy on HIV shedding in semen is known only in a clinical trial setting; however, it is of major importance to public health, particularly if a substantial proportion of patients continues to shed potentially drug-resistant HIV in semen despite receiving therapy. We evaluated the effectiveness of antiretroviral therapy in a community setting. Methods Study Design The study was conducted from November 1996 through May 1998 at the Hospital Universitrio Clementino Fraga Filho (HUCFF), a large teaching hospital of the Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. The institutional review boards of the HUCFF, Johns Hopkins University (Baltimore, Maryland), and the University of Pittsburgh (Pittsburgh, Pennsylvania) approved the study. Ninety-three HIV-infected men were recruited when they decided to start antiretroviral therapy. The decision to start, change, or stop therapy was made by study patients and their physicians according to the guidelines of the Brazilian Ministry of Health, without input from the study investigators. All patients provided informed consent. The first (baseline) visit occurred before the introduction of antiretroviral therapy. Follow-up visits were scheduled for 1, 2, 3, and 6 months after initiation of therapy. A detailed interview and a physical examination were performed at each visit. Patients with genital ulcerations or urethral discharge were excluded from the analysis. Semen was collected by masturbation at the study site after at least 48 hours of sexual abstinence, was processed within 30 minutes of collection, and was frozen at 70 C after liquefaction. Laboratory Methods We used the NucliSens assay (Organon Teknika, Durham, North Carolina) to measure HIV viral load in whole semen and blood plasma after one freezethaw cycle (4). We chose the NucliSens assay because it is less sensitive to inhibition by unknown factors in semen (7). Patients were tested for syphilis with the VDRL test; a positive result was confirmed with a fluorescent treponemal antibody absorption test (FTA-ABS). A commercial polymerase chain reaction kit was used to detect Chlamydia trachomatis infection in urine. We determined CD4+ and CD8+ lymphocyte counts by using the Becton Dickinson FACScan procedure (Becton Dickinson, San Jose, California). Statistical Analysis In the main analysis, we determined the reduction in HIV viral load from the pretreatment visit to each of the four follow-up visits. Because changes in log-transformed HIV viral load in blood and semen seemed to be normally distributed, 95% CIs were calculated to assess the differences between pre- and post-treatment values. If HIV viral load values were below the detection level (400 copies/mL), they were assigned a value of 282.84 (400 divided by the square root of 2). Spearman rank correlation coefficients were calculated to determine the correlation between HIV viral load in blood plasma and whole semen. Results At the 6-month visit, 88 of 93 patients (95%) had a blood sample available and 85 (91%) provided a semen sample. The median age was 33.8 years. Most patients (95%) had a sexual risk factor for HIV infection. Thirty-five patients (38%) had a CD4+ count less than 200 cells/mm3, 29 (31%) had a CD4+ count of 200 to 350 cells/mm3, and 27 (29%) had a CD4+ count greater than 350 cells/mm3. Ninety-one patients were antiretroviral-naive, and 2 had a history of nucleoside monotherapy. Eighty patients (86%) began double nucleoside combination therapy, and 13 (14%) began a triple-drug regimen that contained a protease inhibitor. Seventy-two patients (77%) started with zidovudine and didanosine. At the last follow-up visit (6 months), 19 patients (22%) were receiving a triple-drug combination regimen. Baseline values for viral load in semen and blood plasma did not significantly differ by choice of type of therapy. All urine specimens were negative for C. trachomatis, and one was positive for Trichomonas vaginalis. Serum VDRL and FTA-ABS results were positive in 14 patients. None of these patients reported a history compatible with syphilis in the month before sample collection. Baseline Measurements The median baseline viral load was 47 000 copies/mL in plasma and 7800 copies/mL in semen. Viral load was undetectable in 4 blood plasma samples (4%) and 24 semen samples (26%). At baseline, HIV viral load in semen was positively correlated with HIV viral load in plasma ( =0.44 [95% CI, 0.26 to 0.59]) and negatively correlated with CD4+ cell count ( =0.31 [CI, 0.11 to 0.48]). Effect of Therapy on HIV Shedding in Semen Antiretroviral therapy was associated with a 66% reduction in the proportion of patients who had detectable HIV RNA in semen from baseline to the 6-month follow-up visit (Figure). At baseline, HIV RNA was detected in 69 (74%) semen samples and 89 (96%) blood samples. Six months after introduction of therapy, HIV RNA was detected in 29 (33%) semen samples and 33 (38%) blood samples. As early as 1 month after the start of therapy, a reduction of approximately 1.41 log10 units was observed in patients who had detectable HIV RNA in semen at baseline (Table). The viral burden was reduced in patients receiving a triple-drug regimen that contained a protease inhibitor as well as in those receiving a double nucleoside combination regimen. Of patients who had detectable HIV RNA in semen at baseline, 18 of 48 receiving dual therapy (38%) and 2 of 16 receiving a triple-drug, protease inhibitorcontaining regimen (13%) had detectable HIV RNA in semen after 6 months of therapy (data not shown). Figure. Patients with detectable HIV viral load (>400 copies/mL) in semen ( top ) and blood plasma ( bottom ) before and after introduction of antiretroviral therapy. Table. Mean Reduction in HIV Viral Load in 69 Patients with Detectable Baseline Seminal HIV Viral Load according to Type of Combination Therapy Discussion The main finding of our study is that antiretroviral therapy substantially reduces the concentration of HIV RNA in semen. Seventy-four percent of our patients had detectable HIV RNA in semen at baseline compared with 25% at the last follow-up visit, a reduction of 66%. All but 2 of our patients were antiretroviral-naive, which may explain the profound effect of therapy on HIV shedding in semen. In addition, the clinically significant proportion of patients who were prescribed antiretroviral therapy but continued to shed HIV may have resulted in part from nonadherence to therapy. Of interest, a statistically significant reduction in seminal HIV viral load was seen at all time points in patients receiving a double nucleoside regimen. Although protease inhibitorcontaining regimens are usually recommended for initial therapy for HIV infection (8), it has been suggested that initial therapy with a double nucleoside regimen could be an option for patients who have an intermediate risk for HIV progression (9). Our finding that double nucleoside combination therapy has a positive effect on HIV shedding in semen suggests that this therapy may also be able to reduce the spread of HIV infection through sexual transmission. Our findings have important consequences for the dynamics of the AIDS epidemic. Evidence suggests that reduced HIV viral load in semen is associated with diminished sexual transmissibility of HIV. Previous studies have shown a correlation between blood plasma HIV viral load and heterosexual transmission (2). Furthermore, a high viral load in sexual secretions is positively correlated with risk for sexual transmission of the simian immunodeficiency virus (10). The longitudinal design is a strength of our study. The inconsistent results of earlier studies on this topic may be explained by cross-sectional design or by laboratory methods (11-13). Another strength of our study design is that it was meant to simulate clinical practice. Because we had no control over the decisions to start or change antiretroviral therapy, our results are probably more representative of the true effectiveness of therapy in the community setting. Our results are generally consistent with those of the few small longitudinal studies that were nested in clinical trials (3-5). The results obtained from clinical trials may not be generalizable because these trials tend to select for adherent patients and may therefore overestimate the effect of therapy (14, 15). However, because we do not know the proportion of persons informed about the study who presented for enrollment, we could also have selected for persons who were more adherent than the general population. Therefore, our results could overestimate the impact of antiretroviral therapy on HIV shedding in semen. The main limitation of our study is that the correlation between HIV viral load in semen and sexual infectiousness of HIV has not been established. Replication-competent HIV has been recovered from the semen of HIV-infected men who were receiving antiretroviral therapy and had undetectable HIV viral load in blood plasma and semen (16). In addition, increased high-risk behavior (17) may reduce the effect of antiretroviral therapy on HIV transmission (18). However, strong evidence supports a positive correlation between HIV viral load and risk for transmission (2). From a public health perspective, reduced HIV viral load in semen will probably lead to reduced sexual infectiousness and