Peter Kataaha

HIV prevalence attitudes and behaviour in clients of a confidential HIV testing and counselling centre in Uganda.

Objective.To describe clients, operation and impact of an African public HIV testing and counselling centre. Design and setting.Analysis of samples from clients attending the AIDS Information Centre (AIC) in Kampala, Uganda in early 1991. Subjects.HIV-1-positive and HIV-negative consecutive clients (250 of each), 86 consecutive couples, and 200 consecutive clients who were HIV-negative in 1990 and were attending for their repeat test. Main outcome measures.HIV seroprevalence rates, attitudes, behaviour and behaviour change. Results.HIV-1 prevalence was 28% overall, 24% in men and 35% in women. Reasons for taking the HIV test were a planned marriage or a new relationship (27%; 84% in couples), to plan for the future (35%), distrust of sexual partner (14%) and illness or disease/death (not HIV-specific) of partner (20%). The majority of the reported intentions in response to a positive or a negative HIV test result were positive, demonstrating the ability to cope with this information. Of repeat clients, two (1%) had become HIV-1-positive. The majority of repeat clients reported one sexual partner only (67%) or sexual abstinence (25%). Compared with pre-test information from AIC clients attending for the first time, repeat clients reported casual sexual contacts less often (6 versus 25%) and, of those, the majority used condoms. Conclusions.Our study demonstrates the demand for and the feasibility of confidential HIV testing and counselling services in Uganda, and illustrates the value of these services in achieving behaviour changes. Such services should be considered an additional approach for the reduction of HIV transmission in Africa, especially in areas with high HIV seroprevalence rates.

A rapid manual method for CD4+ T-cell quantitation for use in developing countries

Objective:To evaluate a manual method (Cytosphere) for quantifying CD4+ T-cell numbers. Design:Cross-sectional study of HIV-1-seronegative and HIV-1-seropositive individuals evaluated for absolute CD4 counts by both standardized flow cytometric measurements and manual Cytosphere technology using a hemacytometer. Setting:University research hospitals in both the United States and Africa. Patients, participants:Blood specimens from 382 patients were evaluated. These were broken down into 294 samples obtained from HIV-1-seropositive patients and 88 samples obtained from HIV-1-seronegative patients. Interventions:None. Outcome measured:Absolute CD4 cell number. Results:Evaluation of samples obtained from HIV-1 patients in both the United States and Africa demonstrated an overall correlation of the Cytosphere assay with flow cytometry of 0.912 (95% confidence interval, 0.895–0.928; P<0.001). When samples were stratified based on CD4+ T-cell counts determined by flow cytometry, the Cytosphere assay had a 96% predictive value for correctly identifying individuals with CD4 T-cell counts > 200×106/l and a 92% predictive value for correctly identifying individuals with CD4 T-cell counts < 200×106/l. Conclusions: This assay appears to have the potential for the quantitation of CD4 cells in the limited laboratory facilities in developing countries and to have a strong correlation with standard flow cytometric technology. AIDS 1993, 7:1565–1568

Phase I/II trial of HIV-1 hyperimmune globulin for the prevention of HIV-1 vertical transmission in Uganda

ObjectivesTo assess the safety, tolerance, pharmacokinetics, and virologic and immunologic changes associated with the use of Ugandan HIV hyperimmune globulin (HIVIGLOB) in HIV infected pregnant Ugandan women and their infants. DesignA prospective, phase I/II, three-arm dose escalation trial of HIVIGLOB. MethodsHIVIGLOB was prepared from discarded HIV infected units of blood collected from the National Blood Bank in Kampala. From June 1996 to April 1997, 31 HIV positive pregnant women were enrolled with HIVIGLOB infusions given at 37 weeks gestation and within 16 h of birth for infants. The first 10 mother–infant pairs were infused at a dose of 50 mg/kg, followed by 11 pairs at 200 mg/kg, and 10 pairs at 400 mg/kg. Study participants were followed for 30 months. ResultsThirty-one women and 29 infants were infused with HIVIGLOB. The infusions were safe and well tolerated by the women and their infants at all doses. There were no significant changes in virologic or immunologic parameters after HIVIGLOB infusion. Pharmacokinetic properties of this product were similar to other immune globulin products with a median half-life of 28 days in women and 30 days in infants. ConclusionAn HIV immune globulin product derived from HIV infected Ugandan donors is safe, well tolerated, and has pharmacokinetic properties consistent with other immunoglobulin products. Data suggest that a 400 mg/kg dose of HIVIGLOB would be the most appropriate dose for a subsequent efficacy trial of HIVIGLOB for the prevention of mother to child HIV transmission.

Identification of diverse HIV type 1 subtypes and dual HIV type 1 infection in pregnant Ugandan women.

Vertical (mother-to-child) transmission accounts for the majority of pediatric HIV-1 infections. Many factors are involved in vertical transmission, however it is not clear which factors are most important for determining whether a mother will transmit HIV-1 to her infant. It has been suggested that HIV-1 subtype may influence vertical transmission and that subtype D viruses may be less likely to be transmitted in this setting. We analyzed HIV-1 gp120 V3 region sequences from the plasma of 20 pregnant Ugandan women of known transmission status who did not receive antiretroviral prophylaxis. V3 regions were cloned, sequenced, and subtyped by phylogenetic analysis. Among 11 women who transmitted HIV-1 to their infants, we detected subtypes A, C, D, and G. Two of the transmitters had dual infection with subtypes A and D. In addition, a third was infected with two distinct strains of subtype G viruses. HIV-1 subtype A and D viruses were found in 9 women who did not transmit the virus to their infants. This study reveals that pregnant Ugandan women harbor diverse HIV-1 subtypes, including women who transmit HIV-1 to their infants. Transmission of HIV-1 with subtype D V3 regions was confirmed in 4 of the 11 transmitters, including 2 who had dual infection with subtype A and D HIV-1.

Beta 2-microglobulin, HIV-1 p24 antibody and acid-dissociated HIV-1 p24 antigen levels: predictive markers for vertical transmission of HIV-1 in pregnant Ugandan women.

ObjectivesTo evaluate the clinical utility of plasma β2-microglobulin (β2M) levels, acid-dissociated HIV-1 p24 antigen, and HIV-1 p24-antibody titers in predicting HIV-1 vertical transmission in 227 HIV-1-infected Ugandan pregnant women. DesignPlasma β2M levels, acid-dissociated HIV-1 p24-antigen positivity, and HIV-1 p24-antibody titers were determined using commercial enzyme immunoassays (EIA) in a Ugandan cohort of 52 HIV-1-seropositive transmitting mothers, 175 HIV-1-seropositive non-transmitting mothers, and 52 seronegative mothers within 6 weeks prior to delivery. ResultsTransmitter mothers had significantly higher plasma concentrations of β2M (1.80

HIV-1 ICD p24 antigen detection in Ugandan infants: Use in early diagnosis of infection and as a marker of disease progression

pM 1.13 mg/l) than non-transmitter seropositive mothers (1.32

Dual Transmission of Subtype A and D HIV Type 1 Viruses from a Ugandan Woman to Her Infant

pM 0.81 mg/l; P= 0.0013). Similarly, a significantly higher proportion of transmitter mothers had detectable p24 antigen than non-transmitter mothers [six out of 51 (11.8%) versus six out of 173 (3.5%); P=0.03]. Compared with the vertical transmission rate of 23% in the seropositive group, the positive predictive values of a β2M level > 1.5 mg/l or detectable HIV-1 p24 antigen for vertical transmission were 34 and 50%, respectively. Five of six (83.3%) seropositive mothers with both a β2M level > 1.5 mg/l and detectable p24 antigenemia transmitted HIV-1 infection to their infants compared with 25 of 124 (20.2%) seropositive mothers with values below the cut-off values for both tests (P= 0.00249). However, β2M was not found to be a significant independent predictor of vertical transmission when analyzed in a multivariate model with p24 antigenemia. There was no significant difference in HIV-1 p24-antibody titers in transmitter mothers versus non-transmitter mothers (P= 0.299). Conclusionβ2M levels and acid-dissociated HIV-1 p24-antigen assays may be used to predict which HIV-1-infected pregnant women are at greatest risk for vertical transmission. However, only the p24-antigen test was independently predictive of vertical transmission and its clinical utility is limited.

Non-isotopic polymerase chain reaction methods for the detection of HIV-1 in Ugandan mothers and infants.

The objective of this study was to determine the use of immune‐complex dissociated (ICD) p24 antigen detection for the diagnosis and prognosis of HIV‐1 infection in Ugandan children. Plasma collected prospectively from children born to HIV‐1 infected Ugandan women was stored and later analyzed for the presence of neutralizable HIV‐1 p24 antigen using the Coulter ICD p24 antigen and neutralization kits. HIV‐1 infection status, disease progression, and survival of the children were determined. Specimens from 311 children born to HIV‐1 infected women, including 138 HIV‐1 infected children, and 113 children born to negative women were tested. Sixty‐nine (50%) infected children were p24 antigen positive at least once. For early HIV‐1 diagnosis, the specificity and positive predictive value of the assay were consistently high (>95% and >83% respectively), but the sensitivity was low (6–53%), especially in the first months of life. The presence of p24 antigenemia in the first two years of life was associated with poor survival (20%) by 80 months of age compared with infected children without antigenemia (43%, P < 0.001). Early detection of p24 antigen (≤2 months) was associated with higher mortality than first detection at an older age (>6 months, P < 0.001). The data suggest that ICD p24 antigen detection is not a sensitive method for the determination of infant HIV‐1 status in our cohort of HIV‐1 infected Ugandan children tested in the first two years of life. There was a strong correlation, however, between the presence and time of onset of p24 antigenemia and mortality among HIV‐1 infected children. J. Med. Virol. 62:426–434, 2000.

Plasma viral load in symptom-free women and vertical transmission of HIV-1

217 GROUP M HIV-1 VIRUSES have been categorized into subtypes A±J based on phylogenetic reconstruction using DNA sequences. Sequence variation between these subtypes generally ranges from 25 to 35% within the env gene. In most cases of HIV-1 infection, viruses of a single subtype are detected. While the identification of recombinant viruses derived from different subtypes suggests the occurrence of dual infection, there are relatively few reports in which HIV-1 viruses of two different M group subtypes were detected in a single individual. In most cases, it is not possible to determine the timing of infection with viruses of different subtypes or whether both subtypes originated from the same source. The setting of vertical (mother-to-infant) HIV-1 transmission provides a unique opportunity to study dual transmission. In this setting, the time available for transmission is relatively limited and the mother is usually the only potential source of infection. There is little known about dual HIV-1 infection in the setting of vertical transmission. In one study, subtype A and C viruses were detected in a Rwandan woman; however, only subtype A was detected in her infant. In a second study, subtype B and C viruses were detected in a Brazilian woman and in her infant. To examine the potential transmission of HIV-1 viruses of different subtypes, we analyzed V3 cDNAs from Ugandan women and infants. The presence of diverse HIV-1 subtypes in Uganda increases the potential for dual infection. HIV-1 subtypes found in Uganda include A, B, C, D, and G; subtypes A and D account for the majority of infections. Plasma samples were collected from a Ugandan woman and her infant at the time of delivery. An additional sample was collected from the infant at 6 weeks of age. These samples were collected in 1993 as part of a natural history study of vertical HIV-1 transmission in Kampala, Uganda. Plasma was shipped to the United States for analysis. HIV-1 RNA was extracted from 200 m l of plasma using the Amplicor HIV-1 Monitor test kit (Roche Diagnostic Systems, Branchburg, NJ) according to manufacturer instructions. RNA extracts were resuspended in a final volume of 400 m l. Twenty-five microliters of each RNA extract was used for reverse transcription (RT) with Moloney murine leukemia virus reverse transcriptase. Each 25-m l aliquot contained at least 50 copies of HIV-1 RNA. To minimize the potential for bias due to primer binding, random hexamer oligonucleotide primers [pd(N)6; Pharmacia, Piscataway, NJ] were used in the RT reactions. The V3 region of the env gene was amplified in a nested polymerase chain reaction (PCR) using primers that correspond to highly conserved regions of the env gene.23 To reduce the potential for sample contamination, each step of the RT-PCR amplification was assembled in a separate, enclosed workstation using aerosol-resistant pipette tips and dedicated equipment. Equipment in each workstation was ultraviolet (UV) irradiated after each experiment. Negative controls without template were included for each RT and PCR reaction. Each step of the analysis (including RT, PCR, cloning, and plasmid analysis) was performed on a separate day for each sample. Comparison of V3 cDNA sequences obtained from these plasma samples and from unrelated samples analyzed in our laboratory showed no evidence of contamination. PCR products were purified using a spin column technique and cloned into the pCR2.1 vector, using the TA cloning kit (Invitrogen, Carlsbad, CA). The resulting plasmids were isolated and sequenced using the 2 21 M13 forward primer and the BigDye terminator cycle sequencing ready reaction kit (Perkin-Elmer Applied Biosystems, Foster City, CA). At least 10 cDNAs from each sample were analyzed by DNA sequencing and the predicted amino acid sequences were determined (Fig. 1). V3 sequences were used for subtype analysis. Sequences were aligned manually in the program VisEd (provided by Dr. Ken Peters). For regions of high variability, the alignment was based primarily on the most conservative amino acid substitutions. The full sequence alignment contained 18 unique Ugan-

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and p24 antigen in breast milk of HIV-1-infected Ugandan women and vertical transmission.

Two non-isotopic polymerase chain reaction (PCR) methods were evaluated by testing blood from 41 HIV-1-seropositive and 16 HIV-1-seronegative Ugandan mothers and 56 of their children (aged 0.5-15.0 months). Amplification of HIV-1 sequences was performed in duplicate using a biotinylated primer pair to the gag region (SK 462-431) and nested primer pairs (JA 17-20) to the pol region of HIV-1. gag sequences were hybridized using a microtiter plate coated with the SK 102 probe followed by colorimetric detection using an avidin-horseradish peroxidase conjugate and tetramethylbenzidine/peroxide substrate. pol sequences were detected on agarose gel stained with ethidium bromide. Results of HIV-1 PCR analysis showed that 40 out of 41 (98%) seropositive mothers and 10 out of 29 (34%) seropositive children had detectable HIV-1 gag and pol sequences. None of the 16 seronegative mothers nor 27 seronegative or Western blot-indeterminate children had detectable HIV-1 sequences. Our results suggest that non-isotopic PCR methods are sensitive, specific, and potentially useful in the early diagnosis of HIV-1 infection in developed and developing countries.