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Methods for identification of primary HIV infections seem increasingly important to understand pathogenesis, and to prevent transmission, which is particularly efficient during acute infection. Most current algorithms for HIV testing are based on detection of HIV antibodies and are unable to identify early infections before seroconversion. The efficiency of prospective cohorts, which is a standard approach for identifying primary HIV-1 infection, depends on a variety of epidemiological and cultural factors including HIV incidence and stigma and, not surprisingly, varies significantly in different geographical areas. We report a voluntary counseling and testing (VCT)-based approach to identifying primary HIV-1C infection that was developed as part of a primary HIV-1 subtype C infection study in Botswana. The referral strategy was based on: (1) collaboration with VCT centers at city clinics operated by the Ministry of Health; (2) partnering with the busiest non-government VCT center; (3) educating healthcare workers and the community about primary HIV infection; and (4) pairing with diverse VCT providers, including NGOs and private-sector organizations. Acute HIV-1 infections were defined by a negative HIV-1 serology combined with a positive HIV-1 RT-PCR test. Recent HIV-1 infections were identified by detuned EIA testing according to the classic STARTH algorithm. The VCT-based referral strategy resulted in the successful identification of 57 cases of acute and early HIV infection. A referral strategy of expanded VCT with viral RNA (Ribonucleic acid) testing to a national program in Botswana may be a promising approach for identification of primary HIV infections on a countrywide level. The program should offer VCT with viral RNA testing to the general public, facilitate proper counseling and risk reduction, and allow initiation of early HAART, and may reduce new viral transmissions. [ABSTRACT FROM AUTHOR]

Poster presentation

Background: Clinical laboratories in Botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers' kits and textbooks., Objectives: The aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in Botswana., Method: A total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of Botswana, and immunological and haematological laboratory parameters were measured., Results: The mean age was 28.8 (95% Confidence Interval [CI] 27.7-29.8) years, with a median of 27 years and a range 18-66 years. The mean haemoglobin level was significantly lower for women (12.4 g/dL; 95% CI 12.1% - 12.7%) than men (15.1 g/dL; 95% CI 14.9% - 15.3%). The women's haemoglobin reference values (9.0 g/dL - 15.0 g/dL) levels were lower than observed in predominantly White populations (12.0 g/dL - 16.0 g/dL), but comparable with regional consensus reference intervals (9.5 g/dL - 15.8 g/dL) recently defined for East and Southern Africa., Conclusion: The established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally.

Background: Clinical laboratories in Botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers' kits and textbooks., Objectives: The aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in Botswana., Method: A total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of Botswana, and immunological and haematological laboratory parameters were measured., Results: The mean age was 28.8 (95% Confidence Interval [CI] 27.7-29.8) years, with a median of 27 years and a range 18-66 years. The mean haemoglobin level was significantly lower for women (12.4 g/dL; 95% CI 12.1% - 12.7%) than men (15.1 g/dL; 95% CI 14.9% - 15.3%). The women's haemoglobin reference values (9.0 g/dL - 15.0 g/dL) levels were lower than observed in predominantly White populations (12.0 g/dL - 16.0 g/dL), but comparable with regional consensus reference intervals (9.5 g/dL - 15.8 g/dL) recently defined for East and Southern Africa., Conclusion: The established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally., Competing Interests: The authors declare that they have no financial or personal relationship(s) which may have influenced them inappropriately in writing this article.

Background: Aiming to answer the broad question "When does mutation occur?" this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection., Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations., Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p=0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p=0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3-3.0), dominance (4.8 (3.4-6.8)), and completeness (3.6 (2.3-5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes., Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness.

Background: Estimation of HIV incidence rates is important for timing interventions, planning prevention studies, and monitoring the epidemic. This requires accurate estimation of the "recency period" (also known as the "window period") between seroconversion and achievement of specific detectable levels of anti-HIV antibody titers, such as the standardized optical density (SOD) in the early phase of HIV-1 infection., Methods: To obtain a better understanding of interpatient variation of the recency period, prospective measurements of antiviral antibody titers in the early phase of HIV-1 subtype C infection were quantified by Vironostika-LS. Time of seroconversion was estimated by Fiebig staging., Results: The profiles of SOD values during the first year of infection commonly showed slow initial increase followed by a more rapid increase, although in some patients, SOD values increased rapidly soon after seroconversion. Using an SOD cutoff of 1.0, the average duration of the recency period in subtype C infection in the local epidemic in Botswana was estimated to be 151 days (95% confidence interval: 130 to 172 days) post seroconversion. The recency period was significantly associated (P = 0.007) with the level of viral replication during the first 2-3 months post seroconversion. Reduction of SOD values after initiation of antiretroviral therapy (ART) was a dominant pattern in antiretroviral drug (ARV)-treated subjects., Conclusions: Our data suggest that HIV incidence estimation based on sensitive/less sensitive enzyme immunoassay cross-sectional testing could be potentially improved by incorporation of viral load levels at the time of detection of a recent infection.

The evolution of proviral gp120 during the first year after seroconversion in HIV-1 subtype C infection was addressed in a case series of eight subjects. Multiple viral variants were found in two out of eight cases. Slow rate of viral RNA decline and high early viral RNA set point were associated with a higher level of proviral diversity from 0 to 200 days after seroconversion. Proviral divergence from MRCA over the same period also differed between subjects with slow and fast decline of viral RNA, suggesting that evolution of proviral gp120 early in infection may be linked to the level of viral RNA replication. Changes in the length of variable loops were minimal, and length reduction was more common than length increase. Potential N-linked glycosylation sites ranged +/-one site, showing common fluctuations in the V4 and V5 loops. These results highlight the role of proviral gp120 diversity and diversification in the pathogenesis of acute HIV-1 subtype C infection.

Background: Most knowledge of primary HIV-1 infection is based on subtype B studies, whereas the evolution of viral parameters in the early phase of HIV-1 subtype C infection is not well characterized., Methods: The kinetics of viral RNA, proviral DNA, CD4+ T-cell count, and subsets of CD4+ T cells expressing CCR5 or CXCR4 were characterized in 8 acute and 62 recent subtype C infections over the first year postseroconversion., Results: The viral RNA peak was 6.25 +/- 0.92 log10 copies per milliliter. After seroconversion, heterogeneity among acute cases was evident by patterns of change in viral load and CD4+ T-cell count over time. The patterns were supported by the rate of viral RNA decline from peak (P = 0.022), viral RNA means (P = 0.005), CD4 levels (P < 0.001), and CD4 decline to 350 (P = 0.011) or 200 (P = 0.046). Proviral DNA had no apparent peak and its mean was 2.59 +/- 0.69 log10 per 106 peripheral blood mononuclear cell. In recent infections, viral RNA set point was 4.00 +/- 0.97 log10 and viral RNA correlated inversely with CD4+ T cells (P < 0.001) and directly with proviral DNA (P < 0.001)., Conclusions: Distinct patterns of viral RNA evolution may exist shortly after seroconversion in HIV-1 subtype C infection. The study provides better understanding of the early phase of subtype C infection.

We report the first case, to our knowledge, of antibody-negative human immunodeficiency virus type 1 (HIV-1) subtype C infection, which was identified during screening for acute HIV-1 infection in Botswana. Results of tests for HIV-1 antibodies were consistently negative, including rapid and regular enzyme-linked immunosorbent assay and Western blot. The nonrecombinant HIV-1 subtype C infection was confirmed by viral genotyping within the gag, pol, and env genes. The period between referral of the patient in a clinically stable condition and AIDS-related death was approximately 3 months. The reported case indicates the importance of studying the prevalence of seronegative HIV-1 infection in southern Africa, where subtype C predominates.