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Item An improved PCR method for detection of HIV-1 proviral DNA of a wide range of subtypes and recombinant forms circulating globally(Elsevier Ltd, 2011) Weinder, J.; Cassens, U.; Gohde, W.; Sibrowski, W.; Odaibo, G.; Olaleye, D.; Reichelt, D.; Greve, B.Proviral DNAs are being measured increasingly as a marker of the efficacy of highly active anti-retroviral therapy (HAART) and is accepted for the early diagnosis of perinatal HIV-1 infections. This requires a standardized test which enables the detection of a wide range of subtypes worldwide including O, N and circulating recombinant forms (CRFs). Based on a previous publication, a PCR - Test for HIV-1 provirus detection in peripheral blood mononuclear cells (PBMCs) was developed. Blood samples from 80 individuals infected with HIV-1 and 20 persons negative for HIV-1&2 from Africa and Germany were tested for the presence of HIV-1 provirus DNA. The primer system used enables the detection of proviral DNA despite the high concentrations of human DNA. The limit of detection was determined to be 5 copies per 10(5) cells. All 20 samples from persons negative for HIV were negative for HIV-1 proviral DNA while provirus DNA was amplified from 76 of the 80 (95%) samples from persons infected with HIV. The amplified products were detected by gel-electrophoresis, flow cytometry and real-time PCR. All three detection systems provided the same results.Item Service uptake and performance of the prevention of mother-to-child transmission (PMTCT) programme in Ibadan, Nigeria(2010) Oladokun, R. E.; Awolude, O.; Brown, B. J.; Adesina, O.; Oladokun, A.; Roberts, A.; Odaibo, G.; Osinusi, K.; Olaleye, D.; Adewole, I. F.; Kanki, P.The Prevention of Mother to Child Transmission (PMTCT) programme in the University College Hospital (UCH), Ibadan has been in existence for more than five years and has scaled up to other sites. The study evaluated the service uptake and performance of the programme using national key indicators. Antenatal and delivery records of women enrolled between July 2002 and June 2007 were reviewed. A total of 51952 women attended first antenatal visits and received HIV pre-test counselling. Of these, 51614 (99.5%) accepted HIV test and 49134 (95.2%) returned for their results. Out of the tested patients, 2152 (4.2%) were identified to be HIV positive. Partners of positive patients accepting HIV testing were 361 (16.7%) with 87 (18.6%) testing positive. There were a total of 942 deliveries out of which 39.2% of the mothers and 95.2% of the babies respectively received ARV prophylaxis. In all, 85.8% (788/918) of the mothers opted for formula as the method of infant feeding. Out of the 303 babies eligible for ELISA testing, 68.3% reported for the test and 17 (8.7%) tested positive. There has been progress in the programme, reflected in the increase in the number of new clients accessing the PMTCT service. However, partner testing and follow up of mother-infant pairs remain formidable challenges that deserve special attention.Item Human Immuno-deficiency Virus and Hepatitis B virus coinfection in pregnancy at the University College Hospital, Ibadan(2010) Adesina, O.; Oladokun, A.; Akinyemi, O.; Adedokun, B.; Awolude, O.; Odaibo, G. O.; Olaleye, D.; Adewole, J.Human Immuno-deficiency virus (HIV) and Hepatitis B Virus (HBV) share common modes of transmission which include blood borne and the vertical routes. Although, the natural course of HIV does not appear altered by HBV, the rate of liver-related deaths is several times higher among HIV/HBV co-infected persons. Clinicians providing care for HIV positive individuals, including pregnant women, need to be aware of this problem. This is a 2-year cross-sectional study that commenced in January 2006, among HIV positive pregnant women seen at the University College Hospital, Ibadan. During the study period, 721 HIV positive pregnant women were screened for hepatitis B virus infection. Sixty-four women (8.9%) were positive for HBsAg, 14(1.9%) were HCV positive and 642 (89.2%) were negative for both HBV and HCV. One patient was positive forboth HBV and HCV. There were no remarkable differences between HIV infected and HIV-HBV coinfected patients in terms of the hematological, albumin and bilirubin measurements. Alanine transaminase was however higher in the HIV-HBV co-infected patients than HIV patients and this was statistically significant (17.5 iu/ ml vs. 15.0 iu/ml, p value--0.009). In addition, the CD4 cell count was lower and the viral load marginally higher in the hepatitis B virus positive patients. The differences were however not statistically significant (p value--0.114 and 0.644 respectively). HIV-HBV co-infection in HIV positive pregnant women is not of negligible proportions as demonstrated in this study. Thus, HIV positive pregnant women should be screened for HBV and assisted to access care targeted at preventing morbidity and vertical transmission.Item A new affordable flow cytometry based method to measure HIV-1 viral load(International Society for Advancement of Cytometry, 2009) Greve, B.; Weidner, J.; Cassens, U.; Odaibo, G.; Olaleye, D.; Sibrowski, W.; Reichelt, D.; Nasadala, I.; Gohde.Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5'LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes. A coamplified internal standard warranted the functionality of the PCR and allows reproducible viral load measurements. Two drawbacks of current viral load measurements are overcome by the flow cytometry based test described hereof. First, all known worldwide relevant HIV-1 subtypes including subtypes O, N, and new CRFs are quantifiable with high sensitivity (50 to >1 x 10(6) copies per PCR). Second, the cost per test can be reduced to less than 12 US$ instead of the current 50-100 US$. Additionally, the test described in this report offers the possibility to perform complete monitoring program (CD4 T-cell count, CD4% and viral load) for the first time, with the same device for HIV-infected persons.Item The complexity of circulating HIV type 1strains in Oyo state, Nigeria(Mary Ann Liebert, Inc, 2007) Sankale, J.; Langevin, S.; Odaibo, G. T.; Meloni, S. T.; Ojesina, A. I.; Olaleye, D.; Kanki, P.Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to circulate in west Africa. We undertook a survey of HIVs in Oyo state, in southwestern Nigeria. We analysed 71 samples from Ibadan the capital city, and 33 samples form Saki, 100 miles west of Ibadan. We sequenced part of the gag gene and the envelope C2V3 region from 102 and 89 samples, respectively. In the 87 samples for which both genes were sequenced, subtypes G and CRF02_AG were found in equal proportions (32.2%each). Other samples included CRF06_cpx(8.0%), subtype A (2.3%), C(1.1%), unclassified(1.1%), or discordant sequences suggesting the presence of a large number of recombinants involving CRF02_AG and/or subtype G(20.7%) or other subtypes(2.3%). The subtype/CRF designation was concordant in two gene fragments in the majority of samples evaluated. However, we observed difference in subtype distribution between the two locations with a predominance of subtypes G in Ibadan and CRF02 in Saki. This is first in-depth analysis of HIV variability at a state level in Nigeria. Our analysis revealed a significant level of viral heterogeneity and a geographical difference in subtype distribution, and demostrated that CRF02_AG does not account for the majority of circulating strains.Item Subtypes-specific patterns in HIV type 1reverse transcriptase and protease in Oyo state, Nigeria: implications for drug resistance and Host Response(Mary Ann Liebert, Inc, 2006) Ojesina, A. I.; Sankale, J.; Odaibo, G.; Langevin, S.; Meloni, S. T.; Sarr, A. D.; Olaleye, D.; Kanki, P. J.As the use of antiretroviral therapy becomes more widespread across Africa, it is imperative to characterize baseline molecular variability and subtype-specific peculiarities of drug targets in non-subtype B HIV-1 infection. We sequenced and analyzed 35 reverse transcriptase (RT) and 43 protease (PR) sequences from 50 therapy-naive HIV-1-infected Nigerians. Phylogenetic analyses of RT revealed that the predominant viruses were CRF02_AG (57%), subtype G (26%), and CRF06_cpx (11%). Six of 35 (17%) individuals harbored primary mutations for RT inhibitors, including M41L, V118I, Y188H, P236L, and Y318F, and curiously three of the six were infected with CRF06_cpx. Therefore, CRF06_cpx drug-naive individuals had significantly more drug resistance mutations than the other subtypes (p = 0.011). By combining data on quasisynonymous codon bias with the influence of the differential genetic cost of mutations, we were able to predict some mutations, which are likely to predominate by subtype, under drug pressure. Some subtype-specific polymorphisms occurred within epitopes for HLA B7 and B35 in the RT, and HLA A2 and A*6802 in PR, at positions implicated in immune evasion. Balanced polymorphism was also observed at predicted serine-threonine phosphorylation sites in the RT of subtype G viruses. The subtype-specific codon usage and polymorphisms observed suggest the involvement of differential pathways for drug resistance and host-driven viral evolution in HIV-1 CRF02_AG, subtype G, and CRF06_cpx, compared to subtype B. Subtype-specific responses to HIV therapy may have significant consequences for efforts to provide effective therapy to the populations infected with these HIV-1 subtypes