HIV/AIDS is a global pandemic with 37 million individuals living with HIV infection and approximately 39 million have died from AIDS worldwide. The objectives of this project are to define the unique epidemiological, clinical, virologic, and immunologic features of HIV and its co-infections in developing countries, to determine the viral kinetics associated with sexual transmission, and to characterize the molecular strains of HIV internationally for infectiousness and progression of disease. We continued our research on HIV cross-sectional incidence testing. A major component of our incidence testing algorithms involves the use to the 3rd generation Genetic Systems 1 /2 + O ELISA which is not currently available in many countries (including China and South Africa). Therefore we developed and evaluated an avidity modified 4th generation Genetic systems ELSIA. We analyzed 2,140 samples from 808 individuals from the United States with known duration of HIV infection. The mean duration of recent infection using an avidity index (AI) cutoff of 50% was 135 days and only 2.1% of samples from individuals infected > 2 years were misclassified as recent. All samples from elite suppressors had an AI >80%. We determined that viral suppression and low CD4 cell count were significantly associated with misclassification among individuals infected >2 years. We plan to determine how well this biomarker works in combination with other serologic and molecular biomarkers of recent infection to determine its capacity to accurately estimate HIV incidence as part of a multi-assay algorithm. Using the knowledge developed in developing accurate methods for estimating HIV incidence, we developed similar methodology for HCV incidence estimation. Using 997 samples from 568 individuals of the BBASH and ALIVE cohort who had known duration of HCV infection, we developed an antibody avidity assay which we used in combination with a viral load test to establish an algorithm that could reliably discriminate between recent and non-recent infection. We determined that individuals appeared recently infected for an average of 147 days (95% CI, 125195 days) for our testing algorithm. We also discovered that individuals co-infected with HIV had pronounced decreased HCV antibody avidity. We performed modeling experiments to determine the power of methodology to detect a 50% change in population level incidence. By varying the initial incidence, prevalence, and HIV coinfection frequency, we determined that surveys of 1000 individuals will provide a sample size large enough to see a 50% change in incidence. It is now possible with this methodology to perform population surveys for both prevalence and incidence of HIV and HCV infection and to measure the impact of prevention interventions targeting both infections. The major barrier to curing HIV infection is the persistence of HIV in latently infected resting memory CD4+ T cells. Previous studies have quantified this pool of latently infected cells in North Americans and Europeans but no study had quantitated the latent HIV-1 reservoir (LVR) in sub-Saharan Africans, who make up the largest population of HIV-infected individuals globally. We therefore conducted a study in 60 virally suppressed HIV-infected individuals in Rakai, Uganda to determine the minimum frequency of latently infected cells with replication competent virus using the quantitative viral outgrowth assay (Q-VOA). The median LVR size in this Ugandan population was estimated to be 0.40 IUPM. LVR size correlated negatively with time on suppressive ART, and positively with pre-ART viral load, and having two or more instances of transiently detectable plasma viral load after initial ART-suppression. Preliminary analysis of viral subtype suggests no significant difference in LVR size between individuals infected with subtype A vs. subtype D. Of note, the LVR size in this Ugandan population is over 3-fold smaller than that previously reported in Americans. This represents the first quantification of latently infected resting CD4+ T cells with replication competent virus in an ART treated, virally suppressed sub-Saharan African population. Further immunologic correlates and decay over time analyses are ongoing. The Johns Hopkins Emergency Department has served as an observational window on the HIV-epidemic in inner city Baltimore for over 25 years. We recently analyzed 7 discrete identity-unlinked serosurveys conducted on 18,240 untargeted adult JHH-ED patients between 1987-2013 for demographic trends in HIV prevalence, cross-sectional incidence estimates, viral load and HCV prevalence. HIV prevalence in 1987 was 5.2%, peaked at 11% from 1992-2003, and then declined to 5.6% in 2013. Proportion of undiagnosed HIV declined over time from 77% in 1987 to 12% by 2013. HIV incidence estimates in 2001 were 2.1% and declined steadily to 0.16% by 2013. Proportion of HIV+ individuals with viral suppression increased steadily from 23% in 2001 to 59% by 2013. Consistent with increasing viral suppression, 80% of 214 HIV+ individuals surveyed in 2013 had antiretroviral drugs detected in their sera, a marked increase from 2007 (27%). However, HCV in this population remained at 18-19% from 1988 until 2007 and declined only slightly to 14% by 2013. We have expanded our use of ultra-deep pyrosequencing to identify HIV superinfection and describe its effects on the pandemic. In a study of post-partum women in Malawi we found eight confirmed SIs in women who infected their infants via breast feeding with a rate of SI of 7.5/100pys, and five confirmed SIs in the non-transmitters for a rate of SI of 4.4/100pys (p=0.78), suggesting that SI was common in this group but did not increase transmission via breast feeding. The occurrence of HIV-SI was also shown to increase broadly reactive neutralizing antibody and further studies are warranted to examine the implications for vaccine research. We have expanded our collaborations to work with Dr. Irini Sereti and Dr. Martha Nason to examine the role of immune activation, and particularly increased cytokine levels, on herpetic reactivation after the initiation of ART. This led to the finding that ART initiation increases levels of IL-6, which was associated with HSV-2 and CMV shedding in women from Rakai Uganda. We submitted, and were awarded, a bench to bedside grant to examine this in more detail in Uganda and at the NIH clinical center. We undertook a study to characterize prevalence of HCV, HIV/HCV co-infection and the HCV care continuum among IDUs in 15 cities throughout India. 14,450 IDUs who were 18 years of age and reported drug injection in the prior 2 years were surveyed. The median weighted HCV prevalence across cities was 41.1%; HIV/HCV co-infection prevalence ranged from 2.0 to 28.5%. Of the 5,777 IDUs with HCV infection, only 5.6% had been previously diagnosed, 2.7% were linked to care, 1.4% had ever been treated and 0.38% exhibited sustained virologic response. Engagement in HCV care was significantly better among participants living in states with established injection drug use epidemics. The high burden of HCV and HIV/HCV co-infection coupled with low-access to HCV testing and treatment highlights the urgency to include India in the global HCV agenda especially given recent advances in HCV treatment.