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Background: In June–July 2008 a non-governmental organization (NGO) in Jalalpur Jattan (JPJ), Gujrat, Pakistan arranged two voluntary HIV screening camps after numerous HIV-infected persons reported to their treatment center in Lahore; 88 (35.8%) of 246 persons screened in those camps were positive by rapid test. Intense media coverage made the residents of JPJ hostile to further inquiries. The Pakistan Field Epidemiology Training and Laboratory Training Program (FELTP) was requested by the Provincial AIDS Control Program to carry out an epidemiological investigation. Methods: HIV-positive persons or family members of patients who died of AIDS and consented to be interviewed during the period 15 December 2008 to 2 January 2009 were investigated. Enhanced contact tracing was done to identify additional cases. A structured questionnaire was used to collect data regarding clinical history, risk factors, and HIV knowledge and practices. The national HIV/STI Referral Laboratory collected blood samples for HIV serology and molecular studies independently following pre- and post-counseling. Results: A total of 53 HIV-infected persons were investigated. Out of these, 47 (88.7%) were alive at the time of investigation and 27 (50.9%) of the cases were female with 6 children aged 10 years or less. Median age was 35 years (mean 34.7, range 3–70). Most frequent symptoms were unexplained fever 42 (79.2%), diarrhea 34 (64.15%) and skin infections 27 (50.9%); 13 (24.5%) had co-infection with tuberculosis (TB) and 10 (18.9%) with hepatitis (B or C). Use of injections 51 (96.2%), dental procedures 21 (40%) and barber shop visits among males 18 (72%) were common risk factors. Extramarital sex was reported by 4 (9.4%). Only 19 (35.8%) were aware that HIV can be sexually transmitted and 18 (34%) were aware of HIV transmission by blood transfusion. Phylogenetic analysis revealed HIV infection in this group was HIV-1 Subtype A, transmitted over a decade, and the situation is endemic rather than an outbreak. Conclusion: The investigation indicates high rates of HIV infection in JPJ. Unlike other studies from Pakistan, a high proportion of cases in females and children less than 10 years of age were observed. Socio-cultural norms and stigmatization limited in-depth investigation of sexual and behavioral practices and history of drug abuse. A shift of HIV infection from high-risk groups to the general population was seen and requires vigilant surveillance besides targeted health education, clinical management, lab facilities for diagnosis and monitoring, and voluntary counseling and testing services to limit disease spread.