Phylogenetic Analysis of an HIV Outbreak in a Dialysis Unit at a Tertiary Care Hospital in Multan, Pakistan

Background: In Pakistan, the number of Human immunodeficiency virus (HIV) cases is increasing significantly, attributed to risk factors such as injection drug use, sexual transmission, etc. However, transmission through hemodialysis units is not well documented. In 2024, an outbreak of HIV cases in Multan, Pakistan, drew alarm from local health authorities due to reports linking it to a large public hospital in South Punjab. Here, we report the molecular epidemiological investigation of the outbreak. Methods: Twenty-five hemodialysis patients identified during the outbreak were enrolled. Blood samples were subjected to DNA extraction and polymerase chain reaction (PCR) amplification. Phylogenetic analysis was conducted using the maximum-likelihood approach in IQ-TREE. For dating phylogenetics, a maximum clade credibility tree (MCC) was constructed using the BEAST tool. The MCC tree was constructed using the Bayesian Skyline model with an uncorrelated lognormal relaxed clock. The VESPA program was used to identify amino acid signatures unique to outbreak sequences compared with Pakistani reference sequences. Results: A total of 25 patients (identified as part of the HIV outbreak) were enrolled. 96% (24 out of 25) also tested positive for Hepatitis C, while none tested positive for Hepatitis B. The age range of patients in the study was 23 to 72 years (median age: 44.88 years). In terms of gender distribution, 13 out of 25 were male. All the sequences were identified as HIV subtype CRF02_AG. Phylogenetic analysis revealed that Multan sequences formed a well-supported monophyletic cluster, indicating shared recent origin. Signature pattern analysis identified a unique molecular fingerprint at 26 nucleotide positions, whereas molecular dating placed the emergence of the cluster between 2023 and 2024, consistent with the outbreak timing. Conclusions: Findings provide biologically plausible evidence of a point-source HIV outbreak linked to lapses in infection prevention and control practices at the hemodialysis unit.