Phylogenetic Characterization of HIV-1 Sub-Subtype A1 in Karachi, Pakistan.

(1) Background: HIV-1 sub-subtype A1 is common in parts of Africa, Russia, former Soviet Union countries, and Eastern Europe. In Pakistan, sub-subtype A1 is the predominant HIV-1 subtype. Preliminary evidence suggests that distinct strains of HIV-1 sub-subtype A1 are circulating in Pakistan; however, an in-depth molecular phylogenetic characterization of HIV-1 sub-subtype A1 strains in Pakistan have not been presented. We performed a detailed characterization of the HIV-1 sub-subtype A1 epidemic in Pakistan using state-of-the-art molecular epidemiology and phylodynamics. (2) Methods: A total of 143 HIV-1 sub-subtype A1 gag sequences, including 61 sequences generated specifically for this study from PLHIVs part of our cohort, representing all sub-subtype A1 gag sequences from Pakistan, were analyzed. Maximum-likelihood phylogenetic cluster analysis was used to determine the relationship between Pakistani sub-subtype A1 strains and pandemic sub-subtype A1 strains. Furthermore, we used signature variation, charge distribution, selection pressures, and epitope prediction analyses to characterize variations unique to Pakistani HIV-1 strains and establish the association between signature variations and Gag epitope profile. (3) Results: The HIV-1 sub-subtype A1 sequences from Pakistan formed three main clusters: two that clustered with Kenyan sequences (7 and 10 sequences, respectively) and one that formed a Pakistan-specific cluster of 123 sequences that were much less related to other sub-subtype A1 sequences available in the database. The sequences in the Pakistan-specific cluster and the Kenyan reference strains exhibited several signature variations, especially at amino acid positions 312, 319, 331, 372, 373, 383, and 402. Structural protein modeling suggested that amino acid changes in these positions result in alterations of the Gag protein structure as well as in Gag-specific T-cell epitopes. (4) Conclusions: Our results suggest that the majority of the Pakistan HIV-1 sub-subtype A1 strains were unique to Pakistan and with a specific mutation pattern in Gag.

Analysis of temporal changes in HIV-1 CRF01_AE gag genetic variability and CD8 T-cell epitope evolution.

Currently, little is known about the time-dependent evolution of human immunodeficiency virus-1 (HIV-1) circulating recombinant forms (CRF) 01_AE, a dominant recombinant form associated with HIV-1 epidemics worldwide. Since gag is a highly immunodominant HIV-1 protein, we performed a comparative analysis of the CRF01_AE gag protein's time-dependent changes and evolution. A total of 3105 HIV-1 CRF01_AE gag sequences representing 17 countries from the timeline 1990-2017 were obtained. The sequences' phylogenetic relationship and epidemic dynamics were analyzed through a Maximum Likelihood tree and Bayesian Skyline plot, respectively. Genomic variability was measured through Shannon entropy and time-dependent immunoevolution was analyzed using changes in proteasomal degradation pattern, cytotoxic T lymphocytes (CTL) epitopes, and Human leukocyte antigens (HLA) restriction profile. The most recent common ancestor of the HIV CRF01_AE epidemic was estimated to be 1974±1. A period of exponential growth in effective population size began in 1982, fluctuated, and then stabilized in 1999. Genetic variability (entropy) consistently increased, however, epitope variability remained comparable; the highest number of novel CTL epitopes were present in 1995-1999, which were lost over time. The spread of the HIV-1 CRF01_AE epidemic is predominant in countries within Asia. Population immunogenetic pressures in the region may have contributed to the initial changes and following adaptation/stabilization of epitope diversity within gag sequences.

The emergence of an unassigned complex recombinant form in a Pakistani HIV-infected individual.

In Pakistan, the HIV situation has gone from an outbreak to a concentrated epidemic, and the virus has now crossed into the low-risk population. In addition, several new HIV outbreaks have occurred in different parts of the country. HIV-1 subtype A has been the major epidemic subtype in Pakistan; however, as the epidemic has grown, the emergence of several new subtypes and recombinant forms has been observed. Here, we present the first case and genetic analysis of an unassigned, complex recombinant form in a Pakistani HIV-infected individual with virological failure. Genetic analysis of the sequence indicated that this recombinant form is multi-drug resistant, harboring drug resistance mutations against more than one class of antiretroviral drugs.

Analysis of anti-microbial and anti-biofilm activity of hand washes and sanitizers against S. aureus and P. aeruginosa.

OBJECTIVE: To analyse the biofilm-forming potential of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa, and to assess antimicrobial activity of commonly used sanitizers in hospital and laboratory settings. METHODS: The study was conducted at Aga Khan University Karachi from August 2016 to January 2017. The biofilm-forming potential of Staphylococcus aureus and Pseudomonas aeruginosa clinical isolates were evaluated qualitatively using air-liquid interface tube method, and air-liquid interface cover slip assay. The antimicrobial activity of commonly-used hand-washes and sanitizers were assessed using agar well diffusion method, while the anti-biofilm activity of the hand-washes and sanitizers was qualitatively assessed using air-liquid interface covers lip as s ay. RESULTS: Of the eight hand-washes and sanitizers, 2(25%) showed antimicrobial activity against both Staphylococcus aureus and Pseudomonas aeruginosa, while 2(25%) exhibited antimicrobial activity against either S. aureus or P. aeruginosa. Also, 4 (50%) of them showed no inhibitory activity against S. aureus and P. aeruginosa. CONCLUSIONS: The findings shall have important consequences with regards to infection control in hospital and laboratory settings.

Emergence of HIV-1 Unique DG Recombinant Form in Pakistan.

After the first case of HIV infection in 1987, the number of cases has continuously increased in Pakistan, turning from isolated incidents to outbreaks to the concentrated epidemic. The HIV epidemic in Pakistan is mainly driven by subtype A; however, the overlapping transmission chains facilitate recombination between subtypes and existing circulating recombinants forms (CRFs), leading to the emergence of unique recombinant forms (URFs). In this study, we report the first case of a URF (URF_DG) in a Pakistani HIV-infected patient. Phylogenetic and drug resistance analysis of the patient-derived sequence indicated that Pakistani URF_DG sequence was closely related to the URF_DG sequence reported from the United Kingdom, but had more drug resistance mutations than the U.K. sequence.

Emergence of Circulating Recombinant Form 56_cpx in Pakistan.

In Pakistan, HIV has converted from outbreak to concentrated epidemic and has also bridged into the low-risk population. The HIV epidemic in Pakistan mainly comprises subtype A. Here, we present the first case and genetic analysis of a circulating recombinant form 56_cpx in a Pakistani HIV-infected patient. Genetic analysis of the sequence indicated that Pakistani 56_cpx sequence had more drug resistance mutation than the other 56_cpx sequences available in the database.