Probabilistic measures of HIV-1 transmission in different HIV-1 key population groups of Larkana, Pakistan.

OBJECTIVE: To estimate the probability of human immunodeficiency virus (HIV)-1 transmission from different key HIV population groups using probabilistic modelling. METHODS: This study was conducted in December 2020. A probabilistic model was used to estimate the probability of HIV-1 transmission from different key HIV population groups in Larkana. Our model was run on three probabilistic assumptions: 1) each replication gave two conceivable results: 'true' or 'false'; 2) the chance of giving a 'true' result is the same for each replication; and 3) the replications are independent - 'true' in one will not impact the likelihood of 'true' in another. RESULTS: The results estimated the probability of HIV transmission in key HIV population groups in Larkana to range between 0.42-0.54 per trial, where the highest probability of transmission was predicted for men who have sex with men (MSM; 0.54 per trial), followed by transgender (TG; 0.46 per trial) and people who inject drugs (PWID; 0.457 per trial). CONCLUSIONS: Our results suggest that there is a high likelihood of HIV transmission by key population groups in Larkana, such as MSM, TG, and PWID. Mathematic models, such as one proposed in our study can aid the HIV and acquired immunodeficiency syndrome (AIDS) control programmes in evaluating and optimising the strategies in controlling transmission of HIV from the key population groups.

Low-cost thermotherapy for cutaneous leishmaniasis in Sindh, Pakistan.

OBJECTIVE: To study the efficacy of a low-cost heating pack device used for thermotherapy in the treatment of cutaneous leishmaniasis. METHODS: The study was conducted at the Department of Dermatology, Civil Hospital Sukkur, Pakistan, from April 20, 2012, to January 3, 2013. Thermotherapy with Hand-Held Exothermic Crystallisation Therapy for cutaneous leishmaniasis was performed on each lesion of the participating subjects at an average initial temperature of 51.6°C for 3 minutes daily for 7 days. Patients were followed regularly for 6 months after the therapy. SPSS 20 was used for statistical analysis. RESULTS: Even though all 27 patients completed 1 week of thermotherapy, only 23(85.2%) patients could be evaluated for full treatment response since 4(14.8%) were lost to complete follow-up. By the final 180-day evaluation, 19 (83%) patients had been cured. Applications were well tolerated with no side effects. CONCLUSION: The devise was a convenient, safe, non-toxic and effective treatment for cutaneous leishmaniasis at a fraction of the cost of standard antimonial treatment. Further studies are needed to certify its safety and efficacy as monotherapy for the condition.

Identification of a novel first-generation HIV-1 circulating recombinant form (CRF152_DG) among people living with HIV in Karachi, Pakistan.

The objective of this study was to characterize a novel circulating recombinant form of human immunodeficiency virus type 1 (HIV-1) among people living with HIV in Karachi, Pakistan. We conducted near-full-length genome (NFLG) sequencing on eight samples exhibiting D/G recombination signals in the pol gene region. We successfully obtained NFLG sequences (790-9,614; with reference to the HXB2 genome) from four of the eight samples and then conducted phylogenetic and recombination analyses on them. The four NFLG sequences from our study and one DG unique recombinant form previously identified in the United Kingdom (GenBank accession: MF109700) formed a distinct monophyletic cluster with an Shimodaira-Hasegawa approximate likelihood ratio test node support value of 100%. Bootscan analyses of the five NFLG sequences of DG recombinants showed that all five NFLGs shared the same unique mosaic pattern of recombination breakpoints between D and G clades, with two D fragments in the pol and vif regions inserted into a G backbone. Subregion phylogenetic analyses confirmed these sequences to be a novel circulating recombinant form (CRF) composed of subtypes D and G. The DG recombinant sequences were eventually designated as CRF152_DG by the Los Alamos HIV Sequence Database staff. IMPORTANCE: In Pakistan, the genetic diversity of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly complex, compared to the early years of the epidemic that started after the detection of the first cases of HIV-1 in 1987 in Karachi. Based on the available molecular studies, two dominant HIV-1 clades, sub-subtype A1 and CRF02_AG, have been found to co-circulate with other clades, namely B, C, D, G, CRF01_AE, CRF35_A1D, and CRF56_cpx, in various urban areas of Pakistan. Several novel recombinant forms have also been detected. This first report of CRF152_DG highlights the complex nature of the HIV epidemic in Pakistan and emphasizes the importance of continual molecular surveillance (ideally based on whole-genome sequences) of HIV.

Human Immunodeficiency Virus Type-1 Genetic Diversity and Drugs Resistance Mutations among People Living with HIV in Karachi, Pakistan.

The human immunodeficiency virus type-1 epidemic in Pakistan has significantly increased over the last two decades. In Karachi, Pakistan, there is a lack of updated information on the complexity of HIV-1 genetic diversity and the burden of drug resistance mutations (DRMs) that can contribute to ART failure and poor treatment outcomes. This study aimed to determine HIV-1 genetic diversity and identify drug-resistance mutations among people living with HIV in Karachi. A total of 364 HIV-positive individuals, with a median age of 36 years, were enrolled in the study. The HIV-1 partial pol gene was successfully sequenced from 268 individuals. The sequences were used to generate phylogenetic trees to determine clade diversity and also to assess the burden of DRMs. Based on the partial pol sequences, 13 distinct HIV-1 subtypes and recombinant forms were identified. Subtype A1 was the most common clade (40%), followed by CRF02_AG (33.2%). Acquired DRMs were found in 30.6% of the ART-experienced patients, of whom 70.7%, 20.7%, and 8.5% were associated with resistance to NNRTIs, NRTIs, and PIs, respectively. Transmitted DRMs were found in 5.6% of the ART-naïve patients, of whom 93% were associated with resistance against NNRTIs and 7% to PIs. The high prevalence of DRMs in ART-experienced patients poses significant challenges to the long-term benefits and sustainability of the ART program. This study emphasizes the importance of continuous HIV molecular epidemiology and drug resistance surveillance to support evidence-based HIV prevention, precise ART, and targeted AIDS care.

Determinants of HIV sero-conversion among male injection drug users enrolled in a needle exchange programme at Karachi, Pakistan.

OBJECTIVE: To assess the determinants of HIV sero-conversion among male injection drug users enrolled in needle exchange programme at Karachi. METHODS: An unmatched retrospective case control study was conducted among male injection drug users receiving needle exchange services in Karachi. The cases and controls were identified from one drop in center providing needle exchange services. The data for the study participants was collected retrospectively from the programme. Descriptive statistics, univariat analysis, and multivariate regression analysis for determinants of HIV sero-conversion and Hosmer and Lameshow goodness of fit test for model adequacy were performed. RESULTS: Mean age of the study participants was 34.17 +/- 10.74 years. Average monthly income of the participants was US$ 125.15 +/- 76.32. In unconditional multivariate regression analysis being unmarried (AOR: 3.0 95% CI 1.14-7.9, p=0.02), not living with family (AOR: 2.8 95% CI 1.18-6.79 p = 0.02), family history of addiction (AOR: 2.5, 95% CI 1.01-6.49, p = 0.04), injecting drugs in groups (AOR: 2.8, 95% CI 1.12 7.02 p=0.02), not obtaining syringes from the programme (AOR: 26.45, 95% CI 2.47-282.8 p = 0.007), and history of blood transfusion (AOR: 52.9, 95% CI 1.32-2118.416 p = 0.03) were significantly associated with HIV positive sero-status. Model adequacy was assessed by Hosmer and Lameshow goodness of (J: 4.95, p = 0.7) indicating that the model was accurate. CONCLUSION: Social and drug related risky behaviours are important determinants of HIV among male IDUs in Karachi. The situation calls for programmematic initiatives for addressing the risky behaviours among IDUs for effective control of epidemic in the country.

Genetic and antiretroviral drug resistance mutations analysis of reverse transcriptase and protease gene from Pakistani people living with HIV-1.

BACKGROUND: Antiretroviral therapy (ART) effectiveness is compromised by the emergence of HIV drug resistance mutations (DRM) and can lead to the failure of ART. Apart from intrinsic viral factors, non-compliance with drugs and/or the use of sub-optimum therapy can lead to the emergence of DRMs. In Pakistan HIV currently exists as a concentrated epidemic, however, ART coverage is very low, and drug adherence is poor. ART is selected assuming without baseline genotyping. Pakistan has recently seen a rise in treatment failures, but the country's actual burden of DRM is still unknown. In this study, we perform the genetic and drug resistance analysis of the pol gene from Pakistani HIV-positive ART-naïve and ART-experienced individuals. METHODS: In this study, HIV-1 pol was sequenced from 146 HIV-1 positive individuals, divided into ART-naïve (n = 37) and ART-experienced (n = 109). The sequences were also used to determine HIV-1 subtypes, the prevalence of DRM, and pol genetic variability. RESULTS: DRM analysis identified numerous DRMs against reverse transcriptase inhibitors in both ART-naïve and ART-experienced groups, including a few that are classified as rare. Additionally, the ART-experienced group showed mutations associated with resistance to protease inhibitors. Genetic analysis showed negative selection pressure in both groups, but a higher rate of evolution in the ART-naïve group. CONCLUSION: High prevalence of DRMs, especially against previous first-line treatment in ART- naïve and the accumulation of DRMs in ART-experienced groups is concerning and warrants that a more extensive DRM survey be carried out to inform first-line and second-line ART regimen recommendations.

Viral infections in Pakistan: prevalence, factors affecting spread, and recommendations for control.

Pakistan is endemic to a number of viral infections, owing to its humid climate, topographical variation, soaring population, and lack of education and awareness. These viruses may have several different modes of transmission, including respiratory or airborne transmission, sexual transmission, blood-borne, fecal-oral transmission, vector-borne transmission, and transmission following an organ transplant. Although several different microorganisms are responsible for causing these infections, a few viruses are found more commonly in Pakistan and are primarily responsible for causing infections. In this study, we present a review of the most recent studies on different viruses, transmitted through various transmission routes, found commonly in Pakistan, along with the prevalence of each, and recommend control measures required against these viruses.

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.

Unassigned Complex Unique Recombinant Forms Related to CRF36_cpx in Children Identified in an HIV-1 Outbreak in Pakistan.

In 2019, an outbreak of HIV infection predominantly affecting children occurred in Larkana district, Pakistan. This is the largest outbreak ever reported in this age group in Pakistan. In this study, we report two HIV-1 unique recombinant forms identified during the outbreak. Blood samples were collected from HIV-positive children as part of a case-control study to investigate the outbreak. The pol gene was sequenced and used to detect HIV subtype/recombinant forms using subtype, recombination, and phylogenetic analyses. Drug resistance mutation (DRM) analysis was performed to characterize the DRMs in each sequence. We observed the emergence of two unassigned unique recombinant forms related to CRF36_cpx in 15 individuals of the 344 samples collected. Genotype analysis revealed the presence of multiple DRMs associated with resistance to reverse transcriptase inhibitors. The discovery of these unassigned unique recombinant forms in our population highlights the need for comprehensive molecular epidemiological studies to fully understand the distribution and drug resistance patterns to aid control efforts.

Phylogenetic and drug- and vaccine-resistance profiles of Hepatitis B Virus among children with HIV co-infection in Pakistan.

INTRODUCTION: HIV-1 and hepatitis B virus (HBV) share common routes of transmission and therefore co-infection is common. In 2019, an HIV-1 outbreak that resulted in >1000 children being infected, predominantly through nosocomial transmission, occurred in Sindh, Pakistan. We conducted a phylogenetic and drug resistance analysis of the HBV Reverse Transcriptase (RT) gene in children with HIV-1 and HBV co-infection. METHODOLOGY: Blood samples were collected from 321 children with HIV who were recruited as part of a study to investigate the HIV-1 outbreak. All samples were tested for HBV surface antigen (HBsAg) using an ELISA assay, and positive samples were used to amplify and sequence the HBV RT gene. The phylogenetic relationship between sequences was analyzed, and drug- and vaccine- resistance mutations in the RT gene were explored. RESULTS: Of 321 samples, 23% (n = 75) were positive for HBsAg on ELISA. Phylogenetic analysis of the sequences revealed that 63.5% of HBV sequences were sub-genotype D1, while the rest were sub-genotype D2. Cluster analysis revealed grouping of sub-genotype D1 sequences exclusively with Pakistani sequences, while clustering of sub-genotypes D2 predominantly with global sequences. The 236Y mutation associated with resistance to tenofovir was observed in 2.8% of HBV sequences. Additionally, seven vaccine escape mutations were observed, the most common being 128 V. CONCLUSION: Our study suggests ongoing transmission of HBV D1 and D2 sub-genotypes in the HIV-1 co-infected population, likely nosocomially, given common routes of HVB and HIV-1 transmission. The prevalence of major HBV drug- and vaccine-resistant mutations remains low. Surveillance for further transmissions and the possible emergence of major drug- or vaccine-resistant variants is required.

Prevalence of syphilis among antenatal clinic attendees in Karachi: imperative to begin universal screening in Pakistan.

OBJECTIVES: To assess the prevalence and associated risk factors of syphilis among antenatal clinic attendees by a multi-center cross-sectional study in Karachi, Pakistan. METHODS: We administered a structured questionnaire and obtained a blood sample for syphilis serology (rapid plasma reagin test with Treponema pallidum hemagglutination assay confirmation) from all women giving informed consent over six weeks in 2007. Prevalence was calculated at 95% confidence intervals. Multivariate analysis was adapted to assess risk factors. RESULTS: There were seven (0.9%) confirmed cases of syphilis (95% CI: 0.4, 1.8) in a sample size of 800 women recruited from three urban sites (-1% refusal rate). Women who lived in an area where male drug use is prevalent had 1.5% higher prevalence rates than women from the other two sites 0.5%. CONCLUSIONS: We documented higher-than-expected syphilis seroprevalence rates in a low risk population of antenatal clinic attendees in Pakistan. Bridge populations for syphilis may include drug users, who are usually married, and Hijras or their clients. In accordance with our results, the national policy for syphilis control in Pakistan should be modified to include universal syphilis screening in antenatal clinics with subsequent partner notification.

Pattern of sexually transmitted infections in males in interior Sindh: a 10-year-study.

BACKGROUND: Sexually transmitted infections (STIs) are widespread in Pakistan and have not been fully documented particularly in Sindh Province. The aim of this study is to determine the number and clinical pattern of various types of STIs in general population of Larkana division and its surrounding cities. METHODS: A hospital based prospective study was carried out at Male-STD-Clinic in the Department of Dermatology, Shaheed Muhtarma Benazir Bhutto Medical University Hospital Larkana from January 2000 to December 2009. RESULTS: Among 4,288 patients, 3,947 (92.04%) had the history of extra marital sexual contact and simultaneously had developed the clinical signs of STIs; 341 (7.95%) had history of extra marital sexual contact but did not have the manifestation of STIs. Majority of the patients (3,860, 90.01%) had the history of heterosexual contact with different partners, but only few 171 (3.98%) of them had the history of homosexual contact. According the syndromic diagnosis 1930 (45.00%) patients had genital ulcer (including herpes genitals) with or without skin manifestations, 690 (16.09%) had urethral discharge, 431 (10.05%) had genital warts, 349 (8.14%) had lesions other than STIs related, 304 (7.08%) had more than one syndrome, 193 (4.50%) had scrotal swelling, 46 (1.07%) had inguinal bubo, 3 (0.06%) were human immunodeficiency virus (HIV) positive, and 1 (0.02%) had ophthalmia neonatorum. Based on the clinical and etiological grounds: 2560 (59.70%) had syphilis, 640 (14. 92%) had gonorrhoea, 399 (9.30%) had mixed infections, 40 (0.93%) had chancroid, 431 (10.05%) had genital warts, 40 (0.93%) had lymphogranuloma venerum (LGV) and granuloma inguinale (GI), 3 (0.06%) were HIV positive, 208 (4.85%) had genital herpes, 120 (2.79%) had orchitis, 56 (1.30%) had non gonococcal urethritis (chlamydia were 19), and 1 (0.02%) had ophthalmia neonatorum. CONCLUSIONS: Mode of transmission of STIs in this region is mainly by heterosexual contact and syphilis is the commonest followed by gonorrhoea.

Factors associated with HIV infection among children in Larkana District, Pakistan: a matched case-control study.

BACKGROUND: In April, 2019, an HIV outbreak predominantly affecting children occurred in Larkana District, Sindh, Pakistan. By December, 2019, 881 (4·0%) of 21 962 children screened for HIV had tested positive. We aimed to assess factors associated with HIV infection in this outbreak. METHODS: In this individually matched case-control study, we sampled 406 cases (individuals aged <16 years who had registered for paediatric HIV care at the HIV Treatment Centre at Shaikh Zayed Children's Hospital in Larkana City, Pakistan) and 406 controls (individuals without HIV matched by age, sex, and neighbourhood residence, recruited through doorknocking at houses adjacent to case participants). An interviewer-administered questionnaire was used to collect data on possible risk factors for HIV acquisition and a blood sample was collected from all participants for hepatitis B and hepatitis C serology. Mothers of all participants underwent HIV testing. Odds ratios were estimated using conditional logistic regression to assess factors associated with HIV infection. FINDINGS: 406 case-control pairs were recruited between July 3 and Dec 26, 2019. Five pairs were excluded (three pairs had an age mismatch and two pairs were duplicate cases) and 401 were analysed. The prevalence of hepatitis B surface antigen was 18·2% (95% CI 14·5-22·3) among cases and 5·2% (3·3-7·9) among controls, and the prevalence of hepatitis C antibodies was 6·5% (95% CI 4·3-9·4) among cases and 1·0% (0·3-2·5) among controls. 28 (7%) of 397 mothers of cases for whom we had data, and no mothers of 394 controls, were HIV positive. In the 6 months before recruitment, 226 (56%) of 401 cases and 32 (8%) of 401 controls reported having more than ten injections, and 291 (73%) cases and 78 (19%) controls had received an intravenous infusion. At least one blood transfusion was reported in 56 (14%) cases and three (1%) controls in the past 2 years. HIV infection was associated with a history of more injections and infusions (adjusted odds ratio 1·63; 95% CI 1·30-2·04, p<0·0001), blood transfusion (336·75; 23·69-4787·01, p<0·0001), surgery (399·75, 13·99-11 419·39, p=0·0005), the child's mother being HIV positive or having died (3·13, 1·20-8·20, p=0·020), and increased frequency of private clinic (p<0·0001) and government hospital visits (p<0·0001), adjusting for confounders. INTERPRETATION: The predominant mode of HIV transmission in this outbreak was parenteral, probably due to unsafe injection practices and poor blood safety practices. General practitioners across Pakistan need training and systems support in reducing injection use, and in providing safe injections and transfusions only when necessary. FUNDING: Department of Pediatrics and Child Health, the Aga Khan University, Karachi, Pakistan.

Phylogenetic and Drug-Resistance Analysis of HIV-1 Sequences From an Extensive Paediatric HIV-1 Outbreak in Larkana, Pakistan.

INTRODUCTION: In April 2019, an HIV-1 outbreak among children occurred in Larkana, Pakistan, affecting more than a thousand children. It was assumed that the outbreak originated from a single source, namely a doctor at a private health facility. In this study, we performed subtype distribution, phylogenetic and drug-resistance analysis of HIV-1 sequences from 2019 outbreak in Larkana, Pakistan. METHODS: A total of 401 blood samples were collected between April-June 2019, from children infected with HIV-1 aged 0-15 years recruited into a case-control study to investigate the risk factors for HIV-1 transmission. Partial HIV-1 pol sequences were generated from 344 blood plasma samples to determine HIV-1 subtype and drug resistance mutations (DRM). Maximum-likelihood phylogenetics based on outbreak and reference sequences was used to identify transmission clusters and assess the relationship between outbreak and key population sequences between and within the determined clusters. Bayesian analysis was employed to identify the time to the most recent common recent ancestor (tMRCA) of the main Pakistani clusters. RESULTS: The HIV-1 circulating recombinant form (CRF) 02_AG and subtype A1 were most common among the outbreak sequences. Of the treatment-naïve participants, the two most common mutations were RT: E138A (8%) and RT: K219Q (8%). Four supported clusters within the outbreak were identified, and the median tMRCAs of the Larkana outbreak sequences were estimated to 2016 for both the CRF02_AG and the subtype A1 clusters. Furthermore, outbreak sequences exhibited no phylogenetic mixing with sequences from other high-risk groups of Pakistan. CONCLUSION: The presence of multiple clusters indicated a multi-source outbreak, rather than a single source outbreak from a single health practitioner as previously suggested. The multiple introductions were likely a consequence of ongoing transmission within the high-risk groups of Larkana, and it is possible that the so-called Larkana strain was introduced into the general population through poor infection prevention control practices in healthcare settings. The study highlights the need to scale up HIV-1 prevention programmes among key population groups and improving infection prevention control in Pakistan.

Types and risk factors of violence experienced by people living with HIV, Pakistan: a cross-sectional study.

BACKGROUND: In Pakistan, data are lacking on the violence experienced by people living with HIV. AIMS: This study determined the prevalence and risk factors of violence (physical, psychological and sexual) in people living with HIV in Karachi, Pakistan. METHODS: This was a cross-sectional study in 2016 of people living with HIV attending clinics of Bridge Consultants Foundation, a community-based care provider. Date were collected using an interview-based questionnaire. Multivariate logistic regression analysis was done to assess the risk factors for violence with adjusted odds ratios (aOR) and 95% confidence intervals (CI) presented. RESULTS: The sample included 250 people living with HIV; 183 were men, 60 were women and 7 were transgender. The mean (standard deviation) age of the participants was 30 (6.5) years. The prevalence rates of psychological, sexual and physical violence were 79.6%, 74.8% and 64.4%, respectively. More women experienced physical violence than men (76.2% versus 60.7%). Psychological violence was associated with injecting drug use (aOR = 2.64, 95% CI: 1.27-5.50) and being married (aOR = 0.46, 95% CI: 0.24-0.90). Marriage (aOR = 2.30, 95% CI: 1.27-4.16) and having an HIV-positive partner (aOR = 2.07, 95% CI: 1.09-3.92) were risk factors for sexual violence. Physical violence was associated with young age (aOR = 0.95; 95% CI: 0.91-0.99) and having an HIV-positive partner (aOR = 2.17, 95% CI: 1.10-4.26). CONCLUSION: Violence is an important public health problem affecting people living with HIV in Pakistan. This issue needs to be addressed by the government and nongovernmental organizations.