Genetic Diversity of Human Immunodeficiency Virus Type 1 in Asymptomatic Blood Donors in Islamabad, Pakistan.

Objective The serological testing of human immunodeficiency virus (HIV) is mandatory under the blood safety legislation of Pakistan; hence, data exist on the prevalence of HIV in blood donors. However, little is known about the molecular epidemiology of HIV in the blood donor population. Therefore, the current study was designed to study the genetic diversity of HIV-1 infection in a population of apparently healthy treatment-naive blood donors in Islamabad, Pakistan. Material and Methods A total of 85,736 blood donors were tested for HIV by the chemiluminescence immunoassay. All positive donor samples were analyzed for the presence of various HIV genotypes (types and subtypes). Viral ribonucleic acid was extracted from blood samples of HIV positive donors and reverse transcribed into complementary deoxyribonucleic acid (cDNA). The cDNA of all positive donors was then analyzed for the presence of various HIV genotypes (types and subtypes) by employing subtype-specific primers in a nested polymerase chain reaction. The amplified products were run on ethidium bromide-stained 2% agarose gel and visualized using a ultraviolet transilluminator. A particular subtype was assigned to a sample if the subtype-specific reaction made a band 20% highly intense compared with the band made by the subtype-independent reaction. Results A total of 85,736 blood donors were screened for the presence of antibodies to HIV. Out of them, 114 were initially found reactive for HIV. The repeat testing resulted in 112 (0.13%) positive donors, 95% confidence interval 0.0014 (0.0011-0.0018). These 112 samples were analyzed for molecular typing of HIV-1. The predominant HIV-1 subtype was A ( n = 101) (90.1%) followed by subtype B ( n = 11) (9.9%). Conclusion These findings are key to understand the diversified HIV epidemic at the molecular level and should assist public health workers in implementing measures to lessen the further dissemination of these viruses in the country.

Molecular epidemiology of hepatitis B virus genotypes in blood donors in Islamabad, Pakistan.

BACKGROUND: Hepatitis B virus (HBV) is a major causative agent of early, severe and prolonged liver infection that subsequently leads to cirrhosis of liver and hepatocellular carcinoma. The aim of this study was to evaluate the molecular epidemiology of hepatitis B virus (HBV) genotypes and comparison of serological assay performance versus polymerase chain reaction (PCR) in HBV screening. METHODS: Blood samples of 8517 healthy blood donors were collected during the period of January to June 2017 from Blood Bank of Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad. Samples were screened for HBsAg assay using technique of chemiluminescence immunoassay. PCR of positive samples was carried out using already reported genotype-specific primers by Naito et al. (2001). The results were confirmed by visualizing genotype bands. RESULTS: The study confirmed the presence of HBV in 2.5% of blood donors, and PCR confirmed the presence of HBV-DNA in 92 samples. The genotyping was done by PCR using type-specific primer sequences. PCR was dogged to check six genotypes, i.e., A, B, C, D, E, and F. The results of this study show high levels of Genotype D is this region, i.e., 52.17% with less dominating Genotype C, which is 16.30% with decreasing ratio of Genotype E (14.13%), Genotype A and B (9.78%), and mixed D + E (2.17%). The presence of coinfection is found at lowest rate. Due to the high percentage of HBV/D, it is concluded that D genotype is common in our population. CONCLUSION: The most prevalent HBV genotype in ICT region was genotype D, which is responsible for liver cirrhosis and hepatocellular carcinoma. Efficacy of drugs varies with variation in genotypes of hepatitis B virus and also with geographical distribution.

Evaluation of screening effectiveness of hepatitis B surface antigen and anti-HCV rapid test kits in Pakistan.

OBJECTIVE: The current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with chemiluminescence immunoassay (CLIA) and polymerase chain reaction (PCR). MATERIALS AND METHODS: This single-center, cross-sectional study was conducted at the Department of Pathology and Blood Transfusion Services, Shaheed Zulfiqar Ali Bhutto Medical University, PIMS, Islamabad, from January to April 2019. A total of 10 commercially available immunochromatographic test (ICT) devices and one CLIA kit (LIAISON XL) were tested for their sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using 100 positive and 100 negative samples each for HBV and HCV, respectively. RESULTS: The sensitivities and specificities of ICT kits for hepatitis B surface antigen were 65% and 70% (Hightop), 67% and 85% (RightSign), 62% and 73% (Wondfo), 70% and 80% (Accu-Chek), 68% and 77% (Fastep), 73% and 85% (Abon), 77% and 83% (ImmuMed), 80% and 90% (Insta-Answer), 67% and 81% (BioCheck), and 72% and 83% CTK Biotech, respectively. Similarly, the sensitivities and specificities of different ICT kits for HCV were 69% and 80% (Hightop), 76% and 83% (RightSign), 69% and 81% (Wondfo), 78% and 79% (Accu-Check), 68% and 68% (Fastep), 63% and 73% (Abon), 71% and 70% (ImmuMed), 79% and 68% (Insta-Answer), 62% and 66% (BioChek), and 69% and 78% CTK Biotech, respectively. The sensitivity and specificity of Diasorin Liaison Murex assay for both HBV and HCV were found to be 100% when compared with PCR. The PPV, NPV and Accuracy were determined accordingly. CONCLUSION: Rapid testing ICT devices for both HBV and HCV available in Pakistan were found to have a variable degree of sensitivity and specificity when compared with CLIA and PCR. Comparatively expensive but quality methods are more reliable as compared to rapid devices.

Internal quality control of blood products: An experience from a tertiary care hospital blood bank from Southern Pakistan.

INTRODUCTION: Internal quality control (IQC) is the backbone of quality assurance program. In blood banking, the quality control of blood products ensures the timely availability of a blood component of high quality with maximum efficacy and minimal risk to potential recipients. The main objective of this study is to analyze the IQC of blood products as an indicator of our blood bank performance. METHODS: An observational cross-sectional study was conducted at the blood bank of Liaquat National Hospital and Medical College, from January 2014 to December 2015. A total of 100 units of each blood components were arbitrarily chosen during the study. Packed red cell units were evaluated for hematocrit (HCT); random platelet concentrates were evaluated for pH, yield, and culture; fresh frozen plasma (FFP) and cryoprecipitate (CP) were evaluated for unit volume, factor VIII, and fibrinogen concentrations. RESULTS: A total of 400 units were tested for IQC. The mean HCT of packed red cells was 69.5 ± 7.24, and in 98% units, it met the standard (<80% of HCT). The mean platelet yield was 8.8 ± 3.40 × 109/L and pH was ≥6.2 in 98% bags; cultures were negative in 97% of units tested. Mean factor VIII and fibrinogen levels were found to be 84.24 ± 15.01 and 247.17 ± 49.69 for FFP, respectively. For CP, mean factor VIII and fibrinogen level were found to be 178.75 ± 86.30 and 420.7 ± 75.32, respectively. CONCLUSION: The IQC of blood products at our blood bank is in overall compliance and met recommended international standards. Implementation of standard operating procedures, accomplishment of standard guidelines, proper documentation with regular audit, and staff competencies can improve the quality performance of the transfusion services.

Detection of HBsAg mutants in the blood donor population of Pakistan.

BACKGROUND: Infection with the Hepatitis B virus (HBV) continues to be one of the leading healthcare issues in Pakistan, affecting over 6 million people. The existence of HBsAg mutants is well documented in many countries. In Pakistan, HBV screening in the majority of the blood banks is performed by Rapid Detection Devices or ELISA tests. These tests are designed to detect HBsAg, but may not detect the mutant HBsAg. Failure to detect the HBsAg mutant may result in the transmission of HBV infection from donor to recipient. Hence, there is a need to identify a HBsAg assay which can detect mutants in a country where simple and conventional HBsAg assays with varying sensitivity and specificity are used to detect HBV infections. MATERIAL AND METHODS: Three routinely used diagnostic tests (Rapid Detection Devices, ELISA and CLIA) for HBsAg were compared with the LIAISON® XL Murex HBsAg Quant Assay to determine the prevalence of HBV mutants in the Pakistani blood donor population. The samples of blood donors from different cities of Pakistan were collected. The testing was performed using SD Bioline rapid assay (n = 1500), ELISA (n = 1500), and Abbott ARCHITECT®CLIA system (n = 1500) at the centers where the donations were collected. All samples (n = 4500) were re-tested for comparative analysis on the LIAISON® XL Murex HBsAg Quant assay (DiaSorin S.p.A.). PCR testing was performed as a gold standard on all discordant samples. RESULTS: 119/4500 (2.64%) of the samples were positive for antibodies against HBsAg. The sensitivity of SD Bioline Rapid, GB HBsAg ELISA, Abbott ARCHITECT® and LIAISON® XL Murex HBsAg Quant assay was 17.24%, 43.75%, 90.91%and 100% respectively. The specificity of SD Bioline Rapid, GB HBsAg ELISA, Abbott ARCHITECT® and LIAISON® XL Murex HBsAg Quant Assay was 98.82%, 99.59%, 100% and 100%, respectively. CONCLUSION: LIAISON® XL Murex HBsAg Quant assay is a highly sensitive, specific and accurate screening assay for detecting wild type as well as mutant HBsAg.

Demographic and Clinical Characteristics of Adult Acute Myeloid Leukemia--Tertiary Care Experience.

BACKGROUND: Acute myeloid leukemia (AML) is an acquired clonal frequent malignant disorder of myeloid progenitor cells. Our aim was to study demographical and clinicopathological features of adult Pakistani AML patients at presentation. MATERIALS AND METHODS: In this single centre study extending from January 2010 to December 2014, data were retrieved from the patient records with a predetermined performa and analyzed with SPSS version 22. RESULTS: Overall 125 patients were diagnosed at our institution with de novo AML during the study period. There were 76 males and 49 females (ratio 1.5:1), with an age range between 15 and 85 years and a mean age of 38.8±20.1 years. The major complaints were fever (72.8%), generalized weakness (60%), bleeding (37.6%) and dyspnea (12%). Physical examination revealed pallor in 56.8%, splenomegaly and hepatomegaly in 16% and 12.8%, respectively, and lymphodenopathy in 10.4%. The mean hemoglobin was 8.19±2.12g/dl with a mean MCV of 86.0±9.83 fl, a mean total leukocyte count of 43.1±68.5x109/l, an ANC of 3.09±6.66x109/l and a mean platelet count of 62.3±78.6x109/l. CONCLUSIONS: AML in Pakistani patients is seen in a relatively very young population with male preponderance, compared with the west. However, clinico-pathological features appear comparable to published data.

Acute Myeloid Leukemia: Clinical Spectrum of 125 Patients.

BACKGROUND: Acute myeloid leukemia is an acquired clonal heterogeneous stem cell disorder. Hence, various parameters are sought out to categorize this disease into subtypes, so that as a consequence specific treatment modalities can be offered. Conventionally, the practically used method for classification utilizes French American British (FAB) criteria based on morphology and cytochemistry. The aim of present study was to determine the current spectrum of AML sub types in patients in Karachi. MATERIALS AND METHODS: This single centre cross sectional study was conducted at Liaquat National Hospital, Karachi, extending from January 2010 to December 2014. Data were retrieved from archives were analyzed with SPSS version 22. RESULTS: A total of 125 patients were diagnosed at our institution with de novo AML during five years period, 76 males and 49 females. Median age was 34.5 years. AML-M1 was the predominant FAB subtype (23.2%) followed by M2 (18.4%), M3 and M4 (16% each), M0 (14.4%), M5 (7.2%), M6 (3.2%) and M7 (1.6%). CONCLUSIONS: AML in Pakistani patients is seen in a relatively young population. The most common FAB subtype observed in our study was acute myeloblastic leukemia, without maturation (M1).

HIV prevalence in blood donors and recipients in Pakistan: a meta-analysis and analysis of blood-bank data.

BACKGROUND: The first instances of HIV-antibody detection in donated blood in Pakistan were reported in 1988. Since then, documentation of HIV in blood donors and of rates of transmission via transfusion has been limited. Previously assumed to have a low prevalence, HIV is an increasing health concern in Pakistan. Since there is no national, centralized blood-banking system, there are no reliable data on which to base estimated risks of transfusion-associated HIV infection. This study was therefore conducted to estimate the prevalence of HIV in blood donors and recipients in Pakistan between 1988 and 2012. METHODS: Meta-analyses were undertaken of reported prevalences of HIV in blood donors and recipients published during 1988-2012. Papers were identified by searching PubMed, Google, CINAHL and PakMediNet and the websites of the World Health Organization, the national HIV/AIDS Surveillance Project and the National AIDS Control Programme of Pakistan. In addition, the 1998-2012 records of the Aga Khan University blood bank were analysed. RESULTS: The 254 abstracts identified at the preliminary search were reviewed and, after removal of duplications, case-reports, editorials and reviews, 32 papers were selected that met the inclusion criteria. All studies that reported on HIV antibodies in blood donors/recipients were included, irrespective of the methodology used. Since seroconversion had only been confirmed through supplemental testing in a few papers, the results were analysed separately for reports based on screening only and confirmed cases. A total of 142 of 2 023 379 blood donors and 4 of 3632 blood recipients were HIV positive, giving an overall pooled seroprevalence of 0.00111% in blood donors and 0.00325% in blood recipients. The annual prevalences of HIV in donors at the Aga Khan University blood banks were similar, ranging from 0.013% to 0.116%. CONCLUSION: Very few reports on HIV in blood donors in Pakistan could be retrieved, and the overall pooled prevalence is low. However, the limited data and confounding factors mean that that these results may significantly underestimate the true situation. It is recommended that a complete survey of blood banks should be conducted throughout the country, in order to provide a more reliable estimate of the risk of transfusion-associated HIV infection in Pakistan.

Evaluation of HIV/AIDS diagnostics kits and formulation of a testing strategy for Pakistan.

BACKGROUND: Rapid diagnosis of HIV/AIDS enables the development of prevention and treatment programmes but accurate, reliable and cost effective testing strategies should be used for testing of HIV/AIDS from a large population. OBJECTIVE: To evaluate the performance and effectiveness of three assays for the diagnosis of HIV in comparison with Western blot and to formulate an alternative cost-effective confirmatory approach for HIV diagnosis. STUDY DESIGN: 472 specimens (serum) from a Pakistani population were evaluated. Two rapid HIV testing kits (Capillus, SD Bioline) and one ELISA (Vironostika Ag/Ab) kit were used to detect HIV. Results were compared with Western blot against which sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of all HIV assays were assessed. RESULTS: 280/472 (59.3%) of the samples were positive for antibodies against purified HIV-1 viral proteins. The sensitivity of SD Bioline and Vironostika ELISA was 100% (95% CI; 98-100) while that of anti-HIV Capillus™ kit was 94.6% (95% CI; 91-96.8). The specificity of the Vironostika ELISA and anti-HIV Capillus™ kit was 100% (95% CI; 97-100) while specificity of SD Bioline was 98.4% (95% CI; 95-99). PPV was 100% (95% CI; 98-100%) for the anti-HIV Capillus™ and Vironostika ELISA and 98.9% (95% CI; 96-99%) for SD Bioline. NPV for SD Bioline and Vironostika ELISA was 100% (95% CI; 98-100%) and 92.7% for anti-HIV Capillus™ (95% CI; 88-96%). CONCLUSION: The sensitivity and specificity of all three kits were satisfactory compared to Western blot and could be used for effective diagnosis of HIV/AIDS in Pakistani population.

Trends in transfusion transmitted infections among replacement blood donors in karachi, pakistan.

OBJECTIVE: To determine the prevalence of Hepatitis-B, Hepatitis-C and Human Immunodeficiency infections in replacement blood donors. MATERIALS AND METHODS: From January 2004 to December 2011, 108,598 apparently healthy donors donated blood at our Blood Bank. Screening was done by Microparticle Enzyme Immuno Assay (MEIA) method on Axsym System (Abbott Diagnostic, USA) and in year 2011 by Chemiluminescent Immunoassay (CIA) method on Architect i2000 (Abbott Diagnostic, USA). From 2010 onward, HIV reactive donors were advised for confirmatory tests and reported back with the results. RESULTS: Of the 108,598 total donors, 108,393 (99.8%) were replacement donors with a mean age of 28.92 (17-55) years. Of this, only 164 (0.15%) were females. Among the replacement donors, 4,906 (4.5%) were found to be reactive for Hepatitis-B, C and Human Immunodeficiency Virus. All the reactive patients, except one, were males. HbsAg was positive in 2,068 (1.90%) and anti-HCV in 2832 (2.61%) donors, while 111 (0.10%) were positive for Human Immunodeficiency Virus. Co-infectivity was observed in 103 (0.09%) cases. The prevalence appeared to be higher in younger age group (17-30 yrs). Only 16.6% cases should be patients returned with results of the confirmatory tests for HIV and were found positive. CONCLUSION: Hepatitis-B and C sero-prevalence in our series of replacement donors appears high compared to most studies from neighboring countries and relatively low in comparison to earlier studies from Pakistan. Prevalence of HIV, however, appears low and turn out of HIV positive cases for confirmatory tests is low. CONFLICT OF INTEREST: None declared.

Managing research evidence to inform action: influencing HIV policy to protect marginalised populations in Pakistan.

Abstract Our aim was to demonstrate that an understanding of the process of how research may (or may not) influence policy and practice is crucial to leverage research findings and bring about evidence-informed policy and its implementation. We describe a process of research design and execution, based on theories of the relationship between evidence and public policy-making, which sought to improve the uptake of evidence into the HIV policy-making process in Pakistan. We designed and implemented specific strategies in research methods, management and dissemination to increase the policy influence by recommendations from a multi-disciplinary research project. Research to policy is complex, rarely linear and causal attribution is problematic. Nonetheless, we believe that, in part, some of the current changes in HIV policy and practice in Pakistan may be due to the managed process of research influence. We offer four key recommendations for those concerned with improving the chances of seeing their research incorporated into policy and practice - these are (1) involve stakeholders in research management; (2) set realistic expectations of research impact; (3) invest in long-term research-policy-maker relationships; and (4) build capacity of end users to use research to demand policy change.