Tariq Moatter

Virological and clinical characteristics of hepatitis delta virus in South Asia

Background & AimsThere is a paucity of data on the impact of hepatitis D virus (HDV) in patients with hepatitis B virus (HBV) infection from South Asia. We studied the impact of HDV co-infection on virological and clinical characteristics.MethodsWe collected data of 480 patients with HBsAg positive and a detectable HBV DNA PCR, who presented to the Aga Khan University, Karachi and Isra University in Hyderabad, Pakistan in the last 5 years. HDV co-infection was diagnosed on the basis of anti-HDV. ALT, HBeAg, HBeAb and HBV DNA PCR quantitative levels were checked in all patients. We divided all patients into two groups based on anti-HDV, and compared their biochemical, serological & virological labs and clinical spectrum. Clinical spectrum of disease included asymptomatic carrier (AC), chronic active hepatitis (CAH), immuno-tolerant phase (IP), and compensated cirrhosis (CC).ResultsHDV co-infection was found in 169 (35.2%). There were 164 (34.6%) HBeAg positive and 316 (65.4%) HBeAg negative patients. Mean ALT level was 66 ± 73 IU. 233 (48.5%) had raised ALT. HBV DNA level was ≥ 10e5 in 103(21.5%) patients. Overall, among HBV/HDV co-infection, 146/169 (86.4%) had suppressed HBV DNA PCR as compared to 231/311 (74.3%) patients with HBV mono-infection; p-value = 0.002. Among HBeAg negative patients 71/128(55.5%) had raised ALT levels among HBV/HDV co-infection as compared to 71/188 (37.8%) with HBV mono-infection (p-value = 0.002); levels of HBV DNA were equal in two groups; there were 27/128 (21%) patients with CC among HBV/HDV co-infection as compared to 23 (12%) in HBV mono-infection (p-value = 0.009); there were less AC (p-value = 0.009) and more CAH (p-value = 0.009) among HBV/HDV co-infection patients. Among HBeAg positive patients, serum ALT, HBV DNA levels and the spectrum of HBV were similar in the two groups.ConclusionsHBV/HDV co-infection results in the suppression of HBV DNA. A fair proportion of HBV/HDV co-infected patients with HBeAg negative have active hepatitis B infection and cirrhosis as compared to those with mono-infection.

Estimating window period blood donations for human immunodeficiency virus Type 1, hepatitis C virus, and hepatitis B virus by nucleic acid amplification testing in Southern Pakistan.

Recently, strategic planning was initiated by the National Blood Transfusion Services Pakistan to improve its blood bank facilities. Emphasis has been placed on appropriate screening of blood products. Located in the southern region, Aga Khan University Hospital is a 700‐bed tertiary care academic institute with comprehensive blood banking. Screening of blood donors has been based on verbal screening and serologic testing to date. Additionally, the need of implementing nucleic acid testing (NAT) was considered in 2011 because of an upsurge in hepatitis epidemiology. The aim of this study was to analyze the efficacy of this additional donor screening program and to evaluate the impact of NAT on the yield and residual risk of transfusion‐transmissible viral infections.

Hemoglobin E syndromes in Pakistani population

BackgroundHemoglobin E is an important hemoglobin variant with a worldwide distribution. A number of hemoglobinopathies have been reported from Pakistan. However a comprehensive description of hemoglobin E syndromes for the country was never made. This study aimed to describe various hemoglobin E disorders based on hematological parameters and chromatography. The sub-aim was to characterize hemoglobin E at molecular level.MethodsThis was a hospital based study conducted prospectively for a period of one year extending from January 1 to December 31, 2008. EDTA blood samples were analyzed for completed blood counts and hemoglobin variants through automated hematology analyzer and Bio-Rad beta thalassaemia short program respectively. Six samples were randomly selected to characterize HbE at molecular level through RFLP-PCR utilizing MnlI restriction enzyme.ResultsDuring the study period, 11403 chromatograms were analyzed and Hb E was detected in 41 (or 0.36%) samples. Different hemoglobin E syndromes identified were HbEA (n = 20 or 49%), HbE/β-thalassemia (n = 14 or 34%), HbEE (n = 6 or 15%) and HbE/HbS (n = 1 or 2%). Compound heterozygosity for HbE and beta thalassaemia was found to be the most severely affected phenotype. RFLP-PCR utilizing MnlI successfully characterized HbE at molecular level in six randomly selected samples.ConclusionsVarious HbE phenotypes are prevalent in Pakistan with HbEA and HbE/β thalassaemia representing the most common syndromes. Chromatography cannot only successfully identify hemoglobin E but also assist in further characterization into its phenotype including compound heterozygosity. Definitive diagnosis of HbE can easily be achieved through RFLP-PCR.

Molecular analysis of human leukocyte antigen class I and class II allele frequencies and haplotype distribution in Pakistani population.

AIM: Distribution of HLA class I and II alleles and haplotype was studied in Pakistani population and compared with the data reported for Caucasoid, Africans, Orientals and Arab populations. MATERIALS AND METHODS: HLA class I and II polymorphisms in 1000 unrelated Pakistani individuals was studied using sequence-specific primers and polymerase chain reaction and assay. RESULTS: The most frequent class I alleles observed were A*02, B*35 and CW*07, with frequencies of 19.2, 13.7 and 20%, respectively. Fifteen distinct HLA-DRB1 alleles and eight HLA-DQB1 alleles were recognized. The most frequently observed DRB1 alleles which represented more than 60% of the subjects were DRB1 *03, *07, *11 and *15. The rare DRB1 alleles detected in this study were HLADRB1 *08 and *09, having frequencies of 0.9 and 1.7%, respectively. In addition, at DRB1-DQB1 loci there were 179 different haplotypes and 285 unique genotypes and the most common haplotype was DRB1*15-DQB1*06 which represented 17% of the total DRB1-DQB1 haplotypes. In our population, haplotype A*33-B*58-Cw*03 comprised 2.8% of the total class I haplotypes observed. This haplotype was seen only in the oriental populations and has not been reported in the African or European Caucasoid. CONCLUSION: Our study showed a close similarity of HLA class I and II alleles with that of European Caucasoid and Orientals. In Pakistani population, two rare loci and three haplotypes were identified, whereas haplotypes characteristic of Caucasians, Africans and Orientals were also found, suggesting an admixture of different races due to migration to and from this region.

Distribution of EGFR mutations commonly observed in primary Lung Adenocarcinomas in Pakistan as predictors for targeted therapy

BACKGROUNDnAcquired genetic alterations and presence of sensitizing mutations in the tyrosine kinase domain of EGFR and other signaling molecules have been found in different subsets of primary lung adenocarcinoma. The commonest EGFR mutations are small in frame deletions of exon 19 and a point mutation (L858R) in exon 21, having a combined occurrence of around 90%. The objective of this study was to determine the frequency and types of EGFR mutations in primary lung adenocarcinomas in Pakistan.nnnMATERIALS AND METHODSnEGFR mutations in tumor samples were screened by multiplex real time PCR. Briefly, DNA from formalin fixed paraffin-embedded tissue was amplified with primers and probes specific to 43 different EGFR mutations in a Cobas z 480 instrument. The assay detects mutations in four exons (18-21) of the EGFR gene.nnnRESULTSnOut of 94 patients, 65 were males and 29 females with a M:F ratio of 2.2: 1. The median age was 62 years (range, 28 - 85 years). In our biopsy samples 70 (74%) cases were of primary lung adenocarcinoma, whereas 24 (26%) were confirmed metastatic adenocarcinoma of primary lung origin. EGFR mutation was positive in 29% of the patients. The highest frequency of L858R was observed in 48% of these, followed by deletion in exon 19 (44%). In addition, other rare mutations such as compound G718X:S768I and insertions in exon 20 insertion were detected in approximately 4% of the patients.nnnCONCLUSIONSnThis study showed that Del 19 and L858R are the most frequent mutations in Pakistani lung adenocarcinoma patients and around 29% of the patients were found eligible for erlotinib therapy.

Coexistence of benign schwannoma and lymphoma in a nasal polyp

The existence of a combined benign schwannoma and lymphoma presenting as a nasal polyp has not been described in the English literature. We are reporting this rare combination in a 50-year-old male whose presenting symptoms were nasal obstruction, nasal deformity and headache. Examination of the left nasal cavity revealed a mass which was confined to the nose on computed tomography (CT) scan examination. Histopathology of the mass revealed a major component to be a benign schwannoma and a minor component a large B-cell lymphoma.

Response to Imatinib Mesylate in Patients with Early Chronic Phase Chronic Myeloid Leukemia and Derivative Chromosome 9 Deletion or Clonal Evolution

Objectives: The significance of clonal evolution and derivative chromosome 9 in Philadelphia-positive CML is not fully characterized and studies have yielded conflicting results. After working on emergence of clonal evolution from our region, we continued to find out the response of Imatinib Mesylate on such cases of CML treated in our center. nMaterials and methods: We conducted a cross sectional, prospective analysis on response of Imatinib Mesylate on patients with Philadelphia positive chronic myeloid leukemia with clonal evolution treated from period of September 2007 till 2010. Patients were grouped on basis of cytogenetic analysis performed by conventional cytogenetic and fluorescence in situ hybridization (FISH) techniques and followed for three years to see the response rate of imatinib mesylate. nResults: We reported here the response rate in one hundred and two previously untreated cases of chronic myeloid leukemia (Philadelphia positive). Twelve patients (11.7%) exhibit derivative chromosome 9, three had trisomy 8, one with addition 15 and one had deletion 16. At follow-up of 30 months 78 cases were evaluable and 45% and 61% showed complete and major cytogenetic response respectively. There is no significant association of derivative chromosome 9 with the response of imatinib mesylate in our group. nConclusion: Imatinib mesylate is the first line therapy in chronic phase of CML but the role in patients with clonal evolution need to be established by larger group of patients.

Multiple sclerosis in Pakistan: histocompatibility antigen composition and disability.

Dear Sir, An association between human leukocyte antigen (HLA) composition (especially DRB1 etc) and disability has been debated in the literature. A study of 380 multiple sclerosis (MS) patients showed that alleles *01 and *04 were independent predictors of increased disability,1 whereas an Iranian study of 183 MS patients did not show an association between DRB1*1501, DQA1*0102, DQB1*0602 and higher Expanded Disability Status Scale (EDSS) scores.2 Weinshenker and colleagues3 also did not find a correlation between DR15-DQ6 and DR13-DQ7 haplotypes and disease severity (as defined by EDSS and duration). Another study4 demonstrated an association between HLADRB1*1501, -DQB1*0301, -DQB1*0302, -DQB1*0602, and -DQB1*0603 alleles and more severe disease outcome on MRI. Disability related to MS was prevalent in our previous study of 142 patients;5 almost two-thirds of the patients in our previous series were severely disabled at 5.2 years after onset which is faster than the rate of progression seen in the West. Our present study was conducted to identify the prevalence of various HLA haplotypes among our MS patients as compared with a control population, and to evaluate a possible correlation between haplotypes and disease severity (EDSS). Patients (n = 100) with confirmed MS were prospectively enrolled from nine centers in Pakistan from January 2009 to September 2010; 40% were male, age range was 16–62 years (mean 32 years). Disease was classified as severe if patients had an EDSS score of 6 or more within 5 years of symptom onset. The control group for the distribution of HLA class II DRB1 and DQB1 alleles was composed of 1000 individuals, representing all major ethnic groups in Pakistan.6 HLA alleles were identified using a polymerase chain reaction and sequence specific primers (PCR-SSP) method as described by the manufacturer (One Lambda, Canoga Park, CA). HLA allele and haplotype frequencies were calculated using Pypop Win32-0.7.0 software. A total of 23% of patients developed severe disability (EDSS 6 or more) within 5 years of onset of symptoms. The most important factors associated with this rapid progression included a primary or secondary progressive course and spinal cord involvement. Almost 50% of patients in the severe disability group had a primary or secondary progressive course while 70% of patients in this group had spinal cord involvement. Higher disability scores showed a significant correlation with primary and secondary progressive MS (p = 0.001) and spinal cord involvement (p = 0.03). HLA typing and haplotype analysis of MS patients were compared with controls. There was no statistically significant difference between the two groups. There was a statistically significant association between high disability scores (EDSS 6 or more) and DQB1*0203 haplotype (p = 0.04). The clinical significance and possible mechanisms underlying this finding will have to be explored in future studies. The association between high disability scores and DRB1 alleles was non-significant. There was no significant correlation between haplotype and type of MS (relapsing– remitting versus primary or secondary progressive) and topography of MS (spinal cord versus optic and cranial involvement). Interestingly, the findings showed higher and more rapidly developing disability in Pakistani MS patients when compared to disability levels in Western MS patients. We wish to note that the recruiting centers are all tertiary care centers and the high disability may possibly be a selection bias.

Identification of hemoglobin Q India (α 1–64 Asp–His) through ARMS-PCR. First report from Pakistan

Various hemoglobinopathies have been reported from Pakistan excepting the rare ones like hemoglobin Q India. Our purpose of study was to identify the mutation (α 1 64 aspartate to histidine) through amplification restriction mutation system-polymerase chain reaction (ARMS-PCR) in patients where hemoglobin Q has been detected via high performance liquid chromatography (HPLC) and also to evaluate the cost effectiveness of the two technologies. All patients irrespective of age and gender who underwent HPLC for identification of their hemoglobin variant during January 1, 2006 to January 30, 2007 were studied. The blood samples with unknown peak at a retention time of 4.7xa0min were evaluated at the molecular level. Analysis of HPLC tracings of 11,008 subjects over a thirteen-month period identified ten individuals with hemoglobin Q. Male to female ratio was 1:1.5 and their age was variable ranging from 1 to 49 (mean 22.8) years. The mean hemoglobin level was 11.3xa0g/dl while MCV (fl) and MCH (pg) were 73.0 and 20.8 respectively. HPLC showed an unknown peak of 17.7% which was detected as Hb Q. ARMS based PCR showed Hb Q specific product of 370xa0bp and also an amplified product of 766xa0bp as the control fragment in these samples. This is the first ever report that documents the presence of Hb Q India (α 64 Asp to His) in Pakistani population. We recommend that HPLC be used as a useful screening tool especially in developing countries where PCR facilities may not be accessible.

R990G Polymorphism of Calcium Sensing Receptor Gene Is Associated with High Parathyroid Hormone Levels in Subjects with Vitamin D Deficiency: A Cross-Sectional Study

Single nucleotide polymorphisms (SNPs), R990G and A986S of the calcium sensing receptor (CaSR) gene, are shown to influence response of parathyroid hormone (PTH) in subjects with optimal vitamin D levels. This cross-sectional study was conducted in subjects with vitamin D deficiency (VDD) to observe associations between CaSR polymorphisms, plasma iPTH, and serum calcium levels. Adult females (n = 140) with known VDD, intact parathyroid hormone (iPTH), and calcium levels were recruited for genotype analysis. The frequencies of the 986 alleles GG, GT, and TT were 68%, 25%, and 7%, respectively, whereas the frequencies of the 990 alleles AA, AG, and GG were 80%, 8.9%, and 11.1%, respectively. The subjects with GG genotype of R990G polymorphism had higher iPTH levels (148.65 versus 91.47 and 86.1u2009pg/mL for GG versus AA, AG, resp., P = 0.008) and lower calcium levels (8.4 versus 9.04 and 9.07u2009mg/dL for GG versus AA, AG, resp., P = 0.002). No such association of A986S polymorphism with plasma iPTH or serum calcium levels was observed in the present study. Patients with VDD bearing the GG genotype of R990G SNPs are prone to have higher iPTH levels and lower calcium.