Iram Amin

PCR could be a method of choice for identification of both pulmonary and extra- pulmonary tuberculosis

BackgroundNucleic acid amplification assays including PCR have revolutionized the detection of Mycobacterium tuberculosis (MTB). Tuberculosis spread to almost every organ of the body and is characterized on the basis of localization of infection. Therefore, different types of body fluids and tissues can be used for the detection of MTB.From 2004 to 2010 total 766 different types of smear negative samples from patients, clinically suspected for tuberculosis were received and investigated at Division of Molecular Diagnostics, University of the Punjab Lahore for the diagnosis of tuberculosis. Mycobacterial DNA was extracted followed by PCR amplification.FindingsA total of 356 (46.5%) samples were found positive by PCR for MTB. These included; serum (4.8%), blood (36.3%), urine (46.6%), cerebro spinal fluid (CSF) (42.1%), ascetic fluid (67.6%), pleural fluid (52%), pericardial fluid (30%), pus (38.6%), bone marrow (60%), sputum (38.8%) and bronchoalveolar lavage (BAL) (70%). Over all there was no significant difference in males and females neither in different age groups for the identification of MTB.ConclusionWe conclude that PCR is a useful and sensitive tool for the early diagnosis of MTB in variety of clinical samples.

Mixed genotype infections with hepatitis C virus, Pakistan.

To the Editor: The prevalence of hepatitis C virus (HCV) infection is high (8% of the population) in Pakistan (1). HCV is an RNA virus that has a high mutation rate. This high rate results in extensive genetic heterogeneity, and HCV isolates are found as either quasispecies or genotypes (2). Humans can be co-infected with >1 genotype (mixed genotype infection) of this virus (3). The rate of HCV mixed genotype infections is extremely variable for different regions and for the same group of patients tested by using different assays (4). Thus, it is difficult to determine the prevalence of mixed genotype infections by currently available assays, including direct DNA sequencing, because they are designed to identify only the HCV genotype dominant in that particular population. Consequently, genotypes present at lower frequencies could be missed or mistyped (5). To determine the prevalence of HCV mixed genotype infections, we retrospectively analyzed genotyping data for paired serum samples from 22,125 HCV-infected patients during the past 11 years (March 2000–May 2010) for all regions in Pakistan by using molecular-based genotype-specific methods (6,7). A total of 12,036 (54.4%) were male patients and 10,089 (45.6%) were female patients. The sensitivity and reliability of the assay we used has been assessed and found to be superior to restriction fragment length polymorphism analysis and serotyping methods for detection of mixed genotypes in a viral population. Our method can detect a small amount (8.3%) of HCV RNA in a mixed genotype population (7). Restriction fragment polymorphism analysis can detect 2 genotypes only if 1 of them represents >41.6% of the genotypes in a mixed genotype population. Of 22,125 HCV RNA-positive serum samples, type-specific PCR bands were observed in 18,181 (82.2%) samples and 3,944 (17.8%) were not typeable. A total of 1,007 (5.5%) patients had HCV mixed genotype infections. The distribution of mixed genotype infections in 1,007 patients is shown in Figure A1. Infection with mixed genotype 3a + 3b was most prevalent (43.79%). Age distribution of patients with mixed genotype infections is shown in the Table. Approximately 33% of patients with mixed genotype infections were 31–40 years of age and 22.5% were 41–50 years of age. Table Distribution of mixed genotype infections with hepatitis C virus in 1,007 patients, by age, Pakistan, March 2000–May 2010 Patterns of HCV mixed genotype infections in Pakistan are similar to those reported from India and Iran (8). However, the prevalence of HCV mixed genotype infections was lower (2%) (8) for Iran than for Pakistan. This lower rate may have been caused by use of a genotyping kit that can detect only genotypes 1a, 1b, 2, and 3a. Thus, mixed infections with other genotypes would not have been detected. A recent study in Brazil reported that mixed genotype infections were detected in 3.9% of intravenous drug users and 7.1% of former injecting drug users (9). These rates were similar to those in our study. In contrast, data from Sweden and Russia showed no mixed genotype infections in serum samples of chronically infected intravenous drug users, hemodialysis patients, and patients with hemophilia (10). Women (288/7,390, 3.89%) in Pakistan had significantly fewer HCV mixed genotype infections than men (719/10,791, 6.66%) (p<0.01). This finding might be the result of women having fewer risk factors for contracting mixed genotype infections. Possible risk factors for infection with mixed genotype infections analyzed were blood transfusions and use of blood products (51.3%); multiple use of needles or syringes (18.4%); sharing razors during shaving or circumcision, piercing instruments, nail clippers, and toothbrushes (13.7%); and major or minor dental surgery (9.5%). Mode of transmission was not clear for 7.1% of the patients. In conclusion, the prevalence of HCV mixed genotype infections in Pakistan is higher than previously reported and higher among men (p<0.01). Comprehensive and detailed investigations are warranted to evaluate the clinical role of chronic HCV mixed genotype infections, provide essential information that can be used to determine type and duration of therapy needed, and predict disease outcome.

Correlation of biochemical markers and HCV RNA titers with fibrosis stages and grades in chronic HCV-3a patients.

Introduction Hepatitis C virus (HCV) is one of the most important causes of chronic liver diseases, which include inflammation, fibrosis, cirrhosis and hepatocellular carcinoma. Several factors have been proposed to determine the clinical outcome of HCV infection. The accurate mechanism by which HCV damages the liver remains poorly understood. In chronic hepatitis C patients, the relation between serum biochemical markers, HCV RNA titers and histological liver injury remain controversial. Objectives The aim of this study was to investigate the relation between serum biochemical markers, HCV RNA titers and the degree of liver damage in patients with chronic HCV. Materials and methods Liver biopsies were performed on 79 of a total of 100 enrolled patients. The histological activity was evaluated by the METAVER scoring system. HCV RNA quantification was performed by quantitative real-time PCR, and HCV genotyping was performed by nested PCR. Biochemical markers were measured with biochemical instruments. Results HCV RNA titers were significantly correlated with aspartate aminotransferase (AST) (P=0.004), alkaline phosphatase (ALP) (P=0.001) and total bilirubin (P=0.012) levels. HCV RNA titers were also significantly correlated with a progression of the fibrosis stage (P=0.000), but no correlation was observed with the change in inflammatory grades. It was observed that bilirubin levels were higher in later fibrosis stages as compared with the initial stage (P=0.000). Results revealed that in different fibrosis stages, the levels of AST (P=0.000), ALP (P=0.000) and alanine aminotransferase (ALT) (P=0.008), the age at diagnosis (P=0.000), the present age (P=0.000) and the BMI (P=0.009) were statistically significant. In the case of the inflammatory grade, levels of bilirubin (P=0.000), ALP (P=0.000), AST (P=0.016) and ALT (P=0.000) were statistically different between the inflammatory grades. Conclusion Serum HCV RNA titers were correlated with AST, ALP and total bilirubin. Levels of ALT, AST, ALP and bilirubin had significant relation with the liver fibrosis stage and the inflammatory grade in genotype 3a. Hence, our study suggests that AST, ALP and ALT may correlate with liver damage.

Hepatitis delta virus genotype-1 alone cocirculates with hepatitis B virus genotypes A and D in Pakistan.

Introduction Hepatitis delta virus (HDV) and hepatitis B virus (HBV) have been identified as major causes of morbidity and mortality in Pakistan because HDV causes infection only in the presence of HBV. Coinfection with both hepatitis viruses can lead to a more severe acute form of disease and to an increased risk of fulminant hepatitis. HDV infection differs in its distribution and severity depending on the geographical distribution, and several genotypes of HDV have been identified so far. Objectives The aim of the present study was to establish the HDV and HBV genotypes in chronically infected Pakistani patients and to determine whether there is any correlation between HDV and HBV genotypes. Patients and methods We studied samples from a total of 46 chronically infected HBV and HDV patients for HBV and HDV genotype analysis out of a total of 75 chronic HBV carriers enrolled. HBV and HDV genotypes were determined using type-specific PCR, followed by sequencing of PCR amplified products. Results The results of HBV genotyping showed that 33 of 46 (71.7%) patients had genotype D, five (10.9%) had A+D mixed genotypes, whereas eight (17.3) samples were untypable. We could detect only one HDV genotype (HDV-1) prevalent in the Pakistani population. The HDV-1 genotype isolate was associated with HBV genotype D alone or in combination with A (HBV-A+D). Conclusion The present study concludes that HDV/HBV coinfection is very high in the Pakistani population and was previously underestimated. The most prevalent circulating genotypes of HBV and HDV are HDV-1 and HBV-D, respectively, in the studied area. There is no specific interaction between HBV and HDV genotypes as suggested by HDV-1/HBV-D or HDV-1/HBV-A+D coinfection. Coinfection of HDV-1 and HBV-D simply reflects the most frequent genotypes circulating in this specific geographical region of the world.

Evaluation of three Techniques for detection of IL28B SNP: a prognostic tool for HCV treatment outcome

The study was aimed to evaluate the specificity, cost and turnaround time of three different techniques that can be used for analyzing the single nucleotide polymorphism of interleukin 28B (IL28B) rs129796860.

HCV-induced regulatory alterations of IL-1β, IL-6, TNF-α, and IFN-ϒ operative, leading liver en-route to non-alcoholic steatohepatitis

Over the course of time, Hepatitis C has become a universal health menace. Its deleterious effects on human liver encompass a lot of physiological, genetic as well as epigenetic alterations. Fatty liver (Hepatic steatosis) is an inflammation having multifactorial ancestries; one of them is HCV (steatohepatitis). HCV boosts several cellular pathways involving up-regulation of a number of cytokines. Current study reviews the regulation of some selective key cytokines during HCV infection, to help generate an improved understanding of their role. These cytokines, IL-1β, IL-6, TNF-α, and IFN-ϒ, are inflammatory markers of the body. These particular markers along with others help hepatocytes against viral infestation. However, recently, their association has been found in degradation of liver on the trail heading to non-alcoholic steatohepatitis (NASH). Consequently, the disturbance in their equilibrium has been repeatedly reported during HCV infection. Quite a number of findings are affirming their up-regulation. Although these cell markers are stimulated by hepatocytes as their standard protection mechanism, but modern studies have testified the paradoxical nature of this defense line. Nevertheless, direct molecular or epigenetic research is needed to question the actual molecular progressions and directions commanding liver to steatosis, cirrhosis, or eventually HCC (Hepatocellular Carcinoma).

Molecular epidemiology of hepatitis delta and hepatitis B viruses circulating in two major provinces (East and North-West) of Pakistan

OBJECTIVES HBV and HDV are major public health problems with millions affected globally and Pakistan accounts for a significant proportion of the global Hepatitis burden. This cross sectional study was designed to assess the general epidemiological and virological features of HBV and HDV in the Punjab and Khyber Pakhtunkhawa (KP) provinces of Pakistan. METHODS A total of 1890 HBV patients from March 2016 to May 2017 were recruited in the study and the presence of HDV was retrospectively evaluated in all participants. Most participants were young adults (from 21 to 30 years). Genotyping was based on PCR amplification using primers specific for HBV genotypes A-F, and HDV. 405 nucleotide fragments of HDV were sequenced. MEGA was used for phylogenetic analysis. RESULTS Overall prevalence of HBV was 14.08% (266/1890). Higher prevalence was observed in males (66.85%) as compared to females (33.15%). Co-infection of HDV was found in 39 (14.66%) patients. HBV genotype-D was prevalent in dual infections followed by HDV/A (p < 0.05).While HDV genotype 1 was predominant in all HBV positive samples. Compared to Punjab, coinfection was higher in KP (14.3% versus15.2%; p < 0.05). CONCLUSION The prevalence of HBV and HDV is high in Pakistan. The description of HBV and HDV genotypes circulating in East and North-West Pakistan can contribute to a better understanding of their relevance in regional epidemics. These infections are highly endemic in the KP, where their control is confounded by its vast territorial dimension with small, hard-to-reach municipalities and diverse ethnic populations.

Study of inhibition of interferon inducible genes by dephosphorylation of E2 envelope gene of HCV genotype 1a

Background: Interferon is considered as the first line of defense against all infections including HCV. Unfortunately 50% of the patients do not respond to the existing treatment. Envelope protein 2 of HCV interacts with the interferon-inducible protein kinase (PKR) protein. This protein contains a sequence identical with phosphorylation sites of the PKR and the translation initiation factor eIF2alpha, a target of PKR. Inhibition of the kinase activity of PKR by this interaction is postulated as a mechanism for the resistance to interferon (IFN) therapy.

Role of circulatory microRNAs in the pathogenesis of hepatitis C virus

Abstract Hepatitis C virus (HCV) is associated with one of the major health problem in world that ultimate results in the liver cirrhosis and leads to carcinoma of hepatocellular components round the world. More than 185 million people were found to be infected with HCV. MicroRNAs are small oligonucleotide RNA having 18–22 nucleotides. Circulating mi-RNAs regulate the replication of HCV and HCV-induced liver fibrosis and HCC. By comparing the expression profiles of mi-RNAs of normal individuals with HCV infected patients, aberrant changes in expression of different mi-RNAs have been observed so it can be predicted that these mi-RNAs are associated with and play a central role in the hepatitis C infection and diseases associated with it. This review demonstrates the major role of circulatory microRNAs in the HCV and HCV associated ailments.