Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

Effectiveness of implementation of standard clinical pathway through healthcare professionals among acute myocardial infarction patients undergoing angiography / angioplasty in a public tertiary care hospital, Karachi.

OBJECTIVE: To assess the effect of acute myocardial infarction standard clinical pathway among acute myocardial infarction patients on length of stay in public tertiary care setting. METHODS: The quasi-experimental non-randomised study was conducted at the Department of Cardiology, Dr Ruth Pfau Civil Hospital, Karachi, from September to December 2018, and comprised acute myocardial infarction patients. Those admitted before the implementation of acute myocardial infarction standard clinical pathway formed the control group, while those admitted after the implementation were in the intervention group. Acute myocardial infarction standard clinical pathway was implemented and the interventional clinical practices of healthcare professionals, including cardiologists, postgraduates, residents, nurses and critical care technicians, were assessed using a standard checklist. Data was analysed using SPSS 21. RESULTS: Of the 100 participants, 50(50%) were in the control group; 31(62%) males and 19(38%) females. The intervention group also had 50(50%) patients; 35(70%) males and 15(30%) females. Regarding effectiveness of the implementation of standard clinical pathway, length of hospital stay reduced significantly in the intervention group compared to the control group (p=0.003). CONCLUSIONS: The implementation of acute myocardial infarction standard clinical pathway reduced the length of hospital stay of acute myocardial infarction patients.

Dengue Vaccines: Ongoing Challenges and Current Status in the Advancement of Different Candidates.

Dengue is a vector-borne highly systemic infectious disease of the tropical and subtropical countries and is devastating millions of lives worldwide. It may be self-eliminated like a mild fever or may cause life-threatening fatal complications as dengue hemorrhagic fever and dengue shock syndrome. The lack of specific and effective antiviral drugs and vaccines amplify its transmission rate across the world. The development of the dengue vaccine has been an ambitious task due to the presence of four different dengue serotypes capable of carrying antibody enhancement complex mechanisms. In this review, we have summarized the ongoing challenges in the construction of a dengue vaccine and the current status of the vaccine development. Limited knowledge of immune responses against dengue infection, lack of human or animal model of disease, and suboptimal assay strategies to detect immune responses after infection or vaccination, are some barriers to vaccine and drug development. A tetravalent vaccine with low cost, high efficiency, and capable of eliciting immune responses against all four serotypes is needed to minimize the epidemics. Currently, only one live attenuated chimeric dengue vaccine, the CYD Dengue Vaccine, has completed its third phase and has been licensed. DENVax and TetraVax-DV-TV003 (TV003) are in the third phase while others are still in the first trial phase.

Association of Interleukin 28B Polymorphism with Clearance of Hepatitis C Virus: A Mini Review.

The highly infectious hepatitis C virus (HCV) is the major cause of chronic hepatitis around the globe. Approximately 3% of the world's population has been affected by both chronic and acute HCV. In this study, we highlight the relationship between single-nucleotide proteins (SNPs) and interleukin (IL) IL28B on chromosome 19 with the treatment response of chronic HCV infection along with its sustained virologic response (SVR). Four SNPs are strongly linked with HCV self-clearance: rs8099917 TT, rs12980275 AA, rs8105790 TT, and rs10853728 CC. Most SNPs, including rs12979860, are located upstream of the IL28B gene fragment, encoding interferon (IFN)-λ3. We find that IL28B variants rs8099917 and rs12979860 strongly influence results of combined pegylated (PEG)-IFN/ribavirin (RBV) therapy. In the case of SNP rs12979860, the CC genotype is linked with greater than a twofold higher SVR than that with TT or CT genotypes. These SNPs are associated with expression of intrahepatic IFN-stimulated genes in liver. Past research shows that females are more efficient in resolving HCV infection, regardless of IL28B genotype. Similar results of IL28B polymorphisms associated with spontaneous HCV clearance were also obtained in Chinese and Taiwanese HCV patients. Another report of RBV and PEG-IFN-treated patients revealed that age, viral load, rs8099917 genotype, and fibrosis were major predictors of antiviral therapeutic response. To select favorable antiviral regimes for treatment using IFN, a combination of host genetic data and viral genotyping may be useful in treating chronic HCV. We propose that these predictive factors must be considered before commencing treatment in HCV patients.

Paradoxical Role of Dengue Virus Envelope Protein Domain III Antibodies in Dengue Virus Infection.

Every year, approximately 100 million individuals are infected with dengue viral infections. Severe dengue infection, characterized as dengue hemorrhagic fever, leads to loss of intravascular fluids and severe bleeding. During dengue virus (DENV) secondary infection, the body produces neutralizing antibodies that cause a strong immune response, resulting in severe hemolysis and plasma leakage. DENV infections in humans stimulate production of virus serotype-specific and cross-reactive antibodies. The envelope (E) protein of DENV contains potent antigenic sites, with one known as E protein domain III (EDIII). Studies of DENV EDIII in mouse models have shown that strongly neutralizing mouse monoclonal antibodies (mAbs) are DENV-serotype specific and bind to an epitope on EDIII that is unique to each serotype. Unlike DENV-serotype-specific mouse mAbs, cross-reactive mAbs that bind to EDIII have moderate-to-weak neutralizing activity. Studies with mouse mAbs resulted in identification and mapping of different epitopes on the lateral ridge of DENV EDIII.

Drug Resistance in Hepatitis C Virus: Future Prospects and Strategies to Combat It.

Induction of highly pathogenic hepatitis C virus (HCV) causes chronic hepatitis round the world. This virus is easily prone to developing resistance against antiviral drugs because of two viral polymerases that do not possess the proofreading and overlapping reading frame abilities. There is more than one explanation for how this virus builds up resistance against antiviral drug treatments. Assays are now available to detect HCV-resistant variants, based on phenotypic and genotypic assays, and next generation sequencing. But these assays are of a little value at baseline, because they are not influential enough for making therapeutic decisions in HCV patients. Moreover, HCV monitoring is now an essential part of clinical practice. Special patients, such as those with thalassemia, renal transplant due to renal failure, and the patients undergoing hemodialysis, are at higher risk for acquiring this infection. Management of HCV infection in these patient groups is complicated by multiple side effects, including flu-like symptoms, neutropenia, fever, and neuropsychiatric disorders, thus limiting the use of ribavirin and coexisting iron overload. In HCV patients suffering from depression, the treatment may be discontinued because of some defects in neurochemical pathways caused by interferon, which can enhance the level of depression in these patients. In addition, obesity has been found to be a marker of failure of HCV treatment. There will be many resistance tolerant HCV treatment options available in the near future.

Blood-based gene expression profile of oxidative stress and antioxidant genes for identifying surrogate markers of liver tissue injury in chronic hepatitis C patients.

Oxidative stress is the process by which reactive molecules and free radicals are formed in cells. In this study, we report the blood-based gene expression profile of oxidative stress and antioxidant genes for identifying surrogate markers of liver tissue in chronic hepatitis C (CHC) patients by using real-time PCR. A total of 144 untreated patients diagnosed with CHC having genotype 3a and 20 healthy controls were selected for the present study. Liver biopsy staging and grading of CHC patients were performed using the METAVIR score. Total RNA was extracted from liver tissue and blood samples, followed by cDNA synthesis and real-time PCR. The relative expression of genes was calculated using the ΔΔCt method. The expression profile of 84 genes associated with oxidative stress and antioxidants was determined in liver tissue and blood samples. In liver tissue, 46 differentially expressed genes (upregulated, 27; downregulated, 19) were identified in CHC patients compared to normal samples. In blood, 61 genes (upregulated, 51; downregulated; 10) were significantly expressed in CHC patients. A comparison of gene expression in liver and whole blood showed that 20 genes were expressed in a similar manner in the liver and blood. The expression levels of commonly expressed liver and blood-based genes were also correlated with clinical factors in CHC patients. A receiver operating curve (ROC) analysis of oxidative stress genes (ALB, CAT, DHCR24, GPX7, PRDX5, and MBL2) showed that infections in patients with CHC can be distinguished from healthy controls. In conclusion, blood-based gene expression can reflect the behavior of oxidative stress genes in liver tissue, and this blood-based gene expression study in CHC patients explores new blood-based non-invasive biomarkers that represent liver damage.

HSV-1 Infection: Role of Viral Proteins and Cellular Receptors.

The interaction between herpes simplex virus type 1 (HSV-1) and its host starts with the attachment of the virus for entry and spreading into host cells involving viral glycoproteins and host receptors. Once entered, it remains persistent as a latent infection throughout the host's life as it cannot be cleared completely by the immune system. Viral regulatory proteins and host factors determine whether the virus will enter into the acute or latent mode of infection. Acute viral infection is usually asymptomatic and self-limiting whereas latent infection may remain in the trigeminal ganglion of oropharyngeal mucosa, where it can be activated at any time depending upon the stimulus. Host innate and adaptive immune elements play important roles in limiting HSV-1 infection by interfering with viral replication but are unable to remove the virus completely. In this review, we update how the major proteins involved in entry and pathogenesis of viruses and immune responses against infection.

HCV-induced regulatory alterations of IL-1ß, 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-1ß, 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).

Improving Access to Anti-HDV Testing: Development and Validation of an Affordable In-House ELISA Assay.

In certain low-income nations, the hepatitis Delta virus and hepatitis B virus (HBV) pose a serious medical burden, where the prevalence of hepatitis B surface antigen (HBsAg) is greater than 8%. Especially in rural places, irregular diagnostic exams are the main restriction and reason for underestimation. Utilizing serum samples from a Pakistani isolate, an internal ELISA for the quick identification of anti-HDV was created, and the effectiveness of the test was compared to a commercial diagnostic kit. HDV-positive serum samples were collected, and a highly antigenic domain of HDAg antigen was derived from them. This antigenic HDAg was expressed in a bacterial expression system, purified by Ni-chromatography, and confirmed by SDS-PAGE and Western blot analysis. The purified antigen was utilized to develop an in-house ELISA assay for anti-HDV antibody detection of the patient's serum samples at very low cost. Purified antigens and positive and negative controls can detect anti-HDV (antibodies) in ELISA plates. The in-house developed kit's efficiency was compared with that of a commercial kit (Witech Inc., USA) by the mean optical density values of both kits. No significant difference was observed (a P value of 0.576) by applying statistical analysis. The newly developed in-house ELISA is equally efficient compared to commercial kits, and these may be useful in regular diagnostic laboratories, especially for analyzing local isolates.

Immune-related therapeutics: an update on antiviral drugs and vaccines to tackle the COVID-19 pandemic.

The coronavirus disease 2019 (COVID-19) pandemic rapidly spread globally. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, is a positive-sense single-stranded RNA virus with a reported fatality rate ranging from 1% to 7%, and people with immune-compromised conditions, children, and older adults are particularly vulnerable. Respiratory failure and cytokine storm-induced multiple organ failure are the major causes of death. This article highlights the innate and adaptive immune mechanisms of host cells activated in response to SARS-CoV-2 infection and possible therapeutic approaches against COVID-19. Some potential drugs proven to be effective for other viral diseases are under clinical trials now for use against COVID-19. Examples include inhibitors of RNA-dependent RNA polymerase (remdesivir, favipiravir, ribavirin), viral protein synthesis (ivermectin, lopinavir/ ritonavir), and fusion of the viral membrane with host cells (chloroquine, hydroxychloroquine, nitazoxanide, and umifenovir). This article also presents the intellectual groundwork for the ongoing development of vaccines in preclinical and clinical trials, explaining potential candidates (live attenuated-whole virus vaccines, inactivated vaccines, subunit vaccines, DNAbased vaccines, protein-based vaccines, nanoparticle-based vaccines, virus-like particles and mRNA-based vaccines). Designing and developing an effective vaccine (both prophylactic and therapeutic) would be a long-term solution and the most effective way to eliminate the COVID-19 pandemic.

Herpes Simplex Virus Type 1 and Host Antiviral Immune Responses: An Update.

Herpes simplex virus type 1 (HSV-1) infection activates a rapid stimulation of host innate immune responses and a delicate interplay between virus and host immune elements regulates the whole events. Although host immune elements play well in limiting the HSV-1 infection by interfering viral replication, they are still unable to remove the virus completely, because HSV-1 proteins are efficient enough to bypass the host antiviral immune responses and virus succeed to reactivate again from latency at opportune time. Type 1 interferon signaling pathway is the central point of innate immunity along with some of the activated neutrophils, monocytes, macrophages, and dendritic cells, and some natural killer cells play role, while the CD8+ T cells are crucial in adaptive immunity. In this review, the current knowledge of host and HSV-1 interaction has been described that how the host antiviral immune responses occur and what are the mechanisms of viral evasion adapted by virus to counteract with both arms of immunity.

Short article: Hepatitis C and G virus coinfection in Punjab, Pakistan: incidence and its correlation analysis with clinical data.

INTRODUCTION: Hepatitis G virus (HGV) infection appears to be common in patients with chronic hepatitis C virus (HCV) infection. The aim of this study was to investigate the prevalence of HCV/HGV in patients with chronic hepatitis C (CHC) in Pakistan and to look for possible associations with various clinical and histopathological changes in HCV/HGV coinfection and HCV infection. PATIENTS AND METHODS: The present study included 136 patients. Clinical, biochemical, virological and histological findings were compared between patients coinfected with HCV/HGV and patients with HCV alone. RESULTS: Of the 136 patients with CHC, 16 (11.76%) were coinfected with HCV/HGV. The mean age of coinfected patients was lower than in patients with HCV alone. HCV/HGV coinfected patients did not show significant differences in sex, clinical presentation, biochemical markers, and liver fibrosis as compared to those with HCV infection. Only the mean values of platelets count, mean corpuscular hemoglobin (MCH), and MCH concentration markers were significantly different in HCV/HGV coinfected patients as compare to patients with HCV alone. CONCLUSION: It was found that 11.76% of patients with CHC in Pakistan were associated with HCV/HGV coinfection. No significant differences were observed in clinical and histological features except for platelets count, MCH, and MCH concentration markers between HCV and HGV coinfected patients in comparison with HCV-infected patients.

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.

Role of circulatory microRNAs in the pathogenesis of hepatitis C virus.

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.

Evaluation of three techniques for detection of IL28B SNP: A prognostic tool for HCV treatment outcome.

OBJECTIVE: 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. METHODS: DNA from peripheral blood samples of 111 patients with chronic hepatitis C were genotyped using three types of genotyping methods: direct sequencing, SNaPshot polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Three distinct profiles for IL28B rs12979860 alleles (CC, CT and TT) were obtained with direct sequencing, SNaPshot PCR and PCR-RFLP and the results were consistent among all three methods. CONCLUSION: For routine medical practice, screening IL28B rs12979860 can be performed by PCR-RFLP, which is efficient and reliable as well as cost-effective.

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.

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.

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

BACKGROUND: Nucleic 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. FINDINGS: A 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. CONCLUSION: We conclude that PCR is a useful and sensitive tool for the early diagnosis of MTB in variety of clinical samples.