Evaluation of DNA vaccines encoding M. Tb gene Bfrb and Mpt32 in mice model.

Tuberculosis (TB) is a life threatening infectious disease which is prevalent throughout the world. Mycobacterium bovis based Bacille Calmette-Gue´rin (BCG) is the only vaccine available against TB however, this (single) vaccine is not enough to eradicate it. Furthermore, numbers of researches from different parts of the World have shown its efficacy as variable. Hence other (better) vaccines like DNA vaccines are needed in addition to BCG in order to achieve desired goal of TB eradication. The current study was aimed to develop subunit based DNA vaccines against TB and to check their efficacy. Two constructs Bfrb-pND14 and Mpt32-pND14 were made and used as DNA vaccines. Endotoxin free DNA preparations were made and used in immunization studies. Twenty Balb/c female mice of age eight weeks were used in trial. Two experimental groups each comprising eight animals were used to inoculate Mpt32-pND14 and Bfrb-pND14 vaccines respectively. A group of four animals was used as negative control. Animals were bled through tail periodically and finally through cardiac puncture before euthanization. Antibodies were confirmed through dot blot and Agar Gel Immuno Diffusion test (AGID). All the animals immunized with both vaccines were found positive as tested through dot blot and AGID. The results of this study have indicated that both the M. tb genes have produced strong immune response in mice model through pND14 vector and proved themselves as good subunit based DNA vaccines.

Distribution and clinical significance of hepatitis B virus genotypes in Pakistan.

BACKGROUND: Hepatitis B virus (HBV) genotype and its role in disease progression and patients' response to antiviral treatment, is not well studied in Pakistan. This comprehensive study was aimed to determine the distribution of HBV genotypes in Pakistan and their possible association with phases of HBV infection. METHODS: A total of 840 HBsAg positive samples was collected and tested for HBV DNA quantity. Samples below 100 IU/ml were excluded from the study. A total of 715 samples representing all the six parts of the country were genotyped by type specific primer PCR method. Clinical data of only 384 patients was compared as the remaining 332 were either receiving antiviral treatment or their infection phase was not confirmed. RESULTS: Genotype D was found in 509 samples (71.2 %), genotype A in 55 samples (7.7 %) and mixed infection with genotypes A and D in 124 samples (17.3 %). Genotypes B, C and E were identified in less than 1 % of the total samples. Genotype A, D and their mixture (A + D) were compared for severity of HBV infection. Significant differences were not found in distribution of HBV genotypes among different disease stages. CONCLUSION: HBV genotype D was the predominant infection in all study areas of Pakistan followed by mixed genotypes infection (A + D) whereas genotype A has 10 times lower prevalence than genotype D. Genotypes B, C, E and F altogether make only 1.5 % of the prevalence. Genotype do not appears to show the severity of liver disease.

Evaluation of diagnostic potential of Rv3803c and Rv2626c recombinant antigens in TB endemic country Pakistan.

To overcome and eliminate tuberculosis (TB), definitive, reliable, and rapid diagnosis is mandatory. Presently, the diagnostic potential of acute and latent stage TB specific antigens i.e., Rv3803c and Rv2626c was determined. Immunogenic recombinant genes of Rv3803c and Rv2626c antigens were cloned in bacterial expression vector pET23b and expressed product was purified. The homogeneity and structural integrity was confirmed by Western blot analysis. Diagnostic potential of Rv3803c and Rv2626c antigens was analyzed using the sera of 140 active TB patients (AFB smear positive) by indirect ELISA. Ten patients of leprosy and 94 healthy individuals were taken as disease and normal control respectively. The data was analyzed using R statistical package. The sensitivity and specificity of Rv3803c in active TB patients was of 69.3% and 76.4% respectively with an area under ROC curve of 0.77, whereas sensitivity and specificity of Rv2626c 77.1% and 85.1%, respectively. The area under ROC curve of Rv2626c was 0.89 which was significantly higher than Rv3803c (p < 0.0001). Recombinant antigens Rv3803c and Rv2626c have potential to be used as diagnostic markers for TB and need to evaluate with other antigens for differential diagnosis of TB.

Exploratory study on plasma immunomodulator and antibody profiles in tuberculosis patients.

Host immune responses to Mycobacterium tuberculosis are generally able to contain infection and maintain a delicate balance between protection and immunopathology. A shift in this balance appears to underlie active disease observed in about 10% of infected individuals. Effects of local inflammation, combined with anti-M. tuberculosis systemic immune responses, are directly detectable in peripheral circulation, without ex vivo stimulation of blood cells or biopsy of the affected organs. We studied plasma immunomodulator and antibody biomarkers in patients with active pulmonary tuberculosis (TB) by a combination of multiplex microbead immunoassays and computational tools for data analysis. Plasma profiles of 10 immunomodulators and antibodies against eight M. tuberculosis antigens (previously reported by us) were examined in active pulmonary TB patients in a country where TB is endemic, Pakistan. Multiplex analyses were performed on samples from apparently healthy individuals without active TB from the same community as the TB patients to establish the assay baselines for all analytes. Over 3,000 data points were collected from patients (n = 135) and controls (n = 37). The data were analyzed by multivariate and computer-assisted cluster analyses to reveal patterns of plasma immunomodulators and antibodies. This study shows plasma profiles that in most patients represented either strong antibody or strong immunomodulator biomarkers. Profiling of a combination of both immunomodulators and antibodies described here may be valuable for the analysis of host immune responses in active TB in countries where the disease is endemic.

A study on the association of TNF-α(-308), IL-6(-174), IL-10(-1082) and IL-1Ra(VNTR) gene polymorphisms with rheumatic heart disease in Pakistani patients.

Inflammation is an important contributor to the pathogenesis of rheumatic heart disease (RHD), a disorder of heart valves caused by a combination of immune, genetic and environmental factors. Cytokines are important mediators of inflammatory and immune responses. The aim of this study was to investigate the role of cytokine gene polymorphisms and their potential usefulness as biomarkers in RHD patients from Pakistan. We screened 150 RHD patients and 204 ethnically matched controls for tumor necrosis factor (TNF)-α(-308)G/A, interleukin (IL)-10(-1082) G/A, interleukin (IL)-6(-174) G/C and a variable number of tandem repeats (VNTRs) polymorphism of the IL-1Ra gene using polymerase chain reaction. The results showed that TNF-α(-308) A and IL-6(-174) G alleles were associated with susceptibility to RHD (p=0.000; OR=2.81; CI=1.5-5.14 and p=0.025; OR=1.50; CI=1.04-2.16 respectively). The TNF-α(-308) AA and GA genotypes were associated with susceptibility to RHD (p=0.012; OR=9.94; CI; 1.21-217.3 and p=0.046; OR=1.97; CI=0.98-3.97 respectively) while the GG genotype seemed to confer resistance (p=0.003; OR=0.39; CI=0.20-0.76). The GG genotype for IL-6(-174) was significantly associated with predisposition to RHD (p=0.015; OR=2.6; CI=1.17-5.85). The A1 (four repeats) and A2 (two repeats) alleles at the IL-1Ra VNTR polymorphism were associated with resistance and susceptibility to RHD respectively. However, this polymorphism deviated from Hardy-Weinberg equilibrium in both patients and controls in our population. TNF-α(-308) and IL-6(-174) polymorphisms may be useful markers for the identification of individuals susceptible to RHD in Pakistan. These individuals could be provided aggressive prophylactic intervention to prevent the morbidity and mortality associated with RHD.

Plasma antibody profiles as diagnostic biomarkers for tuberculosis.

Two billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), worldwide. Ten million to 20 million of the infected individuals develop disease per year. TB is a treatable disease, provided that it is diagnosed in a timely manner. The current TB diagnostic methods are subjective, inefficient, or not cost-effective. Antibody-based blood tests can be used efficiently and cost-effectively for TB diagnosis. A major challenge is that different TB patients generate antibodies against different antigens. Therefore, a multiplex immunoassay approach is needed. We have developed a multiplex panel of 28 M. tuberculosis antigen-coated microbeads. Plasma samples were obtained from over 300 pulmonary TB patients and healthy controls in a country where TB is endemic, Pakistan. Multiplex data were analyzed using computational tools by multivariate statistics, classification algorithms, and cluster analysis. The results of antibody profile-based detection, using 16 selected antigens, closely correlated with those of the sputum-based diagnostic methods (smear microscopy and culture) practiced in countries where TB is endemic. Multiplex microbead immunoassay had a sensitivity and specificity of approximately 90% and 80%, respectively. These antibody profiles could potentially be useful for the diagnosis of nonpulmonary TB, which accounts for approximately 20% of cases of disease. Since an automated, high-throughput version of this multiplex microbead immunoassay could analyze thousands of samples per day, it may be useful for the diagnosis of TB in millions of patients worldwide.

Delay in diagnosis of tuberculosis in Rawalpindi, Pakistan.

BACKGROUND: Delay in diagnosis and treatment of tuberculosis (TB) may enhance the chances of morbidity and mortality and play a key role in continuous transmission of the bacilli. The objective of this study was to describe health care seeking behavior of suspected TB patients and initial diagnostic work up prior to consultation and diagnosis at National TB Center (NTC). FINDINGS: Interviews of 252 sputum smear positive patients were taken from NTC, Rawalpindi. The duration between on-set of symptoms and start of treatment was considered as the total delay and correlated with general characteristics of TB patients. The proportion of males and females were 49.6% and 50.4% with median age of 25 and 24 years respectively. A median delay of 56 days (8 weeks) was observed which was significantly associated with age, cough and fever. More than 50% of the current patients had a history of contact with previously diagnosed TB patients. The majority of patients (63%) visited health care providers within three weeks of appearance of symptoms but only thirty five percent were investigated for TB diagnosis. CONCLUSION: Cough and fever are being ignored as likely symptoms of TB by patients as well as health care providers resulting in delay. Engaging private practitioners through public private mix (PPM) approach for expansion of TB diagnosis and increasing public awareness could be more beneficial to reduce delay.

Prevalence of Helicobacter pylori pathogenicity-associated cagA and vacA genotypes among Pakistani dyspeptic patients.

The cytotoxin-associated gene A (cagA), and the vacuolating cytotoxin gene A (vacA) products are considered the most important pathogenic determinants of Helicobacter pylori, a gram-negative bacterium causing gastrointestinal disorders such as duodenal ulcers, gastritis and mucosa-associated lymphoid tissue disease. A higher prevalence of H. pylori has been reported in various regions in the Pakistani population; however, no data are available about the virulence-associated genetic determinants. The objective of this study was to determine the prevalence of virulence-associated genes, cagA, vacA and particularly vacA allelic variants among dyspeptic patients from Pakistan. Gastric biopsy samples were obtained from 78 adult patients presenting dyspepsia symptoms. DNA was isolated and analyzed for the presence of H. pylori and its genotypes by PCR. Genus-specific PCR involving 16S rRNA gene revealed that 66 of the 78 patients were positive for H. pylori, an overall prevalence of 84.6% for this particular study. The most common vacA genotype was s1b/m2 (54.5%) followed by s1a/m1 (19.7%). cagA was positive in 24.2% of the cases and strongly associated with s1a/m1, vacA. The prevalence of virulent cagA, and vacA allelic form s1a/m1 was lower than that reported from neighboring countries.

Passive immunization against highly pathogenic Avian Influenza Virus (AIV) strain H7N3 with antiserum generated from viral polypeptides protect poultry birds from lethal viral infection.

Our studies were aimed at developing a vaccination strategy that could provide protection against highly pathogenic avian influenza virus (AIV), H7N3 or its variants outbreaks. A purified viral stock of highly pathogenic H7N3 isolate was lysed to isolate viral proteins by electrophresing on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by their elution from gel through trituration in phosphate buffered saline (PBS). Overall, five isolated viral polypeptides/proteins upon characterization were used to prepare hyperimmune monovalent serum against respective polypeptides independently and a mixture of all five in poultry birds, and specificity confirmation of each antiserum through dot blot and Western blotting. Antiserum generated from various group birds was pooled and evaluated in 2-week old broiler chicken, for its protection against viral challenge. To evaluate in-vivo protection of each antiserum against viral challenges, six groups of 2-week old broiler chicken were injected with antiserum and a seventh control group received normal saline. Each group was exposed to purified highly pathogenic AIV H7N3 strain at a dose 10(5) embryo lethal dose (ELD(50)). We observed that nucleoprotein (NP) antiserum significantly protected birds from viral infection induced morbidity, mortality and lowered viral shedding compared with antiserum from individual viral proteins or mixed polypeptides/proteins inclusive of NP component. The capability of individual viral polypeptide specific antisera to protect against viral challenges in decreasing order was nucleoprotein (NP) > hemagglutinin (HA) > neuraminidase (NA) > viral proteins mix > viral polymerase (PM) > non-structural proteins (NS). Our data provide proof of concept for potential utilization of passive immunization in protecting poultry industry during infection outbreaks. Furthermore conserved nature of avian NP makes it an ideal candidate to produce antiserum protective against viral infection.