Home > Dna Fingerprinting and Characterization of Mutations Associated With First Line Anti- Tb Drug Resistance in Mycobacterium Tuberculosis Strains Prevalent in Pakistan
Dna Fingerprinting and Characterization of Mutations Associated With First Line Anti- Tb Drug Resistance in Mycobacterium Tuberculosis Strains Prevalent in Pakistan
Tuberculosis (TB) is one of the most devastating infectious diseases that is highly endemic in Pakistan. Pakistan is ranked 5th amongst 22 high tuberculosis burden countries of the world. Global as well as national tuberculosis control program is further challenged by the spread of multiple drug resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis. For controlling the spread of M. tuberculosis isolates, circulating in this region, it is important to explore the transmission dynamics and characteristics of these strains. This information, in turn, can help to implement better treatment and control measures. The study provides the information about the population structure of M. tuberculosis isolates in Pakistan. DNA fingerprinting of the strains was performed by high throughput spoligoriftyping and 24 MIRU-VNTR typing techniques. CAS family constituted the dominant group of the strains followed by the T family and EAI family while Beijing family showed the low prevalence. However, EAI strains were found to show high prevalence in Eastern part of the country. Molecular epidemiological methods play an important role to identify appropriate public health interventions and to measure their impact. Despite of the fact that Pakistan is harboring high disease burden, no molecular epidemiologic studies have yet been conducted to assess the disease transmission. Our study explores, for the first time, TB epidemiology in the Punjab province of Pakistan, using the gold standard tools of molecular epidemiology. We document a relatively low disease transmission rate in the population. The study also provides a good assessment of the discriminatory powers of the various genotyping techniques and suggests the use of duplex format of MIRU-VNTR typing to be used as genotyping technique in this setting because of its low cost and relatively less turnaround time as compared to simplex format. We further suggest the use of Qub 26, MIRU 10, Mtub 04, MIRU 26, MIRU 31 (ETR E), MIRU 16, Qub 4156 and Mtub 21 to be used as preliminary ‘fast lane’ screen to differentiate the M. tuberculosis strains in this particular geographical setting. The use of high throughput techniques like spoligoriftyping is recommended to be used as good tool to help the TB control programs in high disease burden countries. This powerful technique also helped to identify unreliable standards of phenotypic drug sencitivity testing (DST) in some local hospitals. Besides this, an in-house low cost test platform is configured and validated for the rapid screening of multiple drug resistance (MDR) in the present study. Being highly sensitive and specific, not only in the culture isolates but also in clinical samples, it could be used to screen MDR in point of care settings in the developing world where the need is acute. This study for the first time gives the comparative assessment of three freely available databases used to assign lineage to the M. tuberculosis isolates, uncovering the errors and inability of these databases in assigning lineages to isolates. Further, our study also pinpointed the defects in lineage assignation at sublineage level that arose due to the lack of database up gradation. The study also covered the assessment of occurrence of mutations at various target loci and their frequencies in M. tuberculosis isolates, resistant to first line anti- TB drugs. Overall, the profile of the mutations at various loci was similar to that found at other geographical locations worldwide. The most common mutations responsible for the rifampicin resistance were found in codon 531, 526 and 516 of rpoB gene, in isoniazid resistant isolates affecting the codon 315 of the katG and position -15 of the promoter region of inhA gene, in ethambutol resistant isolates affecting the codon 306 of embB gene and in streptomycin resistant isolates, targeting the codon 43 of rpsL gene and codon 512, 513 and 516 of rrs gene. In case of pyrazinamide, very few isolates showed mutations in targeted regions of pncA gene. Besides this, some novel mutations were also observed in this study. The relationship of specific mutations in rpoB and katG genes with M. tuberculosis lineages is also explored. The information about these mutations can be used to develop novel molecular diagnostic method that specifically could be implemented in Pakistan. However, prospective thorough epidemiological studies are needed to monitor continuously changing disease transmission dynamics in the community.
Objective: In order to provide equal educational opportunities, community school networking is an emerging trend to facilitate inclusion of children with mild-moderate Autism. This quantitative research aims to investigate the effectiveness of community networking for children with Autism from Pakistani lower socio-economic stratum of society.
Study Design: Qualitative Research Design
Study Settings and Participants: Six mild-moderate autistic children were enrolled in three mainstreams schools and a liaison between these schools, and a rehabilitation center located in the same area was created to facilitate inclusion. The researchers interviewed six teachers from mainstream schools, three school administrators and one administrator of rehabilitation regarding the effectiveness of community school networking for children with Autism after eight months of this collaboration.
Data Collection Tool: Data were obtained through semi-structured interviews
Results: All participating administrators and teachers underscored the changes in social and behavioral patterns of autistic children which included an imitation of positive behaviors from peers, acceptance, and awareness as strengths of community school networking model. However, major challenges faced were unacceptability from parents of normal children, learning differences, curriculum modifications, time constraints and dependency on the resource teacher. The administrators and teachers recommended that creating awareness programs for parents of normal children, curriculum modifications and in-house psychologists can further facilitate inclusion of children with disabilities.
Conclusion: It was concluded that community school networking model can assist inclusive education and encourage engagement for all children, including those who are autistic.
Probiotics are living microbial adjuncts which has valuable effects on host body incuding improved food digestion, enhancing host‟s immune responses, modification of host associated microbial community and thus, improving the overall ambient environment. Since increase in multidrug resistance pathogens makes the standard treatments in-effective, probiotic have been developed sccessfully as alternate therapeutic drugs in recent era. Commercialized synthetic drugs (e.g. sulfa drugs) have also been interlinked with severe side effects to the hosts which have ultimately lemmatized their use. Bacteriocin (peptide antimicrobial) producing bacteria that can competitively exclude pathogens in the host, is an emrging approach towards novel therapeutics. These defensive proteins are released by 95% of bacterial species in their natural habitat to compete with one another for food and space. Purified bacteriocins of Lactobactericeae (lantibiotics) have been commercialized as prebiotics after successful experiments in laboratory animal. Bacteriocins of Gram negative bacteria (colicins and microcins) are also able to competitively exclude closely related bacterial species when fed to model organism and livestock. The current study was carried out to isolate commensal Gram negative strains that can hinder the growth of common pathogens by producing colicins and microcins against them. Commensal E.coli strains from cattle, sheep and humans were the choice of study since they were expected to better adapt to the host body when administrated as probiotics. Phenotypic inhibitory activity was determined, followed by genomic detection of various colicins, microcins and virulence determinants among the active isolates. Moreover, these strains were also evaluated for antibiotic sensitivity profile, pH tolerance, hemolytic activity and entero-invasive property. The E.coli strains were also sorted into phylogenetic groups of E.coli i.e. A0, A1, B1, B2, D1 and D2. From 300 fecal samples of cattle, sheep and human, 675 lactose fermenting Gram negative colonies were selected, out of which 513 were confirmed as E.coli based on biochemical characterization. For accuracy of further tests, 465 E.coli isolates (n=155/ sample) were evaluated in vitro for growth inhibition of five Gram negative species i.e. E.coli O157:H7, E.coli O26:H11, Salmonella enterica, Klebsiella pneumonia and Pseudomonas aeruginosa. Only 68 isolates (26.4% cattle, 25% sheep and 48.5% human) produced significant growth inhibition among which 41.1% inhibited E.coli O157:H7, 35.2% E.coli O26:H11, 30.7% S. enterica, 24.8% P. aeruginosa and 13% inhibited K. pneumonia. Genomic enumeration of colicin and microcin determinants of these antagonistic E.coli isolates revealed that each of the isolate produced at-least one colicin while the microcins were rarely found in commensal species. The highest prevailing colicin was col E6 (70.4%) followed by col Ib (66.3%), E4 (54.28%), E7 (49.9%), col J (42.9%), col M (35.2%) and col Ia (29.3%). Colicins like S4, D, A, E1, E3 and microcins including mH47, mV, mB17 and mC7 were prevalent at intermediate levels (< 25%) while col E2, 10 and mB17 were rarely found (< 5%) in E.coli isolates. Determinants for col B, K, 5 and mL were not perceived in any E.coli isolate. Phylogenetic grouping revealed that group B2 E.coli was more prevelant (i.e. 27.9%) among human samples followed by 13.2% in sheep and 11.7% in cattle while group D1 was the second most prevalent group i.e. 16%. Almost 13.2 % B1 E.coli, 11.6% A1 and 5.8% A0 E.coli were found among the antagonistic strains from all sources. Virulence gene determinants (i.e. Stx1, Stx2, Hlye, St and eaeA) were detected in 60.2% E.coli isolates while remaining 39.7% were non-pathogenic commensal isolates which were further confirmed by 16SrRNA sequencing. Antibiotic susceptibility profile, hemolysis assay, entero-invasive ability and pH tolerance of all non-pathogenic strains revealed that out of total E.coli strains, four cattle, two human and one sheep derived E.coli strains were the most suitable strains to be considered as probiotic species. However, further insight is suggested to unveil the effects of downstream processes and to discover eminent capsule packaging for these strains so that they can be easily fed to hosts.