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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Molecular characterization of virulent strains of Mycobacterium tuberculosis /

Leong, Wing-man, Hilda. January 2005 (has links)
Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Toll-like Receptor 2-Mediated Recognition of Mycobacterial Lipoproteins and Glycolipids

Drage, Michael Gerald January 2009 (has links)
Thesis (Ph.D.)--Case Western Reserve University, 2009 / Title from PDF (viewed on 19 August 2009) Department of Pathology Includes abstract Includes bibliographical references Available online via the OhioLINK ETD Center

Detection of airborne mycobacterium tuberculosis in an uncontrolled environment

Kirsten, Zubaydah 31 March 2010 (has links)
M. Tech. / Internationally, 9.2 million new cases and 1.7 million deaths occurred from tuberculosis (TB) in 2006. The vast majority of TB deaths occurred in the developing world, that is, Asia and Africa (WHO, 2008). The risk of infection is worsened by overcrowding of healthcare facilities which share re-circulated air without high efficiency particulate arrestance (HEPA) filtration or effective decontamination devices. With emerging and re-emerging infectious agents, the importance of the microbial influence on indoor air quality is gaining momentum around the world. The transmission of Mycobacterium tuberculosis (MTB) is a recognized occupational hazard and the mode of airborne transmission in risk settings needs to be investigated. The current study examined the efficacy of Polymerase Chain Reaction (PCR) for the early detection of airborne MTB using three types of filters: Polytetrafluoroethylene (PTFE), Polycarbonate (PC) and Gelatine, and a sedimentation gel (impaction method). A total of 520 samples, 68 internal positive controls and 68 internal negative controls were tested using two different PCR detection methods. The four different sampling types were each exposed to samples containing an avirulent strain of MTB (H37Ra) and negative controls exposed to aerosolized distilled water in an uncontrolled environment. The air was filtered at a flow rate of 2.5 L/min for a specified time. The filter membranes and sedimentation gel were removed from their respective holders, washed and analysed using conventional and real time (RT) PCR. An additional step using magnetic bead separation was used to assess its performance in overcoming inhibition. The sampling methods used included an in - house preparation of sedimentation gel. This sampling method was used in a study by Vadrot and colleagues in 2004 for detection of airborne MTB using the PCR method. Commercially available filters that were used as sampling methods included PTFE, PC and Gelatine. The detection methods included conventional PCR which detected the MTB complex in three hours, RT-PCR which detected MTB species in 70 minutes and RT-PCR coupled to magnetic bead separation (RT-PCR M) which detected MTB species in 90 minutes. The magnetic bead separation method purified the nucleic acids in the sample by eliminating the inhibitors that were present. The current study showed that by using the magnetic bead capture assay in conjunction with RT-PCR, gave excellent results of 100% sensitivity and 100% specificity when using PTFE, PC and Gelatine sampling methods. The sedimentation gel showed results of 90% sensitivity and 90% specificity. The Gelatine sampling method results showed 100% inhibition with conventional PCR. In conclusion, the use of conventional PCR is limiting for the detection of airborne MTB, possibly due to inhibition factors. In addition, the PTFE filter demonstrated excellent results for all detection methods used. The sedimentation gel did not perform well with PCR and RT-PCR, however gave excellent results with RT-PCR M. The PC filter can be considered the second sampling method of choice after PTFE filter, showing superior results for all diagnostic parameters using RT-PCR M, followed by RT-PCR and then PCR. The greatest application of using this validated method will be in the area of infection control. Environmental practitioners and occupational hygienists would be able to use this method to evaluate environmental control measures and monitor the air quality in healthcare facilities and other workplaces.

Molecular study of mycobacterium tuberculosis complex (MTBC) DNA from Port Elizabeth

Londiwe, Bhembe Nolwazi January 2014 (has links)
Mycobacterium tuberculosis complex (MTBC) is a causative agent of tuberculosis (TB) in humans and animals. The burden of tuberculosis in South Africa is worsened by the concurrent epidemic of HIV. The dynamic of TB epidemics has been investigated and yet little data has been given about the Eastern Cape, particularly Port Elizabeth. The study aimed to investigate the prevalence of drug resistant MTBC and to determine the mutations causing resistance in Port Elizabeth. One hundred and ninety (190) DNA samples isolated from sputum specimen in humans suspected of having TB were amplified using the Seeplex® MTB Nested ACE detection assay. To differentiate Mycobacterium tuberculosis complex (MTBC) members for surveillance purposes a multiplex polymerase chain reaction (PCR) method was done based on genomic regions of differences such as RD1, RD1mic, RD2seal, RD4, RD9 and RD12. Target genes known to confer resistance to first and second-line drugs were amplified and the amplicons sequenced using Big Dye Terminator DNA sequencing kit v3.1 (Applied Biosystems, UK). The patient’s demographic profiles were obtained from the National Health Laboratory Service (NHLS). All hundred and ninety DNA samples tested positive for MTBC using the Seeplex® MTB Nested ACE assay. Results show a high prevalence of extensive drug resistant TB in Port Elizabeth, Eastern Cape Province. One hundred and eighty four (184) DNA isolates were used in the identification of different MTBC species. We ended up working with 184 DNA isolates because we ran out of DNA, and we could not go back to isolate DNA from the affected individuals due to the fact that some patients died, while some have been released to go to their homes. From the 184 DNA isolates 45 (24.5%) isolates were identified to be M. tuberculosis, 94 isolates (51.1%) to be M. bovis BCG and 3 isolates (1.6%) to be M. cannetti. Sequencing results show the position of mutation in each DNA isolate; however in the study we got resistance to MDR to be 100% and 42% pre-XDR while 58% was XDR. These results raise an alarm for the prevalence MDR in MTBC from Port Elizabeth. This is a serious health concern which calls for a need to strategise on the identification of extensive drug resistant TB patients from multi-drug resistant TB patients and ensure monitoring of their treatment.

Molecular and epidemiological characterization of multidrug-resistant Mycobacterium tuberculosis isolates in Johannesburg, South Africa

Mlambo, Charmaine Khudzie 10 January 2012 (has links)
South Africa has a heavy burden of tuberculosis (TB) which is exacerbated by the concurrent epidemic of HIV. Molecular techniques have been used in most developed countries to investigate the dynamics of the TB epidemic, but despite the high prevalence of TB in sub-Saharan Africa, little data on strain types are available outside of the Western Cape. This study aims to provide information on the genotypic characteristics of multidrug-resistant (MDR) Mycobacterium tuberculosis strains in Johannesburg. Patient data obtained from the National Health Laboratory Service (NHLS) referral TB diagnostic laboratory and from Sizwe hospital, a MDR-TB referral hospital, were used to determine the risk factors for treatment outcomes in patients with MDR tuberculosis. Multidrug-resistant M. tuberculosis isolates from over 100 clinics and hospitals in Johannesburg were stored for the study. Spoligotyping and MIRU-VNTR were used to genotype the strains. Drug susceptibility profiles showed that 238 (55%) of the 434 M. tuberculosis isolates tested were resistant to streptomycin and ethambutol, in addition to being resistant to rifampicin and isoniazid. A comparison of spoligotyping results with the international spoligotyping database (SITVIT2) showed a total of 50 shared international types (SITs). Forty-five shared types, containing 417 isolates (96%) matched a pre-existing shared type whereas 5 shared types (containing 11 isolates) were newly created. Diverse strain types were noted, with Beijing, LAM, EAI, T, S, H and X families being dominant. Spoligotype defined families were split into sub-clusters by MIRU typing, resulting in 76 MIRU international types (MITs), containing 389 isolates and 45 orphan isolates. Spoligotyping showed lower discrimination (Hunter-Gaston discriminative index (HGDI) of 0.917) compared with MIRU typing (HGDI = 0.957) but there was no remarkable difference in the discriminatory power of combined spoligotyping and MIRU (HGDI = 0.962) compared with MIRU typing used alone. Twenty-four loci MIRU-VNTR typing was performed on strains from Beijing and CAS, EAI and H families to identify loci with high discriminatory power in our region. The proposed 15 MIRU-VNTR locus combination, together with MIRU 39, was found to be sufficient as a secondary typing method for the routine epidemiological investigation of the Beijing family isolates. Non-Beijing families could be sufficiently differentiated by the 15 MIRU locus combination. This study also describes the treatment outcomes of 351 MDR-TB patients at Sizwe hospital, who started treatment between 2004 and 2007, and investigates possible risk factors associated with poor outcomes. Final treatment outcome was available for 324 (92%) of the patients. Treatment success (completion and cure) was recorded in 158 (48.8%) of patients, while 73 (22.5%) had poor outcomes and 93 (28.7%) defaulted. Eleven (3.1%) patients were transferred out to another health facility and 16 (4.6%) had no recorded final outcome. The proportion of successful treatment increased significantly over time. Univariable and multivariable analysis (P = 0.05) identified the year of MDR-TB diagnosis and spoligotype-defined families as factors associated with treatment outcome. No associations were found between treatment outcome and HIV status, previous TB and additional MDR resistance to either streptomycin or ethambutol. The patient isolates were also characterised molecularly, complementing the study of isolates from Johannesburg alone, and providing information for the Gauteng Province. A sub-study illustrating genotypic diversity of the families constituting extensively drug-resistant TB (XDR-TB) strains in South Africa was conducted subsequent to the nosocomial outbreak in KwaZulu Natal (KZN). The results show that multiple, parallel development of resistance, rather than transmission alone, also plays an important role in the incidence of this extended form of resistance.

IL-4/IL-13-inducible lincRNA-MIR99AHG regulates macrophage polarization and promotes intracellular survival of Mycobacterium tuberculosis

Gcanga, Lona 21 January 2021 (has links)
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Non-coding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long non-coding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains under-studied. Here, we identify an immunoregulatory, lincRNA-MIR99AHG, which is upregulated in macrophages upon IL-4/IL-13 stimulation and downregulated after Mtb infection and in active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we employed antisense GapmeR-mediated lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with LNA-GapmeRs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced proinflammatory cytokine production. In addition, in vivo treatment with MIR99AHG LNA-GapmeRs reduced the mycobacterial burden in the lung and spleen. In vivo LNA-GapmeR treatment experiments demonstrated a role of lincRNA-MIR99AHG as a regulator of macrophage polarization and a host-mediated response post Mtb infection. Further, lincRNA-MIR99AHG translocated to the nucleus and interacts with a high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation to promote Mtb growth and a possible for host-directed targeting or for adjunctive therapeutics against TB.

Pangenome modeling for analyzing the evolution of Mycobacterium tuberculosis.

January 2014 (has links)
泛基因组的概念来源于比较分析同一微生物物种的多个基因组。泛基因组分析已经被用于研究病原微生物基因组的变化,并且揭示了与菌株进化和宿主适应相关的特异基因。目前泛基因组研究主要集中在估计不同物种的泛基因组大小。但是对泛基因组的结构进行进化分析的生物信息方法还有待开发,以便研究不同基因在不同菌株的进化,比如基因的获得或者丢失,或者共同进化的基因簇。这样的分析方法可以把基因和菌株之间的表型关联起来,为进一步的生物学实验提供线索。 / 为了研究结核分枝杆菌种和分枝杆菌属泛基因组进化的规律并揭示其生物学意义,本论文开发了两种泛基因组数据分析的生物信息学方法。第一个是基于局部最大简约计算的祖先状态重构算法。它被用来分析结核分枝杆菌北京型全基因组水平插入/缺失序列(indels)的进化。分析表明基因组退化不仅塑造了该物种不同亚种的形成,并且也塑造了同一亚种不同亚型的分化,比如北京型。该分析还找出了北京型全基因组水平的RD区域和各种被中断的基因,这些基因可能同北京型的毒力进化相关。同时,该算法提供了另一个理解简约分析的视角;该视角可以把统计分析引入到该算法中。本论文提出的第二个模型是基于泛基因组进化的基因聚类模型。通过计算泛基因组中不同基因家族的分布频率,结合基于图论的聚类算法,该模型可以找出泛基因组中共同进化的基因聚类。对分枝杆菌属的泛基因组进行聚类分析发现了不同类别的基因簇,它们与不同分枝杆菌种的表型进化相关。这些结果说明了,一方面结核分枝杆菌在进化过程中丢失大量环境相关的基因;另一方面,它可能通过水平基因转移获得一些基因,特别是PE/PPE基因家族。因此,结核分枝杆菌可能是通过不断的基因组收缩,从一个环境菌种进化为与宿主共进化的病原菌。 / 总地来说,上面的两种方法能够被有效地用于结核分枝杆菌种和分枝杆菌属的泛基因组分析。 将来的工作可以考虑进一步引进随机模型;同时需要建立分枝杆菌的泛基因组数据库,以面对大规模测序的需求。 / Comparative analysis of multiple genomes of the same microbial species has led to the concept of pangenome to characterize the variations of gene content in different strains and to study their relationship to strain phenotype variations. Pangenome studies of microbial pathogens have identified strain-specific genes that may play roles in the evolution and adaptation of the pathogens. In previous studies, much attention was paid to estimate the size of the pangenome of different microbial species. But it is also important to develop bioinformatic methods for analyzing the evolution of the pangenome of a species, such as gene gain and loss or coevolution of clusters of genes, which may help to associate genotype variations with phenotype variations of a microbial species, and thus provides biological insights for further studies. / In this thesis, to analyze the pangenome consisting of complete mycobacterial genomes from public database and additional five Mycobacterium tuberculosis (MTB) Beijing genotype genomes sequenced by our own project, two bioinformatic approaches have been developed. The first is a local parsimony ancestral state reconstruction method, which was used to analyze genome-wide indels evolution of the MTB Beijing genotype. The key finding was that reductive evolution shaped the formation of not only different MTB species, but also different subspecies or genotypes, such as the Beijing genotype, for which genome-wide deletions of large RDs and disruption of individual genes were identified. This finding might have implications for the virulence evolution of the Beijing genotype. The method also provides an alternative perspective to understand parsimony analysis in phylogenetics, which can be used to incorporate statistical analysis into the method. / The second approach developed is a pangenome phyletic model for analyzing the coevolution of genes in the pangenome of a microbial species. This phyletic model calculates coevolution scores of gene frequencies in a pangenome. And graph-based clustering is used to identify coevolved clusters of genes. Applying this method to the genus Mycobacterium helped us to identify various gene clusters, from conserved core clusters of housekeeping genes to species-specific clusters, including genes related to pathogenesis. The key finding was that different MTB species have arose from their mycobacterial ancestor mainly by loss of many environmental related genes. On the other hand, gain of genes has also occurred within the MTB genomes, especially the clusters of the PE/PPE genes. This finding implied that the MTB species have undergone reductive evolution from an environmental species to adapt to and coevolve with their specific hosts. / In conclusion, the two methods were shown to be powerful in analyzing the pangenome of the MTB species and also of the Mycobacterium genus, and have provided useful insights into their genome and virulence evolution for further studies, including both pathogenesis related genes and genotyping genetic markers. Future works in that direction is to introduce stochastic models of gene evolution into these two methods. Finally, this work indicated that pangenome modeling is critical and can provide a good starting point for comprehensive pangenome sequencing of mycobacteria. Therefore, a database of Mycobacterial genomes for integrative pangenome annotation and evolutionary analysis should be developed. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Haokui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 121-135). / Abstracts also in Chinese.

Wild-type minimal inhibitory concentration distributions of secondline drugs in mycobacterium tubercolosis complex clinical isolated in relation to recommended critical concentrations in Limpopo Province, South Africa

Seloma, Ngwanamohuba Mologadi January 2016 (has links)
Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2016. / The reference phenotypic methods for Mycobacterium tuberculosis drug susceptibility testing are qualitative and based on drug critical concentrations. Limitations include lack of standardization and variations in laboratory preparation of drug stock solutions. The recommended critical concentrations are determined by consensus and experience rather than scientific data. Consequently incorrect and inadequate susceptibility breakpoints are used and patients receive ineffective antimicrobial therapy. The determination of wild-type minimal inhibitory concentration distribution is an important tool used by European Committee for antimicrobial susceptibility Testing (EUCAST) to establish clinical breakpoints in Europe. This could be applicable in South Africa. Aim To determine wild-type minimal inhibitory concentration distributions of first and secondline drugs against Mycobacterium tuberculosis complex clinical isolates and compare these with the recommended critical concentration in Limpopo province. Methods A sample of 101 Mycobacterium tuberculosis complex positive cultures were collected from National Health Laboratory Services in Polokwane (Limpopo province) and subcultured on BACTEC MGIT 960 system. The isolates were inoculated on MYCOTB MIC plates to determine the wild-type MIC distributions of first and second-line drugs. The data were compared with currently recommended critical concentrations. DNA was extracted and amplified by PCR. Genotypic drug susceptibility testing was performed using GenoType MTBDRplus version 2.0 and GenoType MTBDRsl version 2.0 for the first- and second-line drugs, respectively. Genotyping of clinical isolates was performed to determine M. tuberculosis strain families using spoligotyping. vi Results Wild-type MIC distributions range reported in this study are as follows rifampin (≤ 0.12 - 0.5 μg/μg/ml), isoniazid (≤ 0.3 - 2.00 μg/ml), rifabutin (≤ 0.12 - 0.25 μg/ml), ethionamide (≤ 0.12 - 5 μg/ml), ethambutol (≤ 0.5 - 2 μg/ml), streptomycin (≤ 0.25 - 0.5 μg/ml), paraaminosalicylic (≤ 0.5 - 4.0 μg/ml), cycloserine (≤ 2 -16 μg/ml), amikacin (≤ 0.12 - 0.5 μg/ml), kanamycin (≤ 0.6 -2.5 μg/ml), moxifloxacin (≤ 0.6 - 0.5 μg/ml), ofloxacin (≤ 0.25 - 1 μg/ml). GenoType MTBDRplus detected (n= 68, 67%) rifampin resistance (MUT 3=26, MUT 2=18, MUT 2B=8) on the rpoB gene. Isoniazid resistant (n=20, 19.8%) was detected katG MUT (n=20, 19.8%) on katG gene (S315T1). Genotypic resistance to second-line drugs determined by GenoType MTBRsl detected no mutations in (n= 98, 97%) isolates on gyrA, gyrB rrs and eis gene and (n=3, 2.9%) isolates non mycobacterium tuberculosis complex were detected. The frequency and percentage of Mycobacterium tuberculosis family strain were identified in (n= 81, 80%) of the clinical isolates which matched 18 pre-existing shared types. The results showed high genotype diversity with the Beijing strain (n= 30, 29.7%) and T family (n= 19, 18.8%) dominating. Twenty isolates (19.8%) had no shared types thus reported as orphan. Conclusion The findings obtained in this study suggest wild-type Minimal Inhibitory Concentration distributions may be considered when setting clinical breakpoints. Discordant results were observed between phenotypic and genotypic DST for rifampin, isoniazid, streptomycin, rifabutin and ethambutol, suggesting that breakpoint concentrations for some drugs are set too high while others are too low. The Mycobacterium tuberculosis clinical isolates displayed diverse family strain with Beijing and T strain predominate breakpoints for first-line and second-line drugs used in Mycobacterium tuberculosis treatments. Poster Presentations Poster presented at faculty of Health science first annual research day on Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using MYCOTB MIC plate. University of Limpopo Tiro hall 16th to 17th September 2014. Poster presented at National Health Laboratory Service Pathology Research and Development Congress (PathReD) on Determination of families strains of Mycobacterium tuberculosis circulating in Limpopo Province, South Africa. Emperors Palace 14th April-16th April 2015.

Perfil molecular de Mycobacterium tuberculosis en muestras biológicas del tracto respiratorio inferior de pacientes limeños con sospecha de tuberculosis

Quispe Huamanquispe, Dora Graciela January 2009 (has links)
La tuberculosis es una enfermedad infecciosa que constituye un grave problema de salud pública a nivel mundial principalmente en países en vías de desarrollo, como el Perú. Las limitaciones de los métodos clásicos de diagnostico (baciloscopía y cultivo) así como la alta frecuencia de ésta enfermedad han creado la necesidad de implementar nuevas estrategias para incrementar la sensibilidad de las pruebas y a su vez reducir el tiempo necesario para establecer el diagnostico confirmatorio. Considerando que los estudios correspondientes a la región del Tracto Respiratorio Inferior (TRI) son escasos, el objetivo de esta tesis fue determinar el perfil molecular de Mycobacterium tuberculosis en muestras biológicas del TRI de pacientes limeños con sospecha de tuberculosis. En el presente trabajo se evaluaron 43 muestras de pacientes limeños con sospecha clínica de tuberculosis las cuales fueron obtenidas a través de lavado bronquial, aspirado bronquial, secreción bronquial, lavado bronco-alveolar, aspirado bronco-alveolar y broncofibroscopía. De estas muestras se extrajo DNA y se realizó la prueba del PCR - Nested, para ello se empleó como secuencia diana el gen de la proteína A (65KDa) de Mycobacterium tuberculosis, los productos amplificados fueron evidenciados mediante electroforesis en gel de agarosa y tinción con bromuro de etidio. Los resultados obtenidos muestran que el 77% de las muestras procesadas poseen DNA de Mycobacterium tuberculosis, demostrándose la alta sensibilidad y especificidad del método empleado, el 82% de estas muestras correspondieron a la región del árbol bronquial superior, debido a que esta región es el punto de partida para la diseminación de esta micobacteria hacia otras regiones del tracto respiratorio y además, a que la mayoría de muestras fueron tomadas directamente de esta región. Asimismo, la aplicación de la prueba PCR- Nested incrementó la sensibilidad de detección 5.5 veces con respecto a un único evento de amplificación, lo cual demuestra la utilidad de esta prueba en el análisis de material biológico con baja carga micobacteriana como las empleadas en este trabajo. Finalmente, se concluye que la prueba PCR- Nested es un método altamente sensible y específico para detectar DNA de Mycobacterium tuberculosis a partir de muestras procedentes del TRI. / Tuberculosis is an infectious disease that constitutes a serious public health problem worldwide, mainly in developing countries such as Peru. The limitations of the traditional methods of diagnosis (smear and culture) as well as the high incidence of this disease have created the need to implement new strategies to increase the sensitivity of tests to reduce time to establish a confirmed diagnosis. Since there are not many researches at the region of the Lower Respiratory Tract (LRT), the objective of this study was to determine the molecular profile of Mycobacterium tuberculosis in biological samples taken from the LRT of patients with suspicion tuberculosis. In the present study, 43 samples from patients with clinical suspicion of tuberculosis were evaluated. The samples were obtained through bronchial lavage, bronchial aspirate, bronchial secretions, bronco-alveolar lavage, and sucked bronco-alveolar broncofibroscopy. DNA extraction was prepared from each sample, and it was used in a PCR- Nested test targeting the gene encoding the protein A (65KDa) from M. tuberculosis. The results showed that DNA from M. tuberculosis was detected in 77% of the processed samples, revealing a high sensitivity and specificity of this method. From the positive samples, 82% corresponded to those obtained from the bronchial tree top region. This last result is explained because of the fact that most of the samples were taken directly from this region, which is the starting point for the mycobacteria dissemination toward other regions of the respiratory tract. In addition, the application of the PCR-Nested test increased the sensitivity of detection by 5.5 fold compared to a single event amplification, demonstrating the usefulness of this test in the analysis of biological material with low mycobacterial load as those used in this work. Finally, we conclude that the PCR-Nested test is a highly sensitive and specific method for detecting DNA from Mycobacterium tuberculosis in samples from the LRT.

Rapid diagnosis of isoniazid resistant mycobacterium tuberculosis by high resolution melting (HRM) assay

Chan, Ming-yan, 陳明恩 January 2012 (has links)
Mycobacterium tuberculosis (MTB) is a major infective agent causing human tuberculosis (TB) in the worldwide. Although tuberculosis can be treated by a six-month course of antibiotics, the prevalence of extensively drug-resistance TB (XDR-TB) made the disease becomes a global health problem. In addition to the conventional MTB detection methods, molecular methods become significant in drug resistant MTB detection which can enhance effective drug treatment. In this study, 200 MTB respiratory specimens were collected from patients with suspected tuberculosis in Tuen Mun Hospital in Hong Kong. Based on the culture method as a gold standard for MTB detection, the presence of MTB in clinical samples was determined by IS6110single tube nested real-time PCR. In addition, by using High Resolution Melting (HRM) analysis, the presence of mutant type KatG315 gene for detecting isoniazid resistant MTB was determined. Among 66 MTB culture positive samples, 10 samples had positive acid fast bacilli (AFB) smears giving the diagnostic sensitivity 15.1%. IS6110 single tube nested PCR was amplified in 51 specimens giving 77.2% MTB detection sensitivity and 97.8% specificity. Among 51 samples positive for IS6110 PCR, 66.7% showed successful amplification in subsequent KatG-HRM assay. Two samples were confirmed to be isoniazid (INH) resistance in Public Health Laboratory Centre (PHLC). However, there was only one sample showing detectable KatG315 mutation in clinical specimen by using HRM while the other was only detected in the corresponding culture isolate. From the result of this study, single tube nested real-time PCR demonstrated MTB detection in clinical samples and INH resistant strain with KatG315mutationcan be detected by HRM analysis. Early detection of mycobacteria allow earlier treatment of the patient, thus transmission of the disease can be controlled. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

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