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Postgraduate Thesis: Rapid diagnosis of isoniazid resistant mycobacterium tuberculosis by hybridization probe based real time PCR
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TitleRapid diagnosis of isoniazid resistant mycobacterium tuberculosis by hybridization probe based real time PCR
 
AuthorsCheng, Wing-suen.
鄭穎璿.
 
Issue Date2012
 
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
AbstractBackground Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available.
 
DegreeMaster of Medical Sciences
 
SubjectMycobacterium tuberculosis - Molecular diagnosis.
 
Dept/ProgramMicrobiology
 
DC FieldValue
dc.contributor.authorCheng, Wing-suen.
 
dc.contributor.author鄭穎璿.
 
dc.date.hkucongregation2012
 
dc.date.issued2012
 
dc.description.abstractBackground Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available.
 
dc.description.naturepublished_or_final_version
 
dc.description.thesisdisciplineMicrobiology
 
dc.description.thesislevelmaster's
 
dc.description.thesisnameMaster of Medical Sciences
 
dc.identifier.hkulb4833361
 
dc.languageeng
 
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
dc.relation.ispartofHKU Theses Online (HKUTO)
 
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.
 
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License
 
dc.source.urihttp://hub.hku.hk/bib/B4833361X
 
dc.subject.lcshMycobacterium tuberculosis - Molecular diagnosis.
 
dc.titleRapid diagnosis of isoniazid resistant mycobacterium tuberculosis by hybridization probe based real time PCR
 
dc.typePG_Thesis
 
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<item><contributor.author>Cheng, Wing-suen.</contributor.author>
<contributor.author>&#37165;&#31310;&#29887;.</contributor.author>
<date.issued>2012</date.issued>
<description.abstract>&#65279;Background 

Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories&#8217; own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH.

Objective 

The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically.

Result 

In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. 

Conclusion 

katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available.</description.abstract>
<language>eng</language>
<publisher>The University of Hong Kong (Pokfulam, Hong Kong)</publisher>
<relation.ispartof>HKU Theses Online (HKUTO)</relation.ispartof>
<rights>The author retains all proprietary rights, (such as patent rights) and the right to use in future works.</rights>
<rights>Creative Commons: Attribution 3.0 Hong Kong License</rights>
<source.uri>http://hub.hku.hk/bib/B4833361X</source.uri>
<subject.lcsh>Mycobacterium tuberculosis - Molecular diagnosis.</subject.lcsh>
<title>Rapid diagnosis of isoniazid resistant mycobacterium tuberculosis by hybridization probe based real time PCR</title>
<type>PG_Thesis</type>
<identifier.hkul>b4833361</identifier.hkul>
<description.thesisname>Master of Medical Sciences</description.thesisname>
<description.thesislevel>master&apos;s</description.thesislevel>
<description.thesisdiscipline>Microbiology</description.thesisdiscipline>
<description.nature>published_or_final_version</description.nature>
<date.hkucongregation>2012</date.hkucongregation>
<bitstream.url>http://hub.hku.hk/bitstream/10722/173941/1/FullText.pdf</bitstream.url>
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