Direct detection of isoniazid-resistant mycobacterium tuberculosis using HybProbe-based real time PCR

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Direct detection of isoniazid-resistant mycobacterium tuberculosis using HybProbe-based real time PCR


Author: Tam, Chiu Mo
Title: Direct detection of isoniazid-resistant mycobacterium tuberculosis using HybProbe-based real time PCR
Degree: M.Sc.
Year: 2016
Subject: Mycobacterium tuberculosis -- Diagnosis
Polymerase chain reaction -- Diagnosis
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: 94 pages : illustrations
Language: English
Abstract: Mycobacterium tuberculosis (MTB) is one of the most tenacious infectious diseases that have been hard to be elucidated for the past century.Not only does the treatment take time for the patients to heal, but drug resistance also easily persists as the treatment goes on. Therefore it is for the interest of the public and the doctors to utilize the best and most suitable method in diagnosing the drug resistance of M. tuberculosis. Isoniazid-resistant MTB is very prevalent due to the mutation of katG315 gene that can be detected by conventional culture and sensitivity test. Even though the traditional microbiology testing is the gold standard nowadays, it is essential to develop a complementary method that can provide a preliminary result for the doctors to decide the suitable treatment at the earliest stage possible. This study aims to examine the possible solution of utilizing HybProbe-based real-time PCR in response to the liability of other related methods. The study involved two parts of experiments aiming to find a plausible solution for mutation detection using direct specimen, while comparing two popular PCR instruments used in clinical laboratories nowadays. The first part was a retrospective study - 28 clinical sputum samples were cultured and susceptibility test was done by the Clinical Microbiology laboratory team from the Queen Mary Hospital. The colonies from the cultured samples were then extracted into DNA, which were followed by melting analysis with duplex HybProbe real-time PCR proposed in this study. This study targeted katG and mabA genes using LightCycler® 480 and Rotor-Gene® Q as the real-time PCR platforms. Melting temperatures of the wildtypes and any mutation detected at the katG and mabA sites were examined for confirmation in the second part of the study. The DNA was also undergone DNA sequencing for comparison.The second part was a prospective study to examine that direct specimen could also be used under the same platforms and conditions. 56 more recent clinical sputum samples underwent culture and susceptibility test, in the same time having DNA extracted directly from the clinical sputum samples, bypassing the step of DNA extraction from colonies as mentioned in the retrospective study. The DNA obtained then underwent DNA sequencing and the exact same melting analysis as the method used in the retrospective study.The study showed that the sensitivity for Rotor-Gene® Q to detect isoniazid resistant sputum samples was 73.68%, while the sensitivity for LightCycler® 480 to detect isoniazid resistant sputum samples was 94.73%. The huge difference in sensitivity was because of a drop-off in false negative results using LightCycler® 480 due to its ability to detect double peak in melting curve - a detection of either heterozygous alleles or a progressing mutation. Therefore, the study provided an achievable solution in using melting analysis to complement routine conventional diagnostics of isoniazid resistant MTB.

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