Detection, susceptibility testing and whole genome sequencing of mycobacterium tuberculosis isolated from cerebrospinal fluid of patients with suspected tuberculous meningitis

Noraini Philip, (2016) Detection, susceptibility testing and whole genome sequencing of mycobacterium tuberculosis isolated from cerebrospinal fluid of patients with suspected tuberculous meningitis. Masters thesis, Universiti Malaysia Sabah.

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Abstract

Tuberculous meningitis (TBM) represents roughly 1% of all cases of tuberculosis (TB) and it is the most severe form. Early diagnosis is difficult as current laboratory tests which are the smear and culture lack sensitivity to detect the few tubercle bacilli in cerebrospinal fluid (CSF). Diagnosis based on molecular methods have been shown to have a higher sensitivity compared to smear and culture. Treatment of TBM requires several months of treatment with anti-TB drugs that cause many side effects. Alternative drug from medicinal plants can be an important source of new antimicrobial agents with little toxicity. Mycobacterium tuberculosis(M. tuberculosis} is the etiologic agent of TB and is shown to have genetic variation between each strain that may contribute to the varied outcome in M. tuberculosis infection. In this study, we evaluated the diagnostic value of smear, culture, multiplex PCR and GeneXpert MTB/RIF to detect M. tuberculosis in CSF samples from patients with suspected TBM registered in Queen Elizabeth Hospital (QEH). We also evaluated the anti-mycobacterial activity of Aloe vera (A. vera) and Allium sativum (A. sativum) against the M. tuberculosis isolate. Whole genome sequencing (WGS) was done to the M. tuberculosis isolate in order to understand the genome feature and genetic variation. CSF samples were centrifuged and the deposits were used for conventional and modified Ziehl-Neelsen (ZN) stain, microscopic observation drug susceptibility (MODS) assay and multiplex PCR targeting MPB64 and IS6110genes. GeneXpert MTB/RIF was performed according to the manufacturer's instruction. Drug-susceptibility testing (DST) was done to determine the susceptibility of M. tuberculosis clinical isolate to isoniazid (INH) and rifampicin (RIF) by using the MODS assay. The M. tuberculosis isolate was further tested with water extract of A. vera and A. sativum at concentration ranged between 0.05 mg/ml - 3.0 mg/ml using the MODS assay. WGS was done using the Pacbio platform and annotation and comparative analysis were done using GeneMarks, RAST and BLAST. Of the 55 CSF samples collected, 12 (21.8%) samples were positive by multiplex PCR, 3 (5.4%) samples were positive by GeneXpert MTB/RIF and only 1 (1.8%) sample was positive by smear and culture.The positive culture was susceptible to both INH and RIF at concentration of 0.4 µg/ml and 1 µg/ml respectively. Anti-mycobacterial activity of A. vera and A. sativum extracts showed minimum inhibitory concentration (MIC) of 1.5 mg/ml and 0.15 mg/ml respectively. Comparative genome analysis showed that the genome of M. tuberculosis isolate has variation compared to the reference genomes. In this study, multiplex PCR showed higher sensitivity for detecting M. tuberculosis in unconfirmed TBM patients and has the potential to be used as a diagnostic method. A. vera and A. sativum can be a new source of anti-mycobacterial agents for the development of drugs in treatment of TB. The genome feature of the M. tuberculosis isolate has variation with the reference genomes.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Tuberculous meningitis (TBM), tuberculosis (TB), diagnosis, molecular methods, treatment, antimicrobial agents
Subjects: R Medicine > RC Internal medicine
Divisions: SCHOOL > Biotechnology Research Institute (BRI)
Depositing User: Munira
Date Deposited: 24 Oct 2017 03:39
Last Modified: 24 Oct 2017 03:39
URI: http://eprints.ums.edu.my/id/eprint/11318

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