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Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

ABSTRACT: Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth Indicator Tube (MGIT 960) culture, respectively. Sensitivity and specificity of GeneXpert was 72.7% and 98.5%, respectively by using MGIT 960 as a gold standard (GeneXpert (n = 20, 6.8%) vs MGIT 960 (n = 22, 7.5%)). All Mtb positive cultures were subjected to 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing, Line probe assay (LPA) and MGIT 960- Drug Susceptibility Testing (DST). The rpoB gene was amplified and sequenced for selected isolates. Among our TBM patients, East African Indian (EAI) lineage (n = 16, 72.7%) was most predominant followed by Beijing (n = 3, 13.6%), S-family (n = 2, 9.1%) and Delhi/CAS (n = 1, 4.5%). Three Mtb strains were found to be Isoniazid (INH) resistant by MGIT 960; however LPA revealed that two strains were INH resistant and one strain was multi drug resistant (MDR) (Resistant to Isoniazid and Rifampicin (RIF)). We identified rifampicin resistant isolate with the mutation D516F in rifampicin resistance-determining region (RRDR) and observed discordant results between LPA, GeneXpert and MGIT 960. In addition, GeneXpert showing false RIF resistance was identified (no mutation in RRDR). We conclude that GeneXpert is useful for the diagnostic confirmation of TBM; however a GeneXpert negative sample should be subjected to MGIT 960 culture or LPA to rule out TBM. EAI lineage was the most predominant among TBM patients in South India and associated with drug resistance. The discordance between GeneXpert, MGIT 960 and LPA with respect to rifampicin resistance has to be ruled out to avoid TB treatment failure or relapse.

SUBMITTER: Krishnakumariamma K

PROVIDER: S-EPMC7535050 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

Krishnakumariamma Krishnapriya K Ellappan Kalaiarasan K Muthuraj Muthaiah M Tamilarasu Kadhiravan K Kumar Saka Vinod SV Joseph Noyal Mariya NM

PloS one 20201005 10

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth ...[more]

PMID: 33017455

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Project description:ObjectivesIndia contributes approximately 25% of the 'missing' cases of tuberculosis (TB) globally. Even though ~50% of patients with TB are diagnosed and treated within India's private sector, few are notified to the public healthcare system. India's TB notification policy mandates that all patients with TB are notified through Nikshay (TB notification portal). We undertook this study in a private hospital to assess the proportion notified and factors affecting TB notifications. We explored barriers and probable solutions to TB notification qualitatively from health provider's perspective.Study settingPrivate, tertiary care, teaching hospital in Bengaluru, South India.MethodologyThis was a mixed-methods study. Quantitative component comprised a retrospective review of hospital records between 1 January 2015 and 31 December 2017 to determine TB notifications. The qualitative component comprised key informant interviews and focus groups to elicit the barriers and facilitators of TB notification.ResultsOf 3820 patients diagnosed and treated, 885 (23.2%) were notified. Notifications of sputum smear-positive patients were significantly more likely, while notifications of children were less likely. Qualitative analysis yielded themes reflecting the barriers to TB notification and their solutions. Themes related to barriers were: (1) basic diagnostic procedures and treatment promote notification; (2) misconceptions regarding notification and its process are common among healthcare providers; (3) despite a national notification system other factors have prevented notification of all patients; and (4) establishing hospital systems for notification will go a long way in improving notifications.ConclusionsThe proportion of patients with TB notified by the hospital was low. A comprehensive approach both by the hospital management and the national TB programme is necessary for improving notification. This includes improving awareness among healthcare providers about the requirement for TB notifications, establishing a single notification portal in hospital, digitally linking hospital records to Nikshay and designating one person to be responsible for notification.

Project description:Genome sequencing plays a key role in understanding the genetic diversity of Mycobacterium tuberculosis (M.tb). The genotype-specific character of M. tb contributes to tuberculosis severity and emergence of drug resistance. Strains of M. tb complex can be classified into seven lineages. The Nonthaburi (NB) genotype, belonging to the Indo-Oceanic lineage (lineage 1), has a unique spoligotype and IS6110-RFLP pattern but has not previously undergone a detailed whole genome analysis. In addition, there is not much information available on the whole genome analysis of M. tb isolates from tuberculous meningitis (TBM) patients in public databases. Isolates CSF3053, 46-5069 and 43-13838 of NB genotype were obtained from the cerebrospinal fluids of TBM Thai patients in Siriraj Hospital, Bangkok. The whole genomes were subjected to high throughput sequencing. The sequence data of each isolate were assembled into draft genome. The sequences were also aligned to reference genome, to determine genomic variations. Single nucleotide polymorphisms (SNPs) were obtained and grouped according to the functions of the genes containing them. They were compared with SNPs from 1,601 genomes, representing the seven lineages of M. tb complex, to determine the uniqueness of NB genotype. Susceptibility to first-line, second-line and other antituberculosis drugs were determined and related to the SNPs previously reported in drug-resistant related genes. The assembled genomes have an average size of 4,364,461 bp, 4,154 genes, 48 RNAs and 64 pseudogenes. A 500 base pairs deletion, which includes ppe50, was found in all isolates. RD239, specific for members of Indo Oceanic lineage, and RD147c were identified. A total of 2,202 SNPs were common to the isolates and used to classify the NB strains as members of sublineage 1.2.1. Compared with 1,601 genomes from the seven lineages of M. tb complex, mutation G2342203C was found novel to the isolates in this study. Three mutations (T28910C, C1180580T and C152178T) were found only in Thai NB isolates, including isolates from previous study. Although drug susceptibility tests indicated pan-susceptibility, non-synonymous SNPs previously reported to be associated with resistance to anti-tuberculous drugs; isoniazid, ethambutol, and ethionamide were identified in all the isolates. Non-synonymous SNPs were found in virulence genes such as the genes playing roles in apoptosis inhibition and phagosome arrest. We also report polymorphisms in essential genes, efflux pumps associated genes and genes with known epitopes. The analysis of the TBM isolates and the availability of the variations obtained will provide additional resources for global comparison of isolates from pulmonary tuberculosis and TBM. It will also contribute to the richness of genomic databases towards the prediction of antibiotic resistance, level of virulence and of origin of infection.

Dataset Information

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

Publications

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

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