Mycobacterium tuberculosis

Presented byARCHANA GAUTAM

3rd,semesterBABA SAHEB BHIMRAO AMBEDKER UNIVERSITY

LUCKNOW

AbstractStrain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineagesthan current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.

Introduction

Dr. Robert Koch discovered

the

tuberculosis bacillus. (1882)

He received the Nobel Prize inphysiology or medicine in 1905 for thisdiscovery

After 100 years later

The bacillus causing tuberculosis,

airborne disease, M.tuberculosis

was identified and described on 24

March 1882 by Robert Koch .

Mycobacterium tuberculosis(MTB)

Basic knowledge about MTB • Mycobacterium tuberculosis, a small, aerobic, non-motile bacillus.• "Gram-positive“• Tuberculosis typically spread through the air when people who have

an active TB infection cough, sneeze etc.symptoms

• The classic symptoms of active TB infection are a chronic cough with blood-tinged sputum, fever, night sweats, and weight loss.

Diagnosis• Radiology (commonly chest X-rays)• microbiological culture.• tuberculin skin test (TST) etc.

M.tuberculosis cont......

M. tuberculosis under

microscope M. tuberculosis under SEM

Kingdom : Bacteria

Phylum: Actinobacteria

Order: Actinomycetales

Suborder:Corynebacterineae

Family: Mycobacteriaceae

Genus: Mycobacterium

Species: M.tuberculosis

M. tuberculosisBacterial colonies

Tb affects many parts

of body but mainly

divided into two parts

i.e. Pulmonary and.

Extra-pulmonary

TB Drug resistance MDR

Cont....

Cont.....

Barcode

Single nucleotide

polymorphism

(SNP)

According

to

research

paper

Why SNPs ? ? ?

The selection of suitable SNPs is crucial for the success of a typing assay.. The need for a suitable sequence .

SNPs for were chosen using following criteria:

SNP is specific for the lineage of interest SNP is not in a region where a genomic deletion has been described (to ensure

binding of oligonucleotides and subsequent allele-calls for all strains) No other SNP less than 50 bp up- or downstream (to avoid mismatches in

oligonucleotide annealing) SNP is not a transition (i.e. no nucleotide change A>G, G>A, C>T or T>C; lower

unspecific signal because no alternate base pairing possible) SNP is in a coding sequence (deletions or insertions less likely) SNP is in an essential gene (deletions less likely) SNP change is synonymous (no amino acid change; therefore lower selective

pressure acting on SNP compared to non-synonymous SNP)

SNPs(molecular marker)

• David Stucki et al (2012) to assess genotype-phenotype associations,

phylogenetically robust molecular markers and appropriate genotyping tools are

required.

Treatment success has recently

exceeded the global target of

85% Case Detection in DOTS areas has recently

attained global target of 70%

TB

in

INDIA

Drug resistance problem in India and globally was worsening

INDIA is the highest TB burden country accounting for more than one-fifth of the global incidence

Indonesia

6%

Nigeria

5%

Other countries

20%

Other 13 HBCs

16% China

14%

South Africa

5%Bangladesh

4%

Ethiopia

3%

Pakistan

3%

Phillipines

3%

India

21%

Source: WHO Geneva; WHO Report 2014: Global Tuberculosis Control; Surveillance, Planning and Financing

Global annual incidence = 9.4 million

India annual incidence = 1.96 million

MDR-TB cases in the hospital

The number of (MDR)-TB cases in the country had increased five-fold between2011and2014.Studies showone-third of theMDR-TB casesare resistant tofluoroquinolones, which arecritical for MDR-TB treatment.

Deshakalyan Chowdhury/Agence France-Presse/Getty ImagesTuberculosis patients at a hospital in Kolkata, April 07, 2008

Problem of TB in India• Estimated incidence

– 1.96 million new cases annually

– 0.8 million new smear positive cases annually

– 75 new smear positive PTB cases/1lakh population per year

• Estimated prevalence of TB disease– 3.8 million bacillary cases in 2000

– 1.7 million new smear positive cases in 2000

• Estimated mortality– 330,000 deaths due to TB each year

– Over 1000 deaths a day

– 2 deaths every 3 minutes

Problem of TB in India (contd)

• Prevalence of TB infection – 40% (~400m) infected with M. tuberculosis (with a 10% lifetime risk

of TB disease in the absence of HIV)

• Estimated Multi-drug resistant TB– < 3% in new cases

– 12% in re-treatment cases

• TB-HIV– ~2.31 million people living with HIV.

– 10-15% annual risk (60% lifetime risk) of developing active TB disease.

– Estimated ~ 5% of TB patients are HIV infected

This Wednesday, the WHO declared drug-resistant TB a public health crisis, saying the airborne disease is rising faster than countries can treat it. The WHO also said many cases are still going unreported

Evolution of TB Control in India

• 1950s-60s Important TB research at TRC and NTI

• 1962 National TB Programme (NTP)

• 1992 Programme Review• only 30% of patients diagnosed;

• of these, only 30% treated successfully

• 1993 RNTCP pilot began

• 1998 RNTCP scale-up

• 2001 450 million population covered

• 2004 >80% of country covered

• 2006-till today Entire country covered by RNTCP

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

120%

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

Annualised New S+ve CDR Success rate

•Population projected from 2011 census•Estimated no. of NSP cases - 75/100,000 population per year (based on recent report)

Annualized New Smear-Positive(NSP) Case Detection Rate (CDR)and Treatment Success Rate(TSR) in DOTS Areas, India, 2002-2014*

2002 2003 2004 2005 2007 2008 2009 2010 2011-14

% TSR

Articles/News on TB in INDIA• Drug-resistant TB cases on rise in north India, reveals

PGI research Express News Service : Chandigarh, Thu May 09 2013, 02:33 hrs

• Study finds doctors also adding to burden of drug-resistant TB cases Times Of India Jul 19, 2014, 01.13 AM

• NEW DELHI: In the wake of multi-drug resistant (MDR)-TB cases increasing in India, Union Health Minister Harsh Vardhan on 6th,september 2014 launched India's first National Anti-Tuberculosis Drug Resistance Survey, 2014-2015. (NDTV REPORT)

• "It often happens that patients who have TB disease stop taking the drugs soon after they start feeling better. They can become sick again as if the drugs are not taken correctly, the TB bacteria may still remain alive and the person may become resistant to those drugs and will have to then given second-line drugs, “(NDTV REPORT)

• In December, India’s director general of health services, Dr. Jagdish Prasad told The Wall Street Journal that India planned to provide free drugs to patients being treated by private doctors so that the government could make sure all patients are properly tested and have an accurate assessment of disease burden (Wall Street Journal )

Discussion

• In conclusion, using the whole-genome sequences of over 1,601 MTBC isolates, we characterize a high-resolution map of polymorphisms consisting of more than ninety thousand SNPs. This genomic variation is used to infer phylogenetic relationships both inter- and intra-lineage to an unprecedented level of resolution, and lead to the development of an extendable nomenclature for sublineages. We identify a panel of 62 robust SNP markers (of 413 suitable alternatives) that can be used to construct high resolution and reproducible phylogenies, which can be incorporated in diagnostic assays and assess genotype–phenotype associations. Future work should focus on other types of lineage-specific polymorphisms (for example, insertions, deletions and large structural variants), which are less common than SNPs, but may have major functional consequences. The proposed system has the flexibility to incorporate novel strain types should they be reported. Incorporating anti-tubercular drug-resistance loci will further enhance the usefulness of the barcode as an important tool for tuberculosis control and elimination activities worldwide.

Result

• A genomic analysis was performed on whole-genome sequences of 1,601

MTBC isolates from eleven independent sequencing studies from different

areas of the world.

• The technique could identify super-spreaders and predict the existence of

undiagnosed cases, potentially leading to early treatment of infectious

patients and their contacts.

• It may be used to classify clinical isolates to evaluate tools to control the

disease.

• In future decreased the rate of multi drug resistance (MDR) TB.

References

Research paper’s references1.David Stucki, Bijaya Malla, Simon Hostettler, Thembela Huna,et al(2012) Two

New Rapid SNP-Typing Methods for Classifying Mycobacterium tuberculosis Complex into the Main Phylogenetic Lineages.

2.Gutacker MM, Mathema B, Soini H, Shashkina E, Kreiswirth BN, et al. (2006) Single-nucleotide polymorphism-based population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites. J Infect Dis 193: 121–128. doi:10.1086/498574.

3.Wang L, Luhm R, Lei M (2007) SNP and mutation analysis. Adv Exp Med Biol

593: 105–116. doi:10.1007/978-0-387-39978-2_11.

Book’s references1.Brock biology of microorganism Madigan et al (2011) 13th,edition

2.Plant biotechnology the genetic manipulation of plants Slater et al (2012) 2nd,edition

3.Prescott 7th, edition

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