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Whole Genome Sequencing For Tuberculosis

Whole Genome Sequencing For Tuberculosis

Posted By HealthcareOnTime Team Posted on 2022-02-02

Tuberculosis (TB) has been highlighted in medical history as The Leading Killer. Some common terms for TB worldwide include 'The Captain of all these Men of Death', 'Phthisis', 'Scrofula', 'The White Plague'. The emergence and spread of Drug Resistant (DR) Tuberculosis is a major concern that threatens global health.

Whole Genome Sequencing For Tuberculosis

India has the highest estimated deaths among countries reporting TB statistics to WHO, with 4,36,000 deaths in HIVnegative adults and 9,500 in HIV-positive adults. Rapid diagnosis and identification of DR strains are prerequisites for eradicating TB. Accurate diagnosis aids in customizing individualized treatment, thus reducing infectiousness, morbidity and mortality. Without treatment, about 5 to 10% of infected people develop TB disease at some point in their lives.

Although, there exists a number of technologies for early detection of threats, constant improvisation has elevated the degree of usage for a wider population. Let's look at some interesting detection techniques, with a focus on sequencing, a technological advancement in the field of diagnostics.

Sites of TB Infection
Based on site of infection, TB is classified as,
Pulmonary TB - It is a contagious bacterial infection that affects the lungs. This disease is indicated by symptoms like cough and abnormal chest radiograph.
-Extrapulmonary TB - It affects organs other than the lungs, including larynx, lymph nodes, pleura, brain, kidneys, bones and joints. Infected individuals usually are not infectious, unless they have pulmonary along with extrapulmonary disease or have an infected oral cavity or larynx, or an infected open abscess or lession.
Miliary TB-A potentially fatal form of active TB, occurs when bacteria enters the bloodstream and disseminates to all parts of the body, causing infection in multiple sites.

Drug resistance
After tissue invasion, the concern that adds to severity of TB infection is the property of Mycobacterium tuberculosis (MTB) to develop resistance against the antimicrobial drugs used for treatment. It limits the treatment options thereby putting the patient at higher risk of incurable TB.

Types of drug resistant TB
-Multi drug resistant TB (MDR-TB) MDR-TB is caused by TB bacteria resistant to two of the most important anti-TB drugs- Isoniazid and Rifampicin. It occurs either when a person is infected with a resistant strain or when improper treatment leads to drug resistance in a particular infecting strain. In 2018, India had 58,347 (44%) MDR-TB cases out of 1,30,000 drug-resistant TB cases.
-Extensively drug resistant TB (XDR-TB)
XDR-TB is caused by rare type of MDR-TB that is resistant to Isoniazid, Rifampin, as well as Fluoroquinolones and second-line injectable drugs like Amikacin, Kanamycin, and Capreomycin. Among the MDR-TB patients, XDR-TB rate was found to be 1.3%
-Transmission MTB spreads through infectious droplet nuclei (1-5 microns in diameter) via air, when a person with pulmonary or laryngeal TB disease coughs, sneezes, shouts, or sings.

Types of TB diagnosis
It is necessary to choose appropriate screening or diagnostic testing for detecting mycobacteria, as accurate diagnosis will help in customizing a treatment, thus reducing infectiousness, morbidity and mortality, WHO recommended TB diagnostics tools

-TB Screening Test - Mantoux tuberculin skin test (TST) and interferon-gamma release assays (IGRAs) are two widely used tests for detecting Latent TB infection. The IGRAs include tests like QuantiFERON-TB Gold Plus (QFT-Plus) and T-SPOT.TB test (T-Spot)
-Smear Microscopy - Sputum microscopic examination is the most conventional diagnostic test for pulmonary TB. It is widely used in low-income countries due to its rapidity, low cost and high positive predictive value for TB.
-Chest Radiograph - A posterior anterior radiograph aids in detecting chest abnormalities. These abnormalities may suggest pulmonary TB, but cannot be used to make a definitive diagnosis.
-Lipoarabinomannan Urine Strip Test - Lateral flow urine Lipoarabinomannan strip test (LF-LAM) developed for TB diagnosis is based on detection of mycobacterial lipoarabinomannan antigen in urine. It is used to detect active TB disease in HIV patients.
-Culture (MGIT) and Drug Susceptibility Testing (DST) - Culture is World Health Organization (WHO) recommended gold standard technique for TB diagnosis. Organism isolated on solid and liquid culture medium i only important for definitive diagnosis but also for determining phenotypic DST. Identifying drug resistance at the earliest ensures effective treatment.
-TB-PCR - Nucleic acid amplification tests (NAAT) and alternative methods like ligase chain reaction, strain displacement amplification, loop-mediated isothermal amplification (LAMP), transcription mediated amplification and real time PCR are used for detection and identification of MTB.
-CB-NAAT/ GeneXpert MTB/RIF Test - Cartridgebased nucleic acid amplification test (CB-NAAT) or Xpert MTB/RIF assay, is an automated semi-quantitative nested real-time PCR that uses molecular beacon technology to detect M. tuberculosis complex (MTBC) and rifampin resistance. It is a rapid, highly sensitive and specific assay that generate results in less than 2 hours.
-Line Probe Assay (LPA) - LPA is novel technique designed to identify MTBC and drug sensitivity to Rifampicin and Isoniazid. It uses PCR and reverse hybridization methods for detection of mutations associated with drug resistance. Resistance of second line of drugs can be detected by second line LPA.
-Sequencing Based Technique - DNA sequencing of variable genomic regions allows for the rapid and accurate identification of drug resistant TB. It is based on determination of species-specific nucleotide sequences, which are then compared to known quality-controlled sequences from in-house or commercially available databases.

TB genome sequencing/WGS-TB
Advances in diagnosis and drug susceptibility testing methods for MTBC are required to control transmission as well as provide better individualized treatment against MDR/XDR-TB. One of the important criteria for successfully treating TB patients is to administer the right treatment at an early stage.

This state-of-art sequencing approach overcomes many challenges associated with conventional phenotypic testing as well as the limitations of other less comprehensive molecular tests by providing rapid, detailed sequence information for multiple gene regions or whole genomes of interest. Whole genome sequencing (WGS) is a WHO-approved probe-based method for TB diagnosis and detection of drug resistance, genetic diversity as well as transmission dynamics of MTBC. Additional information obtained by Whole genome sequencing on transmission would help in controlling the outbreak.

WGS-TB can identify mutations in genes that predict phenotypic resistance against anti-TB drugs within the entire region of microbial genome. It also allows for more robust MTBC classification into lineages and sublineages, via the identification of single-nucleotide polymorphisms (SNPs). This emphasizes the importance of Whole genome sequencing in making informed decisions prior to antituberculosis therapy.

Benefits of WGS-TB
-Single genome-wide test would replace multiple panel tests
-Detect drug resistance mutations for a broad range of antibiotics
-Reliable and highly sensitive diagnosis
-Speed up TB diagnosis from months to days
-Rapid diagnosis allows immediate action, avoiding wastage of resources

Testing @ HealthcareOnTime
TB genome sequencing offered by Thyrocare is a WGS based test that combines genome sequencing and analysis for a faster and more sensitive detection of tuberculosis. It is a comprehensive single test solution for DST profiling against 18 anti-TB drugs.


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