The Path Travelled in TB Diagnosis- Milestones achieved!
Physical examination– was the only way to diagnose TB in ancient times. Classical symptoms– hemoptysis, night sweats, and fever formed the diagnostic base.
Discovery of Mycobacterium tuberculosis (MTB)– a breakthrough achieved with Robert Koch’s ﬁndings. Acid-fast staining– allowed microscopic identiﬁcation; a test that remains unchanged from its inception. Tuberculin skin test– gave insight into patients’ previous exposure to tuberculosis.
X-rays– are used to assist in the diagnosis of tuberculosis.
solation of bacteria using culture media– a gold standard, despite its time-consuming nature.
Interferon-gamma release assay– facilitated assessment of T-cell activation when exposed to antigens speciﬁc to MTB
Fluorescent dyes– made it much easier to screen specimens for MTB.
Moleculardiagnostictests– TB diagnosis took a leap of faith with tests facilitating speciﬁc ampliﬁcation of nucleic acids from the organism.
Whole-genome sequencing(WGS)– can identify MTB as well as mutations pertaining to drug resistance. It can be explained by the analogy that WGS compares all the words of two books to check if the books are the same or different, unlike other nucleic acid ampliﬁcation tests which match only the number of chapters.
A State of Art Technique!
The sequence of nitrogen bases (A, T, G, C) is unique to an organism. If this sequence of bases can be known, the unique DNA ﬁngerprint of the entity is identiﬁed. WGS is one such high-end laboratory tool that determines the order of bases in the genome of a living being in a single test. In contrast to other molecular tests that use a few bases to compare genomes, WGS compares millions of bases (entire genome). WGS, the high-resolution, powerful, fast, and affordable way to obtain high-level information can hence, be used to generate accurate data on MTB.
This perilous bacterium has been infecting humans since before recorded history from the hunter-gatherer period that is, more than 70,000 years ago. It has always been a struggle to detect MTB; notorious bacteria that can adapt itself to become resistant to multiple drugs; however, nucleic acid ampliﬁcation tests have given us an edge over it. WGS tops it all by giving the ability to sequence MTB genomes directly from clinical specimens thereby, omitting dependency on the time and labor-consuming in vitro culturing methods for its detection. Not limiting to this, next-generation sequencing (NGS) based assays as with WGS, is an excellent tool for comprehensive drug sensitivity testing (DST). Unlike probe-based assays wherein probe-speciﬁc targets are detected, WGS provides detailed sequence information of the whole genome. Hence, it can even identify single nucleotide polymorphisms (SNPs), allowing analysts to predict the susceptibility of the identiﬁed MTB strain to anti-TB drugs.2,3
Fascinating Molecular Workings
WGS is a comprehensive and accurate genome analysis tool. Sequencing technology involves breaking down the genome into smaller DNA fragments, sequencing them, and then putting them back together in the correct order by adopting bioinformatics. This can be done
either by clone-by-clone or whole-genome shotgun methods. WGS remained quite unaffordable for a long period of time, but with the use of NGS instrumentation, it became cost-effective. The workings of WGS, now widely used in diagnostics, research, and epidemiology are stated below-3,4
4DNA Extraction– It is the ﬁrst step that includes rupturing of cells and extraction of genomic DNA from the organism of interest by using commercially available extraction kits and spin columns.
4Quantiﬁcation of DNA– The concentration of the DNA extracted is measured using a spectrophotometer.
4Tagmentation– It is the tagging and fragmentation of extracted DNA using transposon-mediated reaction. DNA shearing is done using molecular scissors to cut the DNA into small pieces that can be read by sequencing machines. Following this, DNA tags/ bar codes are added so as to identify which piece of sheared DNA belongs to which bacteria.
4PolymeraseChainReaction(PCR)– Ampliﬁcation of DNA, that is, multiple copies of each DNA fragment is generated using PCR. This is done to produce enough quantity of the DNA of interest for sequencing.
4DNALibrary– The pool of fragments produced after PCR is called the DNA library. This is loaded onto the sequencer for further processing.
4WholeGenomeSequencing– From the fragments of DNA, the combination of nucleotides making up an organism is determined and the result obtained is called a DNA read. The tags/barcodes added earlier are used by the sequencer to keep track of which bases belong to which bacteria.
4Bioinformatics Analysis– Millions of DNA reads are produced after sequencing. Bioinformatics makes it possible to put them together in the correct order like pieces of a jigsaw puzzle. These computer analysis tools are used to compare and identify differences amongst bacterial sequences.
Offering an edge in TB diagnosis
MTB has been singly able to keep the medical fraternity on their toes, trying to ﬁnd ways of combating it. Increasing numbers of multidrug-resistant TB cases have made the list of global public health problems. This leaves healthcare professionals with the only one option;
i.e. early and timely detection for effective treatment, and prevention of the emergence of drug-resistant strains. Thus surfaced the requirement and demand for fast yet
reliable diagnostic tests. Molecular tests revolutionized MTB diagnosis, allowing healthcare personnel to identify the bacilli as well as know if it is sensitive to ﬁrst line anti-TB drugs. With the advances in research, a plethora of such tests came into the picture. GeneXpert MTB/RIF assay, loop-mediated isothermal ampliﬁcation (LAMP), MTBDRplus, line probe assay (LPA), WGS, and NGS are to name a few. Although nucleic acid ampliﬁcation tests (NAATs) that are WHO-endorsed include Xpert (GeneXpert), LPA, LAMP, and Truelab, many newer methods are under development or evaluation. The newer technologies include Xpert XDR (can detect extensively drug-resistant TB), point-of-care devices (GeneXpert Omni), and high throughput technologies (WGS and NGS).
WGS offers a high-resolution view of the entire genome and hence, plays multiple roles in TB diagnosis, giving it an advantage over other available NAATs. Few advantages are-2,5
Full genomes of the organism can be sequenced 4Does not need pre-speciﬁed targets
Gives a comprehensive solution with extensive information
A suitable alternative to WHO-endorsed probe-based methods
Detects rare mutations and heteroresistance
Can identify TB and detect drug resistance simultaneously
Has a wide DST range and can efﬁciently identify resistance determining regions
4Can uncover patient harboring XDR-TB isolates 4Gives information on transmission dynamics of MTB
4Reveals information on the genetic diversity of the organism
Transforming Disease Detection- Beyond TB
Detection of a myriad of health conditions that were otherwise difﬁcult to diagnose, misdiagnosed or undiagnosed came in handy with the use of WGS. Enlisting some of them as under-6
4Rare diseases– chronically debilitating and life-threatening diseases that face diagnostic and therapeutic challenges started getting reported with WGS. Single nucleotide variants, copy-number variations, and structural variations could be spotted in WGS. This allowed identiﬁcation of mutations causing rare diseases. Example– mitochondrial disease.
4Inherited neurological disorders– WGS can robustly detect common inherited neurological disorders which otherwise are diagnosed using multiple tests. Repeat expansion disorders like intellectual disability and frontal lobe dementia wherein short repetitive DNA sequences are seen can also be diagnosed using WGS.
Genetic diseases in neonates– Monogenic illnesses are often undifferentiated at birth due to genetic heterogeneity and can be differentially diagnosed using WGS.
4Cancers– WGS allows healthcare professionals to explore all types of genomic alterations in the cancer genome. This facilitates real-time analysis of disease progression, in turn, planning/changing therapy in accordance to the response/progression.
4New disease-causing variants– While studying the entire genome sequence in a particular disease, new mutations at sites, other than the reported ones are observed. Sometimes, these mutations may potentially be associated with the same disease.
4Tracking of disease outbreaks– Bacterial/viral strains that genetically diverge even at one single nucleotide can also be precisely identiﬁed by WGS. Thus, enabling identiﬁcation and classiﬁcation of newer pathogenic strains.
Numbers Speak Volumes!
4After COVID-19, TB is the second leading infectious killer.
It is the 13th leading cause of death globally and in 2020 alone, around 1.5 million people succumbed to it.
4In 2020, an estimated 10 million people worldwide suffered from TB. Of these, 1.1 million were children,
3.3 million were women and 5.6 million were men.7
TB is Preventable and Curable!
The ﬁght to tackle MTB seemed to have become unceasing. With each breakthrough, it was felt that victory over TB was almost achieved. But this bacterium never fails to surprise mankind by creating newer obstacles. Owing to the increasing drug resistance, it has become a need of the hour to make available fast and reliable point-of-care diagnostic tools. Prompt diagnosis and assessment of drug resistance patterns lay the foundation of early and effective treatment; minimizing the emergence of drug-resistance strains. Therapeutic plans are in place to treat it, once diagnosed. Anti-TB drug therapy is made accessible by governing bodies to people from all strata of society. Although mortality rates have come down by large folds with all these advances, there are miles to travel before we eliminate TB. However, WGS is a silver lining making this ongoing battle conquerable.