Clinicians worldwide rely heavily (almost 70%) on the laboratory results, for diagnosing the illness, recommending the appropriate treatment and follow-ups. Hence it becomes an onus on laboratorians like us, to give an early and precise diagnosis. With highly infectious diseases like Tuberculosis (TB), it is all the more important to diagnose the patient at the earliest, for initiating appropriate treatment, thereby breaking the chain of spread. 

Despite remarkable advances made over the recent decades, to control this debilitating and life-threatening disease, gaps still exist in its complete eradication. Approximately 3 million people with TB are deemed ‘missing’ due to under-diagnosis and under-reporting to national TB surveillance programs. In order to achieve the goal of TB eradication by 2030, the World Health Organization (WHO), appeals to trace these missing millions. The picture is more complex, due to the emergence and rapid spread of multi-drug-resistant TB (MDR-TB) strains.
Being a laboratorian, we always strive to give the required diagnostic information by developing and utilizing tests with high sensitivity and specificity. Sensitivity of a test is the probability (in percentage) that the sample tests positive given the patient has the disease, whereas specificity is the ability of the test to detect a true negative case. Initially the diagnosis of TB was mostly carried out by smear microscopy and imaging techniques which were not as effective as the gold-standard culture drug- susceptibility test (DST). However, DST being time-consuming, molecular assays which are based on the DNA (genetic material) isolated from the TB bacilli like line probe assay (LPA) and GenXpert, TruNAATs and sequencing were introduced in the recent decades. GenXpert, a revolutionary method, facilitated in rapid detection of presence of TB causing pathogen Mycobacterium tuberculosis (MTB) and also identified the presence of rifampicin drug resistance. However, since MTB managed to acquire resistance towards other drugs other assays such as LPA were utilized as it could detect the resistance towards first-line and second –line anti-TB drugs. The LPA’s are based on binding of the mycobacterial DNA to probes which were specific to certain regions only and so there was always the possibility of missing some resistant strains which were harbored in the patient. Taken as a whole, these diagnostic tests though very sensitive and specific, lacked completeness and thus have limited use. Over the period, the TB pathogen still manages to evolve resistance towards most of the drugs being used against it and this poses a great dilemma to the laboratorians as well as the clinicians, not to forget the patients dealing with it first-hand! These issues led to the introduction of sequencing method into TB diagnosis. 

What do we know about sequencing?

Since the discovery of DNA, way back in 1953, research and diagnostic fields have progressed immensely. The nucleotide sequence of DNA often called the alphabet of life (adenine, guanine, cytosine, thymine) carries the information regarding the gene, that can give better understanding of its functions. Any change from the normal sequence, gives rise to mutation and these occurring in the MTB genome, causing emergence of various drug-resistance. Identifying these mutations is vital for understanding how the disease progresses.    

The most recent development in sequencing method called as next generation sequencing (NGS), which is based on the same premise of DNA sequencing albeit in a high through-put manner (that means it can sequence several genes as well as several samples at the same time) has rapidly moved into our laboratories. 

NGS can facilitate rapid diagnosis compared to the traditional practices and provide detailed and accurate sequence information. Using this technology, the entire genome of the Mycobacterium or the whole genome can be scanned to look for mutations in all the genes through which the TB drugs act (WGS is used as an acronym for whole genome sequencing).  WGS can rapidly and comprehensively identify the culprit and the underlying drug-resistance mechanism for both existing and novel drugs and that too in a single run! Since the entire DNA is being sequenced, the WGS also gives me really important information if there are more than one pathogen known as co-infection or if there are two different types of the Mycobacteriun strain itself (mixed infections) and it could be that one of them is sensitive and one is resistant in the same individual. Some of the previously mentioned tests are unable to pick this and often can result in poor treatment outcomes as optimally effective drug will not be given to this individual.  

As a laboratorian I now rely on TB-whole genome sequencing to give rapid, comprehensive, detailed diagnosis and drug resistance profile.  This way I am contributing to achieving our goal to ENDTB soon and I foresee this reality as more and more laboratories are adopting this technology to give crucial information about drug resistance in TB patients.