Molecular Testing – Closing the Gap Between TB Detection and Treatment

Mycobacteria, belonging to the genus Mycobacterium of the family Actinobacteriaceae, are immobile, rod-shaped, Gram positive bacteria. These bacteria can be divided into three groups for the purpose of diagnosis and treatment: Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis (TB); nontuberculous mycobacteria (NTM), with varying pathogenic potential; and Mycobacterium leprae, the causative pathogen of leprosy.

Mycobacteria are widespread organisms, typically living in soil, water, animal tissue, and food sources, and can colonize their hosts without inducing any pathogenic signs or can cause latent infections that can lead to severe diseases. Although the genus Mycobacterium was first introduced in 1896, infections by MTBC organisms and NTM continue to challenge physicians and microbiologist across the globe. The unique cell wall characteristics of mycobacteria prevent them from being stained by the standard Gram stain procedure. Due to this “acid-fastness”, specific diagnostic tests are required such as auramine-rhodamine or Ziehl-Neelsen staining.

Differentiation between MTBC and NTM can be achieved by immunochromatography, but exact species identification requires more sophisticated techniques such as molecular diagnostics. The infections resulting from MTBC are difficult to treat due to intrinsic resistance to antibiotics like penicillin G, sulfonamides, tetracycline, erythromycin, and chloramphenicol. ^[1] Mycobacteria can survive exposure to acids, alkalis, detergents or oxidative bursts, but fortunately most NTM are susceptible to the antibiotics clarithromycin and rifamycin, although antibiotic resistant strains have also emerged, presenting a new threat to global public health.

The global TB epidemic

MTBC species include M. tuberculosis, M. bovis, M. affricanum, M. canetti, M. microti, and M. pinnipedii, with the most common TB-causing pathogen in humans considered to be M. tuberculosis. These pathogens are transmitted via droplet infection through the air by coughing, sneezing, and speaking. In high incidence countries, the risk of infection is greatly increased in densely populated areas, with infants and immunocompromised patients at heightened risk.

TB is one of the top 10 causes of death throughout the world and is the leading cause of death from a single infectious agent (above HIV/AIDS and malaria). In 2017, it was estimated that 10 million people per year developed the disease,^[2] and approximately 1.7 billion people (23% of the world’s population) are estimated to have a latent TB infection and are therefore at risk of developing active TB disease during their lifetime.^[3]

Early diagnosis and rapid, appropriate medication are needed to improve TB treatment success, which remains low at around 55% globally. Treatment efforts are also hampered by the persistence of drug-resistant TB strains. Recent estimates demonstrate that, in 2017, 558,000 people developed rifampicin-resistant TB (RR-TB) – TB with resistance to the most effective first-line drug – and that 82% of these cases were resistant to the second first-line drug, isoniazid, and are therefore described as multidrug-resistant TB (MDR-TB). ^{[3] [4]}

Closing the gap between detection and treatment requires more specific drug susceptibility testing among those diagnosed with TB. The therapy for MDR-TB is more time consuming and is characterized by more frequent and severe side effects. This has led to a lower compliance rate among treated patients, resulting in a further increase in drug resistances. If the infecting organism is resistant to the two first-line antibiotics (rifampicin and isoniazid) and at least one drug from each of the classes fluoroquinolones and second-line injectable agents (amikacin, capreomycin or kanamycin), it is classed as extensively drug-resistant (XDR)-TB. ^[5] Identifying the drug resistance pattern is therefore crucial to establishing a successful treatment plan to cure the patient and stop the transmission of resistant strains.

Challenges of mycobacteria detection

The distinction between TB and NTM is essential for diagnosis and treatment, and the course of action depends on the respective mycobacteria species. Genotypic (molecular) methods for species differentiation and resistance testing are valuable tools in TB and NTM diagnostics and offer considerable advantages compared with time-consuming conventional methods, such as biochemical testing and phenotypic resistance testing.

The process for identifying TB using traditional culture methods often takes 2-4 weeks for a positive result, and 6-8 weeks for a negative result (which then requires further testing) depending on whether solid or liquid media have been used. This is considerably slower compared with genetic testing, where identification for TB and NTM can be done in under 3 hours. Genetic testing offers a clear advantage for patients, as resistance patterns are available earlier for adequate treatment.

Additionally, there is no requirement for a laboratory to have biosafety level 3 laboratory (BSL3) credentials for molecular methods, because these tests can be performed from direct decontaminated samples. Via the direct sampling method, any laboratory can run genetic testing on samples to determine the presence or absence of M. tuberculosis and conduct further testing to identify NTM.

In May 2016, the World Health Organization (WHO) issued new recommendations on the use of a rapid diagnostic test – the line probe assay (LPA) GenoType MTBDRsl – to detect resistance to second-line anti-TB drugs (SL-LPA). ^[6] Compared with traditional culture-based techniques, LPAs are a more reliable method for ruling out resistance to the second-line antibiotics fluoroquinolones and the injectable drugs capreomycin, kanamycin and amikacin, enabling clinicians to set patients on the proper regime at the earliest time.

Detection of any second-line resistance by the SL-LPA means that MDR-TB patients should not be enrolled on the shorter regimen, as this could jeopardize their treatment outcome and fuel the development of XDR-TB. Patients detected with XDR-TB should also not be enrolled on the shorter regimen and require carefully designed individual regimens to optimize successful treatment.

Clinical impact of molecular testing

Professor Robert Warren, Unit Director and Chief Specialist Scientist, South African Medical Research Council (SAMRC), focuses his research on molecular epidemiology and has continually challenged assumptions related to TB. His findings have demonstrated that transmission of TB occurs largely outside of the household and that the drug resistance epidemic is driven by transmission, especially in previously treated patients, implying reinfection.

South Africa is considered the largest consumer of molecular diagnostic testing for TB, and in Cape Town alone, more than 15,000 LPAs are estimated to be carried out per year. Prof Warren’s experience with this type of testing is extensive.

“The picture in South Africa is very different in terms of the incidence of TB,” explains Prof Warren, continuing: “The country already had a high incidence rate but with the onset of HIV in the 1980s, this rapidly accelerated until our rates were among the highest in the world. The focus of our health system in the early 2000s was to halt the increase of drug-susceptible TB, but the downside of this is that drug-resistant TB got overlooked. Our figures now show that we appear to be on the downward trend for susceptible TB incidence, but now the focus needs to shift onto drug-resistant TB.”

Molecular tools such as the SL-LPA have changed the landscape for testing in South Africa. Convincing physicians to move away from the traditional phenotypic testing to genetic testing was an initial barrier to the introduction of this method but once the benefits became clear, the adoption was swift.

Prof Warren describes his role in developing TB identification methods: “At the research center, we have been responsible for evaluating the diagnostic tools that are available on the market before passing our findings to an independent panel, who then make recommendations to the Department of Health. From the work that we have carried out, the line-probe assay is now one of our most used testing methods for second-line TB identification.”

“The future for molecular testing in the area of TB, in my opinion, will need to focus on the low to middle income countries that don’t have the necessary infrastructure in place for complex or specialized testing”, explains Prof Warren. “There are two different schools of thought here – to develop a test that will deliver results against a defined set of drugs, or to develop a test that will provide results against a comprehensive set of drugs. Each solution has its place but either way, the test needs to be as simple as possible and the results easy for a lab technician to understand without specialist knowledge.”

Future outlook for mycobacteria

The ability to optimize patient treatment by providing a rapid, reliable mycobacteria identification is improving health outcomes of TB and NTM infections worldwide. The future of TB and NTM testing is changing. As higher rates of TB are being detected in undeveloped countries where investment in laboratories and detection infrastructure is lower, the methods of testing need to be adjusted accordingly. There will be fewer trained technicians in these locations and molecular testing will need to become more intuitive to meet this throughput requirement.

The WHO recommendation of the SL-LPA molecular test to detect resistance to second-line anti-TB drugs indicates a step-change in the treatment of this disease. For these diagnostic tests to be applicable into the future, a more comprehensive outlook is required for assay development. In its “End TB Strategy”, the WHO has a shared vision of a world free of TB with zero deaths, disease and suffering by 2035.^[3] This requires a 95% reduction in the absolute number of TB deaths and a 90% reduction in incidence rate compared with the 2015 baseline. Early diagnosis of TB, including universal drug-susceptibility testing and systematic screening of high-risk groups, will be a key component in achieving this target.

References

(1)  Jarlier V and Nikaido H (1994) Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol. Lett. 123:11–18.

(2)  Porvaznik I, Solovič I, Mokrý J (2017) Non-Tuberculous Mycobacteria: Classification, Diagnostics, and Therapy. Adv Exp Med Biol, 944: 19-25.

(3)  Tuberculosis (TB) – Data & Statistics, Centers for Disease Prevention and Control (CDC), 2018, https://www.cdc.gov/tb/statistics/default.htm.

(4)  TB Elimination: Extensively Drug-Resistant Tuberculosis (XRD TB), Fact Sheet, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention (CDC), 2013, https://www.cdc.gov/tb/publications/factsheets/drtb/xdrtb.pdf.

(5)  World Health Organization Global Tuberculosis Report 2018: https://www.who.int/tb/publications/global_report/en/

(6)  The use of molecular line probe assays for the detection of resistance to isoniazid and rifampicin, Policy Update, World Health Organization, 2016, https://apps.who.int/iris/bitstream/handle/10665/250586/9789241511261-eng.pdf?sequence=1.