Think TB

Tuberculosis (TB) caused by Mycobacterium tuberculosis; remains a major public health problem despite noteworthy socio-economic development, advances and availability of technology. It is a preventable and curable disease but still millions of people suffer every year and a number of them die from this infectious disease resulting in devastating social & economic impact. Many countries think of TB as a disease of the past and this lack of awareness results in shortfalls in funding, resulting in the lack of aggressiveness to combat the disease. But it is a disease of the future as well, especially in the lower income countries.

End TB Strategy

WHO’s global strategy for TB prevention, care and control for 2015–2035; End TB Strategy calls for the early diagnosis of TB and universal drug-susceptibility testing (DST), highlighting the critical role of laboratories in the strategy. In order to meet the End TB Strategy targets, Rapid TB diagnostics is a right of all person with signs or symptoms of TB, all bacteriologically confirmed TB patients should receive DST at least for rifampicin, and all patients with rifampicin-resistant TB (RRTB) should receive DST at least for fluoroquinolones (FQs) and second-line injectable drugs (SLIDs).

Right Test –First Time

In recent years, rapid and sensitive tests based on molecular methods, including Xpert MTB/RIF (Cepheid, Sunnyvale USA), the loop-mediated isothermal amplification (TB-LAMP) test (Eiken Chemical, Tokyo Japan), and line probe assays (LPAs), have become available to replace or complement existing conventional tests for detecting Mycobacterium tuberculosis complex bacteria (MTB) and for detecting drug resistance. Despite the advantages of these newer tests, conventional microscopy and culture remain necessary for monitoring the response of a patient to treatment.

TB patients care starts with a quality assured diagnosis. Current DOTS expansion, as well as programmatic management of TB and DRTB therefore require at its core; a strong network of TB laboratories with acceptable biosafety, modern methods for diagnosis, standard operating procedures and appropriate quality assurance and quality control. Laboratory services have historically been grossly neglected, under-staffed and underfunded. Diagnostic capacity is therefore a major bottleneck for scaling up management and control of TB and DRTB. Strengthening TB laboratory services offers one of the best opportunities for overall laboratory improvement as an essential health system activity.

In 2015, WHO published the Policy framework for Implementing Tuberculosis Diagnostics and the Global Laboratory Initiative (GLI) published the GLI Guide for providing technical support to TB laboratories in low- and middle-income countries to assist with the implementation of the latest diagnostic technologies. As new diagnostic tests are implemented, testing algorithms will need to be modified. Algorithms should be designed to use existing laboratory services so that specimens can be referred to the appropriate level for tests that are not available at the peripheral level laboratories. The landscape of TB diagnostics is rapidly changing, and new tests may be recommended by WHO in the near future.

Some of the WHO recommended technologies are given below.


Mycobacteria are distinguished from other micro-organisms by thick lipid-containing cell-walls that retain biochemical stains despite decolourisation by acid containing reagents (so called ‘acid-fastness’).
Advantages: Microscopy of sputum smears is simple and inexpensive, quickly detecting infectious cases of pulmonary TB; Sputum specimens from patients with pulmonary TB especially those with cavitary disease often contain sufficiently large numbers of acid fast bacilli (AFB) to be readily detected by microscopy.
Disadvantages: Direct smear microscopy is relatively insensitive. Smear sensitivity is further reduced in patients with extrapulmonary TB, those with HIV coinfection, and those with disease due to non tuberculous mycobacteria (NTM).
Limitations: Microscopy for AFB cannot distinguish Mycobacterium tuberculosis from NTM, nor viable from non-viable organisms, or drug-susceptible from drug-resistant strains.

Conventional light microscopy

Ziehl-Neelsen (ZN) light microscopy performed directly on sputum specimens is suitable for all laboratory service levels, including peripheral laboratories at primary health care centers or districts hospitals.
Light-emitting diode (LED) fluorescent microscopy
LED technology allows the use of fluorescent microscopy with a LED light source. LED microscopes or attachments require less power, are able to run on batteries, the bulbs have a very long half-life and do not release potentially toxic products if broken.

Culture and species identification7

Liquid culture and DST systems are more complex and sensitive than solid culture and DST media. Increased bacterial contamination and an increased frequency of NTM isolation must be addressed. A rapid method to differentiate Mycobacterium tuberculosis complex from other mycobacterial species is essential.
Advantages: Mycobacterial culture and identification of M. tuberculosis provide a definitive diagnosis of TB, significantly increases the number of cases found and can detect cases earlier. Culture also provides the necessary isolates for conventional DST.
Disadvantages: Culture is much more complex and expensive than microscopy to perform, requiring facilities for media preparation, specimen processing, and growth of organisms, specific laboratory equipment, skilled laboratory technicians, and appropriate biosafety conditions.
Limitations: Specimens have to be decontaminated prior to being cultured to prevent overgrowth by other microorganisms. Good laboratory practices maintain a delicate balance between yield of mycobacteria and contamination by other micro-organisms.
Confirmation is usually done by a combination of biological characteristics of the culture growth and selected molecular or biochemical tests or rapid immunochromatographic assays.

Drug Suspectibility Testing

Phenotypic DST methods are performed as direct or indirect tests on solid or liquid media. In direct testing, a set of drug-containing and drug-free media is inoculated directly with a concentrated specimen. Indirect testing involves inoculation of drug containing media with a pure culture grown from the original specimen. Phenotypic tests have been extensively validated and are currently regarded as the gold standard.
Advantages: DST provides a definitive diagnosis of DRTB. A number of different DST techniques are available. Phenotypic methods involve culturing of M.tuberculosis in the presence of anti-TB drugs to detect growth (indicating drug resistance) or inhibition of growth (indicating drug susceptibility).
Disadvantages: DST methods are suitable for use at central/national reference laboratory level only, given the need for appropriate laboratory infrastructure (particularly biosafety) and the technical complexity of available technologies/methods.
Limitations: The accuracy of DST varies with the drug tested.

First-line DST

DST is most accurate for rifampicin and isoniazid and less reproducible for streptomycin, ethambutol and pyrazinamide.
Second-line DST9
Second-line DST is complex and expensive. Automated liquid systems for second line DST are recommended as the current gold standard.
Aminoglycosides, polypeptides, and fluoroquinolones have been shown to have relatively good reliability and reproducibility, allowing a quality-assured diagnosis of XDR-TB. Routine DST for other second-line drugs (ethionamide, prothionamide, cycloserine, terizidone, P-aminosalicylic acid, clofazimine, amoxicillin-clavulanate, clarithromycin, linezolid) can be performed.

Molecular Testing

The ultimate aim should be to implement molecular assays (such as the line-probe assay, Xpert MTB/RIF or other WHO-endorsed molecular platforms in the future) for rapid first step identification of MDR-TB or HIV associated TB.
Molecular line probe assays (LPAs) focused on rapid detection of rifampicin resistance (alone or in combination with isoniazid) have been endorsed by WHO in 2008 with detailed policy guidance on its introduction at country level. Xpert MTB/RIF assay was endorsed by WHO in December 2012 and supported by a document describing practical considerations for rapid implementation and operational.
Genotypic (molecular) methods have considerable advantages for scaling up programmatic management and surveillance of drug resistant TB, offering speed of diagnosis, standardized testing, potential for high throughput, and fewer requirements for laboratory biosafety.
Advantages: Genotypic methods have considerable advantages for scaling-up programmatic management of drug-resistant and HIV associated TB, in particular with regard to speed, standardized testing, potential for high throughput, and reduced biosafety needs.


It detects both TB and rifampicin resistance in a single test. Rifampicin resistance is a good and reliable proxy for MDRTB in high burden settings. For the first time, a molecular test is simple and robust enough to be introduced outside conventional laboratory settings. The assay provides results directly from sputum in less than 2 hours.Xpert MTB/RIF can be used as a stand-alone diagnostic test in individuals at risk of MDR TB.
Disadvantages: Xpert MTB/RIF requires uninterrupted and stable electrical power supply and yearly calibration of the cartridge modules.


It does not eliminate the need for conventional culture and DST capability. Currently available LPAs are registered for use on smear positive/ smear negative sputum specimens and M.tuberculosis isolates grown from smear negative specimens by conventional culture methods for first line(Rifampicin and Isoniazid).
Limitations: Conventional culture (solid or liquid) is required to monitor treatment response (culture conversion) of DR-TB patients.

What’s New?

In 2012, the FDA approved Bedaquiline, the first new drug to treat TB in 40 years. Another drug, Delamanid, has been approved in the European Union. Both drugs were developed to treat MDR TB. Clinical trials have shown that the drugs clear TB from sputum effectively. Indiscriminate use can lead to drug resistance. Health authorities also tend to take a cautious approach to new drug deployment because of fears of unexpected side effects.