DiagnosisIn potentially reducing the diagnostic time from

DiagnosisIn the last decade, enormous progress has been made resulting in the appearance of promising tools for diagnosis of tuberculosis (Palomino, 2011).

Still, the lack of available specific and sensitive diagnostic test poses the greatest challenge for the treatment of the disease. Age old diagnostic methods for laboratory confirmation include acid fast bacilli (AFB) smear microscopy, culture and lung x-ray. Although rapid and inexpensive, smear microscopy producing results within 24 hours has poor sensitivity thereby posing big difficulty. Moreover, sputum smear examination is negative in early stages of infection and poor positivity staining non-tuberculous mycobacteria further complicates the diagnosis. Lung pathology on x-rays films although almost instantaneously are not seen until several months after infection. These limitations led to the need for developing more specific and sensitive molecular tests potentially reducing the diagnostic time from weeks to days.

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These molecular tests include two Nucleic acid amplification techniques(NAA); the enhanced Mycobacterium tuberculosis Direct Test (E-MTD; Gen-Probe) (Abe et al., 1993) for diagnosis of TB in either smear negative or smear positive cases and the PCR based Amplicor Mycobacterium tuberculosis Test (Amplicor; Roche Diagnostic System) (D’Amato et al., 1995) for testing smear positive respiratory samples (Barnes, 1997).In addition, Ligase chain reaction (Tortoli, Lavinia, ; Simonetti, 1997) for direct detection and insertion sequence IS6110 (Thierry et al., 1990) based detection are also used.

Although the sensitivities of PCR and E-MTD test are comparable to that of century old culture test, the NAA based is very rapid contrary to the culture that takes 2-6 weeks to produce any results because of its slow growing nature (Abe et al., 1993).Even better reliable, rapid, sensitive and specific test for early diagnosis of tuberculosis has been used recently combining techniques of nucleic acid sequence based amplification (NASBA) and reverse hybridization method (GTMD; Genotype Mycobacteria Direct) (Bicmen et al., 2011). Another highly sensitive cartridge based nucleic acid amplification test GenXpert (real time PCR) takes around 2 hours.

It also detects multidrug (rifamycin) resistance especially in smear positive samples. However, its limitation comes with high cost and less sensitivity towards smear negative pulmonary and extrapulmonary samples (Boehme et al., 2011; Hillemann, Rusch-Gerdes, Boehme, ; Richter, 2011; Nurwidya, Handayani, Burhan, ; Yunus, 2018; Zeka, Tasbakan, ; Cavusoglu, 2011). Another assay for rapid detection of RIF resistance that is based on reverse hybridization is line-probe assay(Soini ; Musser, 2001).

Even more economically friendly assay yielding excellent diagnosis in low bacillus load samples is fluorometric PCR-based TB diagnostic test-called Orange G3TB (Garberi et al., 2011). Its low cost makes it a good candidate to be used in poor regions of the world. dna probes (AccuProbe;Gen-Probe Inc.), pcr-based sequencing and dna microarrays are gold standard for identification of mycobacterial species (Katoch, 2003; Soini ; Musser, 2001).

Another method that surpass NAA in diagnosis of tuberculosis is loop-mediated isothermal amplification (LAMP). Its low complexity and high efficiency makes it a method of choice for tuberculosis diagnosis in endemic developing countries (Boehme et al., 2007; Notomi et al.

, 2000).The diagnosis of latent TB infection (LTBI) relies in century old tuberculin skin test (TST). However, high burden of latency globally, variable specificity, and presence of many non-tuberculosis mycobacteria (NTM) in the environment leading to cross-reactivity with BCG and NTM poses a major hurdle to immune based diagnosis of tuberculosis in high TB burden countries like India (Andersen, Munk, Pollock, ; Doherty, 2000; Lifschitz, 1965; Pai, Kalantri, ; Dheda, 2006). More advanced, more specific and less cross reactive invitro T cell based assay, specific for early secretory antigenic protein (ESAT-6) and culture filtrate protein (CFP-10), Interferon-gamma release assay (IGRA) is a method of choice in recent years in contrast to the purified protein derivative (PPD) used in TST (Pai et al., 2006; Sester et al., 2010).

However, both TST and IGRA failed to discriminate between LTBI and active TB in areas with high burden of the disease (Chegou, Black, Kidd, van Helden, ; Walzl, 2009) indicating there is no gold standard for the differential diagnosis of active and latent form of disease.Conclusively, conventional diagnostic methods, NAA tests cannot replace AFB smear or the culture as NAA only detect live M. tuberculosis. Cultures are still needed for identification of nontuberculous mycobacteria.

The overview of the progress in the diagnosis of TB is given by WHO (WHO TB Report 2017). Concluding remark: a good immunological test for diagnosis need to differentiate between latent and active form of disease and should not show any cross-reactivity with M.bovis, BCG and other NTM.


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