**3.1. Phenotypic methods**

Significant effort has been invested into further development of simple, alternative pheno‐ typic methods such as the nitrate reductase assay (NRA), thin-layer agar (TLA), colour test (Color Test), the microscopic observation drug susceptibility assay (MODS), the colorimetric redox indicator (CRI) method and phage-based assays, most of which can be set up directly on specimens [6,7,8]. These methods can detect MTB and resistance to INH and RMP. While MODS, NRA and CRI have been endorsed by the WHO, current evidence was considered to be insufficient for recommending the use of TLA or phage-based assays [8].

Line-probe assays and XPERT MTB/RIF: Line probe assays (LPAs) are actual molecular tests. Three main LPAs for the rapid diagnosis of TB and/or rapid detection of RMP resist‐ ance and MDR-/XDR-TB are currently available on the market: INNO-LiPA Rif. TB (Innoge‐ netics, Belgium), GenoType®MTBDR/MTBDR*plus* and Geno-Type® MTBDR*sl* (both Hain Lifescience, Germany). These assays are based on the targeted amplification (PCR) of specif‐ ic fragments of the MTB genome, followed by hybridisation of PCR products to oligonucleo‐ tide probes immobilised on membranes. INNO-LiPA Rif TB detects only RMP resistance, GenoType MTBDR/MTBDR*plus* detects both RMP and INH resistance, and GenoType MTBDR*sl*detects resistance to flouroquinolons, injectable second-line drugs and ethambutol. These tests are designed for detection the MTB isolates in respiratory specimens. Xpert® MTB/RIF (Cepheid Inc, USA) is a fully automated RT-PCR based assay for the detection of

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The recovery of mycobacterium from peripheral blood and bone marrow samples may be improved by lyses- centrifugation blood culture method. In this method, blood is put into a tube containing an anticoagulant and an agent to effect rupture of both erythrocytes and neutrophils. Following centrifugation of the tube, the sediment is inoculated into the appro‐ priate culture media. This method has increased both the yield and shortened the time of

This is a bacteriophage based test to detect MTB in sputum. Non-pathogenic mycobacteria (sensor cells) were used for control bacteria in the test. The phage replicate, infect and lyses the sensor cells leaving zones of clearing (holes) in the agar. The areas of clearing indicates that the patient sputum contain viable MTB. It is fast with a turnaround time of 2 days. It is cheap, requires few equipments, sensitive (detection as low as 100 tubercles per ml of spu‐ tum). It can be adapted for sensitivity testing. Limitations are applicability to sputum speci‐

Immunodiagnostic tests can provide indirect evidence current or past infections of MTB. Ex‐ ception of tuberculin skin test, immunodiagnostic tests are of limited application due to

TB bacteria and resistance to RMP in direct clinical specimens [20].

**4. Lysis-centrifugation blood culture system**

recovery of mycobacteria from blood cultures [21].

**5. Phage Amplification Technique (PAT)**

men only and technically demanding [22].

**6. Immunological methods**

cross reactivity and poor sensitivity.

MODS is an extensively validated method that has almost perfect agreement with conven‐ tional DST for INH, RMP and MDR-TB (100%, 97% and 99%). The results are available with‐ in a median of 7 days; the method is cheap, non-commercial and works well on all types of primary specimens as well as on isolates. However, it requires relatively long, detailed staff training. [6,7,9,10]

TLA recently demonstrated a good performance of the MDR-/XDR-TB colour test for the identification of MTB complex and detection of resistance to INH, RMP and ciprofloxacin in cultures [11].

### **3.2. Genotypic methods**

Molecular techniques are aimed at the nucleic acid of the mycobacterium as the analyte. Ri‐ bosomal rRNA is useful genetic target for the identification of organisms, since it often con‐ tains spesific sequences and is present in the cells and media in high quantity due to the growth of the mycobacteria. There are various applications of molecular tecniques for the detection and identification of MTB.

PCR is the common format of nucleic acid amplification tests (NAAT); other methodologies include ligase chain reaction, strain displacement amplification, loop-mediated isothermal amplifi cation (LAMP) and transcription mediated amplification. More recently, real-time (RT) PCR technologies based on fluorescent- probe detection or melting-curve analysis have been developed [12-16].

These molecular techniques also aimed detecting resistance genes. Example includes; DNA probe and DNA sequencing of MTB gene such as catalase (katG) or RNA polymerase (rpoB). Mutations in these genes have been associated with resistance to isoniazid and ri‐ fampicin respectively. The using of molecular primers in real- time PCR reaction can differ‐ entiate between the presence of the wild- type sequence and mutated sequence associated with drug resistance. Molecular tests are rapid (within few hours), highly sensitive and spe‐ cific, but expensive, requires expertise and may not differentiate active infection as DNA from a dead organism during antibiotic treatment can be detected and amplified by PCR [17]. Genotypic methods are not routinely used in the mycobacterium laboratory; they are essentially for research purposes [18,19].

Line-probe assays and XPERT MTB/RIF: Line probe assays (LPAs) are actual molecular tests. Three main LPAs for the rapid diagnosis of TB and/or rapid detection of RMP resist‐ ance and MDR-/XDR-TB are currently available on the market: INNO-LiPA Rif. TB (Innoge‐ netics, Belgium), GenoType®MTBDR/MTBDR*plus* and Geno-Type® MTBDR*sl* (both Hain Lifescience, Germany). These assays are based on the targeted amplification (PCR) of specif‐ ic fragments of the MTB genome, followed by hybridisation of PCR products to oligonucleo‐ tide probes immobilised on membranes. INNO-LiPA Rif TB detects only RMP resistance, GenoType MTBDR/MTBDR*plus* detects both RMP and INH resistance, and GenoType MTBDR*sl*detects resistance to flouroquinolons, injectable second-line drugs and ethambutol. These tests are designed for detection the MTB isolates in respiratory specimens. Xpert® MTB/RIF (Cepheid Inc, USA) is a fully automated RT-PCR based assay for the detection of TB bacteria and resistance to RMP in direct clinical specimens [20].
