**RIFTM (rifampicin DST) (Biotech Labs Ltd, Ipswich, UK))**

This test has sensitivity and specificity of 31.2% and 94.9%, respectively in all anti-TB drugsusceptible and resistant TB patients, sensitivity and specificity of 33.3% and 93.9%, respec‐ tively in all anti-TB drug-susceptible and resistant TB patients with HIV-infection/AIDS.

### **1.4.2 Fluoromycobacteriophage assay (Figures 2, 3)**

This method has 94% sensitivities for rifampicin and isoniazid and 98% sensitivity for streptomycin and specificities of 97% for isoniazid, 95% for rifampicin, and 98% for strepto‐ mycin (resazurin microplate technique), sensitivity of 94% for all three anti-TB drugs (rifam‐ picin, isoniazid, and streptomycin) (*EGFP*-phage technique) and specificities of 93% for rifampicin, 90% for isoniazid and 95% for streptomycin (*EGFP*-phage technique).

#### **1.4.3 Luciferase reporter phage assay**

This test has 100% sensitivity and 89-100% specificity for culture isolates.

#### **1.5 Nitrate reductase assay**

This method has sensitivity and specificity of 100% and 100% for rifampicin, 93% and 100% for isoniazid, 76% and 100% for streptomycin, and 55% and 99% for ethambutol, respectively.

#### **1.6 Microcolony method (Thin-layer agar (TLA) method)**

This test has sensitivities and specificities of 100% for both rifampicin and ofloxacin, and sensitivity of 100% and specificity of 98.7% for kanamycin, 100% overall accuracy for rifam‐ picin and isoniazid resistance.

#### **1.7 Colorimetric redox indicator methods**

This method has sensitivities of 100% for rifampicin, ofloxacin, kanamycin and capreomycin and 99.1% for isoniazid, specificity of 100% for rifampicin, isoniazid, ofloxacin and kanamycin, and 97.9% for capreomycin, overall accuracy of 98.4% for rifampicin, 96.6% for isoniazid, 96.7% for ofloxacin, 98.3% for kanamycin and 90% for capreomycin.

#### **2. Genotypic detection (Nucleic Acid Amplification)**

#### **2.1 Line-probe assay (LPA)**

#### **2.1.1 INNO-LiPA Rif.TB® assay (Innogenetics, Ghent, Belgium)**

This test has higher than 95% sensitivity and 100% specificity, sensitivity of 82.2% and specificity of 66.7% for MDR-TB detection.

#### **2.1.2 GenoType® MTBDRplus kit (Hain Lifescience, Nehren, Germany)**

This method has nearly 91% sensitivity for MDR-TB, possibly detects rifampicin and isoniazid resistance and confirms TB infection simultaneously.

#### **2.1.3 Genotype MTBDRsl assay**

This test has ethambutol, 89% sensitivity for ofloxacin, 87% sensitivity for capreomycin, 75% sensitivity for amikacin.

#### **2.2 Real-time PCR**

This technique has sensitivity of 89% and specificity of 99% (molecular beacons), overall sensitivity of 98-100% with 72% sensitivity in smear-negative specimens and specificity of 100% for Xpert MTB/RIF assay (Cepheid Xpert MTB/RIF®, Sunyvale, CA).

#### **2.3 PCR sequencing**

This technique has sensitivities of 96.7% for rifampicin-resistant isolates (*rpoB* gene), 64% for isoniazid-resistant isolates (*katG* gene) and 70% of ofloxacin-resistant isolates (*gyrA* gene) (PCR pyrosequencing).

#### **2.4 DNA Microarrays (DNA biochip)**

This technique has specificity of 97% and 95% for rifampicin, 91% and 60% for isoniazid, 96% and 67% for kanamycin, 93% and 73% for streptomycin, and 98% and 89% for ethambutol, respectively, simultaneous detection of multiple genetic sequences (oligonu‐ cleotide microarray).

Although genotypic methods have potentially fastest results, the massive cross-contamination is still the main risk. To prevent its, strict internal controls, special technique, and separation of working areas are required. The reproducibility of the results of the Xpert MTB/RIF assay under actual field conditions, the strength of the laboratories, and the manner and extent of its introduction are the impact of this new assay. The sources of error for genotypic methods are incomplete coverage of rifampicin-resistance gene-core region and mixtures (multiple mutations, wild-type strain/emerging mutant) and silent mutations. These sources of error may contribute to 1% false-resistant and 5% false-susceptible results [31].

Schematic representation of *phAE87* : *hsp60-EGFP* construction. Shuttle phasmid *phAE87* is a conditionally replicating derivative of phage TM4 in which the cosmid moiety is flanked by Pac I restriction sites. A plasmid derivative of *pYUB854* containing the *EGFP* gene (pMP14) was used to replace the cosmid in *phAE87* followed by lambda packaging and recovery in *E. coli*.

**Source :** Rondo′n L, Piuri M, Jacobs WR Jr, Waard Jde, Hatfull GF, Takiff HE. Evaluation of fluoromycobacteriophages

**Source** : Piuri M, Jacobs WR Jr, Hatfull GF. Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic sus‐ ceptibility testing of *Mycobacterium tuberculosis*. PLoS ONE 2009; 4(3) : e4870. doi: 10.1371/journal.pone.0004870

Drug-Resistant Tuberculosis – Diagnosis, Treatment, Management and Control: The Experience in Thailand

http://dx.doi.org/10.5772/54852

247

**Figure 3.** A, B). Two strains of *Mycobacterium tuberculosis* were incubated separately in 7H9-OAD with 2 µg of rifam‐ picin/ml for 24 hours, infected with the *EGFP*-phage, killed with paraformaldehyde, and then fixed on microscope slides. The images, obtained with a fluorescence microscope, show a susceptible-to-rifampicin strain (A) and a resist‐

for detecting drug resistance in *Mycobacterium tuberculosis*. J Clin Microbiol 2011; 49 (5) : 1838-1842.

ant-to-rifampicin strain (B).

**Figure 2.** Fluoromycobacteriophages construction

Drug-Resistant Tuberculosis – Diagnosis, Treatment, Management and Control: The Experience in Thailand http://dx.doi.org/10.5772/54852 247

**Source** : Piuri M, Jacobs WR Jr, Hatfull GF. Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic sus‐ ceptibility testing of *Mycobacterium tuberculosis*. PLoS ONE 2009; 4(3) : e4870. doi: 10.1371/journal.pone.0004870

**Figure 2.** Fluoromycobacteriophages construction

This test has higher than 95% sensitivity and 100% specificity, sensitivity of 82.2% and

This method has nearly 91% sensitivity for MDR-TB, possibly detects rifampicin and isoniazid

This test has ethambutol, 89% sensitivity for ofloxacin, 87% sensitivity for capreomycin, 75%

This technique has sensitivity of 89% and specificity of 99% (molecular beacons), overall sensitivity of 98-100% with 72% sensitivity in smear-negative specimens and specificity of

This technique has sensitivities of 96.7% for rifampicin-resistant isolates (*rpoB* gene), 64% for isoniazid-resistant isolates (*katG* gene) and 70% of ofloxacin-resistant isolates (*gyrA* gene) (PCR

This technique has specificity of 97% and 95% for rifampicin, 91% and 60% for isoniazid, 96% and 67% for kanamycin, 93% and 73% for streptomycin, and 98% and 89% for ethambutol, respectively, simultaneous detection of multiple genetic sequences (oligonu‐

Although genotypic methods have potentially fastest results, the massive cross-contamination is still the main risk. To prevent its, strict internal controls, special technique, and separation of working areas are required. The reproducibility of the results of the Xpert MTB/RIF assay under actual field conditions, the strength of the laboratories, and the manner and extent of its introduction are the impact of this new assay. The sources of error for genotypic methods are incomplete coverage of rifampicin-resistance gene-core region and mixtures (multiple mutations, wild-type strain/emerging mutant) and silent mutations. These sources of error

Schematic representation of *phAE87* : *hsp60-EGFP* construction. Shuttle phasmid *phAE87* is a conditionally replicating derivative of phage TM4 in which the cosmid moiety is flanked by Pac I restriction sites. A plasmid derivative of *pYUB854* containing the *EGFP* gene (pMP14) was used to replace the cosmid in *phAE87* followed by lambda packaging

**2.1.2 GenoType® MTBDRplus kit (Hain Lifescience, Nehren, Germany)**

100% for Xpert MTB/RIF assay (Cepheid Xpert MTB/RIF®, Sunyvale, CA).

may contribute to 1% false-resistant and 5% false-susceptible results [31].

specificity of 66.7% for MDR-TB detection.

246 Tuberculosis - Current Issues in Diagnosis and Management

**2.1.3 Genotype MTBDRsl assay**

sensitivity for amikacin.

**2.2 Real-time PCR**

**2.3 PCR sequencing**

pyrosequencing).

cleotide microarray).

and recovery in *E. coli*.

**2.4 DNA Microarrays (DNA biochip)**

resistance and confirms TB infection simultaneously.

**Source :** Rondo′n L, Piuri M, Jacobs WR Jr, Waard Jde, Hatfull GF, Takiff HE. Evaluation of fluoromycobacteriophages for detecting drug resistance in *Mycobacterium tuberculosis*. J Clin Microbiol 2011; 49 (5) : 1838-1842.

**Figure 3.** A, B). Two strains of *Mycobacterium tuberculosis* were incubated separately in 7H9-OAD with 2 µg of rifam‐ picin/ml for 24 hours, infected with the *EGFP*-phage, killed with paraformaldehyde, and then fixed on microscope slides. The images, obtained with a fluorescence microscope, show a susceptible-to-rifampicin strain (A) and a resist‐ ant-to-rifampicin strain (B).
