**5. Laboratory investigations for MDR-TB**

2000 have been firstly initiated at tertiary care hospitals in northern Thailand. Data collecting on MDR/XDR-TB control has been tracked through the special project paper-based recording and reporting systems set by the 10th Zonal Tuberculosis and Chest Disease Centre which separated from the routine DOTS recording and reporting systems. Currently, a new computer programme has been developed by staff of this centre to efficiently record and report the MDR/ XDR-TB data in the area of northern Thailand and attempt to gradually extend the use of this

**Figure 1**. **Mechanism of development of anti-tuberculosis drug resistance** 

Development of anti‐tuberculosis

Development of anti‐tuberculosis drug resistance due to related‐ chromosomal mutation

Susceptible strain of *Mycobacterium tuberculosis* in a new host

drug resistance

treatment is the most significant predictor of development of MDR-TB [19]. High prevalence of HIV/AIDS co-infection and inadequate resources for case detection and management have contributed to the emergence of untreatable XDR-TB [20]. Unfortunately, the presence of XDR-TB in non—HIV-infected patients with MDR-TB is independent poor prognostic factors [11]. Prevalence of XDR-TB is globally accounted for approximately 5.4% of MDR-TB prevalence [21]. Drug-resistant TB and drug-resistant gram-negative bacterial infection and disease are associated with the most serious health problems in developing countries [22]. Estimated 81,000 patients with MDR-TB (18.4% of the estimated MDR-TB patients worldwide in 2011) live in the 53 countries of the WHO European Region [23]. This European MDR-TB problem contributed to launching of a new WHO Regional Office for Europe Action Plan to fight MDR-TB to contain the spread of drug-resistant TB in the region by the end of 2015 [23]. The new action plan set the targets to be achieved by the end of 2015, are : 1) decreasing 20% of the proportion of MDR-TB cases among previously treated patients 2) diagnosis of at least 85% of the estimated MDR-TB cases and 3) treating successfully at least 75% of notified MDR-TB cases [23]. If this plan is fully implemented and expected , by 2015, to successfully treat 127,000 MDR-TB cases, and to prevent the emergence of 250,000 new MDR-TB cases and 13,000 new XDR-TB cases [23]. This would interrupt the

transmission of MDR-TB and save 120,000 lives in this region [23].

*Mycobacterium tuberculosis*

Development of human immunity, formation of granuloma and calcification during chronic infection

Changes of specific protein structure in anti‐tuberculosis drug‐resistant‐related

**Figure 1.** Mechanism of development of anti-tuberculosis drug resistance

4

The Thailand' s 2012 National Tuberculosis Management Guidelines [24] set the criteria for

**1.** patients with history of TB retreatment, especially within 6 months after completeness of

**2.** patients with interruption of 6 month-short course chemotherapy (2HRZE/4HR, H=iso‐ niazid, R=rifampicin, Z=pyrazinamide, E=ethambutol) and having continuously positive-

**3.** patients with history of multiple TB treatments, irregular anti-TB drug taking, and

**4.** patients having positive-sputum smear examinations at the end of second and fifth

sputum smear examinations after treatment interruption,

months of the 6-month short course chemotherapy (2HRZE/4HR),

persistently positive-sputum smear examinations,

computer programme throughout the country.

**4. Presumptive diagnosis of MDR-TB**

probable or suspected MDR-TB, are :

treatment or cure,

242 Tuberculosis - Current Issues in Diagnosis and Management

 

> Several plans have been announced for the WHO Stop TB Department to collaborate with the Foundation for Innovative New Diagnostics (FIND) to initiate and introduce rapid-culture technology and new rapid drug-resistant tests in the southern African countries and the world including the international standards for the second-line drug-susceptibility testing [11]. The Thailand' s 2012 National Tuberculosis Management Guidelines [24] set the laboratory investigations for MDR-TB which are direct acid-fast bacilli (AFB)-sputum smear examina‐ tions with sputum culture and drug susceptibility testing (DST). For reduction of the diagnosis time for unrecognized drug-resistant TB, new rapid diagnostic technologies for drug resistance from sputum smear or positive culture for smear-negative and extra-pulmonary TB must be prioritized [10]. All TB control programmes in moderate- and high-MDR TB prevalent settings should consider the promotion of culture and DST including implementation of use of algorithms for the diagnosis of pulmonary and extra-pulmonary TB [10]. Rapid DST is preferred to the conventional DST due to 1-2 days of resulting. Recently, the 2011 WHO Guidelines recommends " Xpert MTB/RIF® " and line-probe assay which are new molecular diagnostic technologies and can detect drug resistance to both isoniazid and rifampicin or only rifampicin [24], but their disadvantage is inability to detect the resistance to every drugs used in MDR-TB treatment for detecting probable XDR-TB, required expertise and expensive technologies/equipments which limit their wider uses [25]. The conventional DST takes 1-3 months of the results that take markedly longer than the new molecular methods and is laborintensive [24]. Other alternative phenotypic methods based on the *Mycobacterium tuberculosis* metabolism such as CO2 (carbon dioxide) production, oxygen uptake, ATP (adenosine triphosphate) bioluminescence, etc. have been experimented and demonstrated promising in overcoming this obstacle of longer time resulting [25]. These methods also have impressive sensitivity and specificity compared to the conventional DST [25]. Currently, molecular line probe assays and automated liquid culture systems are recommended by the WHO as the gold standard for the first-line and second-line DSTs. Liquid culture DST has been demonstrated to have relatively good reliability and reproducibility for aminoglycosides, fluoroquinolones, and polypeptides for detecting XDR-TB [26]. However, liquid culture DST for other secondline drugs such as para-aminosalisylic acid, linezolid, clarithromycin, amoxicillin-clavulanate, cycloserine, clofazimine, terizidone, ethionamide, and prothionamide is not recommended by the WHO [26]. A recent study reported the use of spoligotyping and sequence 6110 restriction

fragment length polymorphism insertion analysis of genomic DNA which demonstrated that MDR-TB cases (74.0%) were more likely to be identified in clusters than anti-TB drug suscep‐ tible cases (33.6%) [27]. Xpert MTB/RIF assay recently has been introduced which meets the requirements of effective diagnosis of pulmonary TB as the following : allowing detection of both the *Mycobacterium tuberculosis* complex and resistance to the principal anti-TB drugs, especially rifampicin (RIF or R), availability on a global scale with standardized-easy use and robust diagnostic tools recently has been introduced [28]. This assay is a nucleic acid amplifi‐ cation test for detection of rifampicin resistance-associated mutations of the *rpoB* gene and *Mycobacterium tuberculosis* complex DNA in sputum [28]. It can be designed for use with other systems to automate and integrate sample processing, nucleic acid amplification, and detection of target sequences using reverse transcriptase polymerase chain reaction (PCR) and real-time PCR [28]. Between 2007-2009, the WHO has approved several drug-resistant TB diagnostic tests such as liquid culture (MGIT®, an automated liquid culture, developed by BD Diagnostic Systems, 2007) which has been used at the 10th Zonal Tuberculosis and Chest Disease Centre, Chiang Mai, Thailand, line-probe assays (INNO-Lipa®, line-probe assay that requires culture, developed by Innogenetics, 2008), noncommercial culture and drug susceptibility testing (Microscopic Observation Drug Susceptibility (MODS), developed by Academic Laboratories, 2009; Nitrate reductase assay, developed by Academic Laboratories, 2009; and Colorimetric drug susceptibility testing, developed by Academic Laboratories, 2009) [29]. GeneXpert MTB/ RIF®, a new automated nucleic acid amplification technique which was developed by Cepheid, The Foundation for Innovative New Diagnostics (FIND) and University of Medicine and Dentistry of New Jersey (UMDNJ) was reviewed by the WHO in 2011 [29] and currently has been recommended to measure the *Mycobacterium tuberculosis* DNA and the rifampicinresistance sequence worldwide. This new technique has been set in Thailand at least 6 sets including the one set at the 10th Zonal Tuberculosis and Chest Disease Centre, Chiang Mai, Thailand in collaboration with the Unites States Centres for Disease Control and Prevention (US-CDC) for reference laboratories in Thailand. GeneXpert test's sensitivity is moderate at 67.2% in AFB-smear negative cases at one-time smear staining of the specimens, and increases to 80% when is performed three times [28]. This test provides the results within two hours and requires minimal training of the laboratory workers [29]. The limitations of the test are requirement of a consistent source of electricity that will limit its use outside of the settings where a regular electric power supply can be guaranteed, its expensive cost of the instrument, and cost per test cartridge [29]. Mishra B *et al*. used the automated BACTEC 460 TB system in study the emergence of drug-resistant TB at an urban tertiary care hospital in South India which revealed that 37.2% were MDR-TB isolates whereas 42% of the pulmonary *Mycobacte‐ rium tuberculosis* isolates and 20.4% of extra-pulmonary isolates were MDR [30]. Phenotypic and genotypic detections of anti-TB drug resistance are described as the following [31- 49]:

This test has sensitivities of 100% for rifampicin, isoniazid, ethambutol and streptomycin, specificity of 100% for rifampicin, 97.7% for isoniazid, 98.0% for ethambutol and 89.8%% for

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

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

245

This method has sensitivity of 72.7% and specificity of 99.7% for rifampicin, 72.6% and 97.9%

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.

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

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.

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‐

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%

**1.3 Microscopic observation broth-drug susceptibility assay (MODS)**

**1.4.1 Commercial FASTPlaque assay (FASTPlaque TB test and FastPlaque TB-**

rifampicin, 90% for isoniazid and 95% for streptomycin (*EGFP*-phage technique).

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

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

for ofloxacin, 98.3% for kanamycin and 90% for capreomycin.

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

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

for isoniazid, and 77.8% and 99.7% for MDR-TB.

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

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

**1.4 Mycobacteriophage-based method**

**1.4.3 Luciferase reporter phage assay**

**1.5 Nitrate reductase assay**

picin and isoniazid resistance.

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

**1.7 Colorimetric redox indicator methods**

streptomycin.

#### **1. Phenotypic detection**

#### **1.1 Slide DST**

This method has less equiptment, suitable for decentralization, 93% rifampicin susceptibility at 88% predictive value of resistance.

#### **1.2 Mycobacteria Growth Indicator Tube (MGIT) Systems**

This test has sensitivities of 100% for rifampicin, isoniazid, ethambutol and streptomycin, specificity of 100% for rifampicin, 97.7% for isoniazid, 98.0% for ethambutol and 89.8%% for streptomycin.

#### **1.3 Microscopic observation broth-drug susceptibility assay (MODS)**

This method has sensitivity of 72.7% and specificity of 99.7% for rifampicin, 72.6% and 97.9% for isoniazid, and 77.8% and 99.7% for MDR-TB.
