**4. Diagnosis of multidrug resistant tuberculosis**

Understanding the mechanisms of TB latency is crucial to development of better control strategies. Infection with Mtb occurs initially in alveolar macrophage, in which the bacteria replicate and induce cytokines that initiate the inflammatory response in the lungs, leading ultimately to the formation of granuloma [23]. Granuloma is defined as an immune structure consisting of connective tissue, lymphocytes and activated macrophages, which has a central necrotic core containing extracellular bacteria. Within the granuloma the bacterium is exposed to multiple stresses that include, among others, hypoxic, nutrient limiting, oxidative, nitrosa‐ tive and acidic conditions [24,25], which trigger a genetic program controlled by the transcrip‐ tion factor DosR [25]. The later regulates the development of a quiescent physiological state, which maintains viability of non-dividing bacteria for extended periods of time. The granu‐ loma contains the infection and prevents its spread to other organs [26]. However, dormant bacteria are capable of reactivation controlled by Rpf (resuscitation promoting factor) genes, which is associated with reversal of the non-replicating state into a metabolically active growing and dividing bacteria [27]. Thus, life-long immunity is not gained by a first episode of active TB disease and the disease may develop again at a later stage, either through relapse

Deciphering the molecular basis of dormancy and reactivation is therefore necessary for developing more efficient TB therapies. Adjuncts of agents that would block transitions between active growth, dormancy, and resuscitation or kill effectively dormant bacteria can significantly enhance the efficacy of current treatments for latent infection. Such agents would

The frequency of spontaneous mutations that confer resistance to an individual TB drugs *in*

*Resistance to INH:* INH is a drug precursor that is activated by Mtb catalase-peroxidase enzyme (KatG) to generate a range of highly reactive species [29]*.* Active INH targets essentially enoylacyl carrier protein reductase (InhA enzyme), which is involved in mycolic acid synthesis [29]. Resistance to INH occurs more frequently than for most anti-TB drugs, at a frequency of 1 in

 bacilli *in vitro* [13]. Clinical isolates of INH-resistant Mtb often lose catalase and peroxidase activities due to KatG S315T mutation [30]. Resistance to INH can also occur through mutations in the promoter region of *inhA*, causing overexpression of InhA, or by mutations at the InhA active site, lowering InhA affinity for INH [31]. *katG* mutation can be associated with *inhA*

*Resistance to RMP:* RMP interferes with RNA synthesis by binding to the β subunit of myco‐ bacterial RNA polymerase, which is encoded by *rpo*B. Mtb resistance to RMP occurs at a frequency of 10−7 to 10−8 as a result of mutations in *rpo*B. Mutations at positions 531, 526 and

*Resistance to PZA*: PZA requires conversion to its active form, pyrazinoic acid (POA), by the pyrazinamidase/nicotinamidase enzyme encoded by Mtb *pncA*, which then permeates

516 in *rpo*B are among the most frequent (96%) in RMP-resistant strains [33].

(EMB) to 1 in 1010 (RMP) [28].

with the same strain or reinfection with a new strain.

206 Tuberculosis - Current Issues in Diagnosis and Management

also shorten the treatment duration of active TB.

*vitro* are well known and vary from 1 in 105

106

**3. Molecular basis of Mtb resistance to SSC drugs**

mutations*,* leading to higher levels of INH resistance [32].

Conventional culturing of the etiologic agent combined with drug susceptibility testing (DST) is the 'gold standard' for diagnosing drug resistant TB in order to initiate adequate treatment. However, this approach is rarely used because it requires 3 to 4 months to produce results. Indeed, only 7% of all MDR-TB cases are detected globally [1]. Hence, the deficiency in tools for rapid DST is associated with inadequate treatment regimens, which tragically increase transmission and spread of drug resistant TB, especially in HIV-infected individuals [38]. This alarming situation stimulated the development of a great number of rapid culture- and molecular-based methods that are currently being evaluated in TB diagnosis laboratories. The Nitrate Reductase Assay (NRA) is based on detection of nitrate reduction into nitrite by Mtb organisms capable of growth in the presence of the test antibiotic [39]. Whereas the Microscopic Observation of Drug Susceptibility (MODS) uses inverted microscope to detect the formation of cord-like structure by Mtb isolates resistant to the test drug [40]. The commercial Mycobac‐ terium Growth Indicator Tube 960 (MGIT 960) is a drug-containing culture system based on the fluorescence detection of resistant bacteria [41]. The Genotype MTBDR*plus* is a molecular line-probe assay that detects simultaneously mutations in the rpoB gene that confers resistance to RMP as well as mutations in the katG gene and the inhA promoter, which are associated with resistance to INH [42]. The Alamar blue and resazurin assays are liquid-based colori‐ metric tests [43]; a color change in wells containing drug-exposed bacteria reflects resistance*.* The MTT assay relays on the ability of drug-resistant (viable) bacteria to cleave the tetrazolium rings of MTT, which produces a violet-purple color [44]. Many of these assays gave excellent detection of MDR-TB, within a significantly shorter time frame when compared to conven‐ tional culturing methods (Table 1).

INH, RMP, PZA and EMB are used during the 6-month treatment period [52]. RMP-resistant TB often carries a much more ominous prognosis, as the outcome of SCC treatment is poor in terms of both disease status at the end of the treatment and relapse [13]. Moreover, RMP monoresistance in Mtb is rare and usually reflects resistance to INH as well, i.e., MDR-TB [53]. In fact, SCC cures less than 60% of MDR-TB, with a recurrence rate of about 28% among patient

Management of Drug-Resistant TB http://dx.doi.org/10.5772/55531 209

The current recommendation for individualized treatment regimens is a combination of at least four drugs to which the Mtb isolate is likely to be susceptible [55]. Drugs are chosen with a stepwise selection process through 5 groups of TB drugs (Table 2) on the basis of efficacy and safety [55]. More than 5 drugs can be used if the sensitivity to a given drug is unclear or if the regimen contains few bactericidal drugs. The duration of the intensive phase of treatment (when an injectable drug is given) should be at least 6 months (or 4 months after culture conversion). The continuation phase (without the injectable drug) should last until 18 months

Although the effectiveness and feasibility of MDR-TB management in resource-limited settings have been demonstrated, less than 2% of all estimated MDR-TB patients currently receive appropriate treatment [5]. Thus, the growing MDR-TB epidemic globally requires moving beyond the pilot project stage in order to scale up DOTS-plus based TB management as a routine component of national TB control programmes. However, there are potential difficulties with implementing DOTS-Plus in low-income countries as it can absorb a large part of resources dedicated to existing DOTS programmes, and subsequently decrease the overall standard of care [56]. Note that the emergence of drug resistant TB in these countries is actually

A major barrier to the management of drug resistant TB in low-income countries is the prohibi‐ tive price of second-line drugs. Therefore in an attempt to address this issue, in 2000, the WHO and its partners established the Green Light Committee (GLC) initiative to facilitate access to quality-assured second-line TB drugs at reduced prices [57,58]. Evaluation of the first GLCendorsed pilot projects of MDR-TB management in five resource-limited countries showed treatment success rates of 59%–83% [59]. During 2012, the number of patients with MDR-TB approved for treatment by the GLC Committee was only 42,033 with 13,000 actually starting treatment.Itisclearthatthesenumbersremainsmallcomparedtotheestimatedannualincidence (440,000 cases) of MDR-TB [1]. Therefore, substantial funding through public-private partner‐

Other than the price of second-line drugs, frequent adverse events and the long duration of the regimen further compromise adherence to TB treatment, even in the most advanced industrialized countries. These drawbacks have resulted in resurgence in research efforts during last decade to develop new TB drugs. In recent years, a number of new drug candidates with novel modes of action and excellent activity against Mtb have entered clinical trials [60]. OPC-67683 (nitro-dihydro-imidazooxazole) and diarylquinoline TMC207 are the most promising of these new drugs since both are highly active against drug-resistant and suscep‐ tible Mtb strains and possess excellent sterilizing activity [61]. These and other drugs under

the result of limited resources to implement the simple DOTS programme.

ships is desperately needed to scale up the availability of second line drugs.

with apparent success [38,54].

after culture conversion [55].

The effective implementation of these rapid diagnostic tests for TB and drug resistance will increase the proportion of patients promptly placed on appropriate therapy, and therefore will improve substantially management and control of TB disease globally. However a major limitation to the use of these rapid tests is their affordability and the availability of equipped laboratories in resource-constrained countries, which unfortunately tend to have the highest burden of MDR-TB cases. Thus, global initiatives are needed to make new diagnostics accessible to low-income countries.


MODS = microscopic observation drug susceptibility; NRA = nitrate reductase assay; AB =Alamar Bbue ; MTT = 3-[4,5 dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide; MGIT = Mycobacterium Growth Indicator Tube; LJPM = Löwenstein-Jensen proportion method.

**Table 1.** Time to results and percentage of results obtained within 8 and 10 days. Reprinted from Ref. 45 with permission of the International Union Against Tuberculosis and Lung Disease. Copyright © The Union.
