## 1 Introduction  
Tuberculosis (TB) is a disease affecting 10.0 million people globally each year [1], with drug resistance becoming a growing threat. TB remains one of the major causes of disease and death worldwide, exacerbated by HIV-TB co-infection [2] and the emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR) TB strains in both industrialized and developing countries [2; 3]. TB is a curable disease; however, the mortality rate associated with this medical condition has considerably increased during the last years. The newly emerging MDR mycobacteria strains lead to a doubling of the MDR TB cases that are hard to treat. The early and correct identification of the drug susceptibility of the TB strain affecting a patient is crucial for a positive anti-tuberculous treatment outcome. Drug susceptibility testing for _Mycobacterium tuberculosis_ (MTB) can be performed with various assays, such as the radiometric BACTEC 460 TB method [4; 5], the automated non-radiometric mycobacteria growth indicator tube (MGIT) 960 system [6; 7; 8] and the microtiter-based alamar blue assay [9; 10], among others. Phenotypic assays based on cell cultures in Lowenstein-Jensen dry medium are still considered to be the gold standard [11; 12]. Nevertheless, mycobacteria present a slow growth under dry conditions and the time for the result of the later assay can take up to three weeks for detection and an additional two to three weeks for drug susceptibility testing. Furthermore, although in high income countries the available financial resources assure the access for the patients to optimal diagnosis and individualized treatment schemes, in low resource regions, MDR-TB and XDR-TB cases with medical care requirements extending up to two years are hard to assess and treat. In the search for cost and time effective assays, the microscopic observation drug susceptibility (MODS) testing was introduced more than 15 years ago [13; 14]. Even though the MODS testing requires highly trained personnel, the necessary laboratory infrastructure is reduced to a centrifuge, an incubator and an inverted microscope. Furthermore, bacteria are cultured in sealed containers under liquid conditions assuring optimal growth conditions and providing information on drug susceptibility in less than two weeks [15]. The most common first line agents administered for drug-susceptible TB are rifampicin, isoniazid, ethambutol and pyrazinamide (PZA) [16]. PZA is the only drug with different activity in vivo and in vitro [17]. Its importance is based on its in vivo sterilizing effect that permits killing of persistent bacilli [18]. PZA is frequently used in both first- and second-line treatment regimens [19; 20; 21]. The incorporation of PZA into TB treatment regimens in the 1960s enabled the duration of first-line treatment to be reduced from nine to 12 months down to six months [22]. Among the drugs rifampicin, isoniazid, ethambutol and PZA, PZA susceptibility testing is the most challenging and it is not routinely performed [23; 24; 25]. The compound itself is not active against mycobacteria. It is administered as a pro-drug and its metabolite, pyrazinoic acid (POA), kills semi-dormant MTB in the acidic regions of acute inflammation that are inaccessible to other anti-TB drugs [22; 24; 26]. The conversion of the pro-drug to the metabolite is realized by the bacterial enzyme nicotinamide/ pyrazinamidase. The known major mechanism of PZA resistance is associated with a mutation of the pyrazinamidase enzyme resulting in no or a very low conversion rate to POA. Therefore, an indirect way of assessing PZA resistance is to determine the presence of POA in the extracellular environment of an in vitro liquid culture in the presence of PZA, such as the MODS-Wayne assay [27]. Colorimetric methods based on the reaction between ammonium iron sulfate and POA have been employed in order to assess the presence of the metabolite in the culture and to confirm the enzyme activity [27; 28].