**1. Introduction**

Tuberculosis is still one of the leading causes of death by infectious diseases with 2 million deaths per year and 9.2 million new cases of tuberculosis disease annually [1-3]. Besides, more than 2 milliard people are infected with latent tuberculosis infection (LTBI) [1-3]. Despite continuous effort in the prevention, monitoring and treatment of tuberculosis, the disease remains a major health problem in many countries [4-6], particularly in developing countries like Indonesia [7]. National tuberculosis programs and other programs conducted by foreign organizations still fail to eliminate the transmission and incidence of tuberculosis. Transmis‐ sion is even on the rise in developing countries despite the availability of effective therapies for tuberculosis, whereas the spread and the incidence of tuberculosis in Europe and North America are under control. Several reasons may be responsible for this failure, such us the difficulty of providing adequate anti-tuberculosis medication in many developing countries due to cost issues, the emergence of multi-drug resistant (MDR) strains of *M. tuberculosis*, and the dramatically high co-incidence of tuberculosis in HIV-infected patients [2, 7]. Another important issue is delay of diagnosis due to the lack of a proper method to identify tuberculosis agents [1, 8].

Smear is the cheapest and most widely available detection method for *M. tuberculosis*. In this technique, the diagnosis of tuberculosis is based on identification of acid-fast bacilli (AFB) in a patient`s sputum [9, 10]. Many staining techniques are available for AFB smear, the most common one of which is the modified Ziehl-Neelsen stain. Unfortunately, the sensitivity and the specificity of those techniques are low due to difficulty in the identification and differen‐ tiation of the various species of *M. tuberculosis* [10]. Two studies found that the AFB smear was positive in only half of patients with subsequent culture positive for *M. tuberculosis* [9, 10]. Another worldwide available detection method is the conventional culture method on

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Lowenstein-Jensen (LJ) medium. This method is the gold standard in the identification of *M. tuberculosis* and still serves as the reference method due to its high sensitivity (89%) and specificity (98%) [4, 7, 9, 10]. However, this technique requires equipments or materials that are often unavailable in resource-poor settings. In addition, this technique is time consuming; the results only can be obtained after 6–12 weeks. In addition, the incidence of other bacterial contamination on culture tends to be high [7, 11]. Even a modern culture method such as the BACTEC MGIT 960 culture system, which uses the modified Middlebrook 7H9 broth and a fluorescent signaling system, allows for earlier detection of growth, but still takes at least 10 days to give any result [9].

*avium, M. intracellulare, M. kansasii,* and *M. gordonae* and also other nontuberculous mycobac‐ teria (NTM) in culture because the result can be obtained after 2 hours [10, 12]. But the sensitivity and specificity of this probe technology will only approximate 100% if there are more than 100 *mycobacteria* present in the sample, except for *M. kansasii* (87%) [12]. Thus, these probes are not sensitive enough to be used directly in clinical specimens like sputum. Also, it still needs to be confirmed by other conventional detection methods such as biochemical test and molecular tests to able to identify the species identity within the *M. tuberculosis* complex, such as for *M. microti, M. bovis, M. bovis of* BCG,*, M. canettii,* and *M. africanum* [10]. There has been extensive research to design an identification system for ribosomal RNA/DNA finger‐ printing and for development of probes that targeting specific rRNA, ribosomal DNA, spacer and flanking sequences of various types of mycobacterium species including *M. tuberculosis, M. leprae, M. avium, M. gardonae,* etc [12, 13]. Those rRNA targeting probes are 10-100 fold more sensitive than DNA targeting. However, since the lowest detection limit is still around 100 organisms. it still needs more evaluation before it can be applied to clinical specimens [12].

Diagnostic Evaluation of Tuberculosis http://dx.doi.org/10.5772/54630 155

Several techniques based on polymerase chain reaction (PCR) and isothermal amplification assay have been developed [7-10, 12]. Various researchers have described the rapid detection of *M. tuberculosis* by PCR, and many have reported a high sensitivity in detecting *M. tubercu‐ losis* in clinical samples by means of DNA amplifications [7, 14]. Such techniques involve amplification of specific gene regions followed by hybridization with species specific primers, and also frequently followed by sequencing and or restriction fragment length polymorphism (RFLP) analysis [12]. RFLP is still most widely used in clinical microbiology laboratories due to its simplicity and lower costs than PCR Sequencing [12]. Multiplex PCR has been used to detect *M. tuberculosis* complex bacteria and other mycobacterium. This technique is based on the amplification of the most widely used specific insertion sequences IS6110 and 16S [7-9]. Based on our experience, multiplex PCR has sensitivity up to 81.62% with negative predictive value up to 79.51% [7]. Nevertheless, taking into account the "simple and economical" issue this technique is probably not suited for most of the countries with a high tuberculosis burden [11]. Other rapid molecular amplification detection method which is being used in our laboratory is multiplex PCR-reverse cross blot hybridization, which can be modified to identify multiple species of mycobacteria at one time by using a specific probe for each species. Compared to the culture and microscopic method, this technique had a sensitivity of 86.03%, negative predictive value of 82.41% and it can be applied to detect NTM [7]. The multiplex PCR reverse cross blot hybridization technique is more complicated than conventional multiplex PCR; but it can detect considerably more NTM species such as *M. avium, M. intracellulare, M. kansasii, M. fortuitum, M. chelonae, M. genavense and M. smegmatis* (Fig. 1) [7].

In term of accuracy and duration time that it needs to get a result, Raman spectroscopy is one of the most promising techniques. This vibrational spectroscopy-based detection method can detect and differentiate various molecular compositions of microorganism [15-18] and therefore is suitable to identify the species and strains of microorganism. Buijtels et al., demonstrated that Raman spectroscopy differentiated between *M. tuberculo‐ sis* with NTM with accuracy up to 100% and with 92.5% correct species identification. This

The goal of tuberculosis control programs is to identify and to cure as many cases as possible; therefore the critical role of early diagnosis is obvious [11]. Under-diagnosis may lead to further spread of the disease because undiagnosed patients can spread the disease unnoticeably [11]. Accurate and early diagnosis is the first important step to effective management. Several new methods for the identification of tuberculosis are available, which including serologic tests and also various molecular methods developed as a result of major advances in understanding the genetic aspects of tuberculosis [8, 9, 11]. Those detection methods can be grouped into two types First, by detection of mycobacteria or its components directly; second by measurement of immunologic responses to mycobacte‐ rium infection [9]. In this chapter we present a short review of some these promising detection methods used in the laboratory to identify tuberculosis.
