**2. Direct detection methods**

The genus mycobacterium consists of almost 100 different species, which all appear similar on AFB staining and culture [7, 10, 12]. Many of these can be isolated from humans, although many also can be found in the environment including in animals. It is not easy, however, to distinguish between pathogen and saprophyte species. Each mycobacterium isolate must be evaluated individually regarding its potential to cause a disease; therefore identification of mycobactera is a lengthy and tedious effort. Since the introduction of nucleic acid amplification assays as diagnostic tool for mycobacteria identification, several probes/gene amplification systems for tuberculosis have been developed for rapid and specific identification of *M. tuberculosis* and other mycobacteria [12, 13]. These techniques allow for the confirmation of identity of isolates, direct detection of gene sequences from the clinical specimens and also for molecular detection of drug resistance [12]. Many previous publications have shown the sensitivity and specificity of several molecular detection assays such as BDProbeTec ET, (Becton Dickinson), COBAS AMPLICOR (Roche), Amplified *M. tuberculosis* Direct Test AMTDT (Gen Probe, USA) for identification of mycobacteria[9].

The use of nucleic-acid probe identification systems was a one step ahead in the rapid identification of mycobacterium species of *M. tuberculosis* complex, *M. avium* complex, *M.* *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].

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

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

The genus mycobacterium consists of almost 100 different species, which all appear similar on AFB staining and culture [7, 10, 12]. Many of these can be isolated from humans, although many also can be found in the environment including in animals. It is not easy, however, to distinguish between pathogen and saprophyte species. Each mycobacterium isolate must be evaluated individually regarding its potential to cause a disease; therefore identification of mycobactera is a lengthy and tedious effort. Since the introduction of nucleic acid amplification assays as diagnostic tool for mycobacteria identification, several probes/gene amplification systems for tuberculosis have been developed for rapid and specific identification of *M. tuberculosis* and other mycobacteria [12, 13]. These techniques allow for the confirmation of identity of isolates, direct detection of gene sequences from the clinical specimens and also for molecular detection of drug resistance [12]. Many previous publications have shown the sensitivity and specificity of several molecular detection assays such as BDProbeTec ET, (Becton Dickinson), COBAS AMPLICOR (Roche), Amplified *M. tuberculosis* Direct Test

The use of nucleic-acid probe identification systems was a one step ahead in the rapid identification of mycobacterium species of *M. tuberculosis* complex, *M. avium* complex, *M.*

detection methods used in the laboratory to identify tuberculosis.

AMTDT (Gen Probe, USA) for identification of mycobacteria[9].

days to give any result [9].

154 Tuberculosis - Current Issues in Diagnosis and Management

**2. Direct detection methods**

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 technique is also are much faster; results can be obtained within 3 hours since positive automated cultured system is obtained [18]. In view of the importance of early diagno‐ sis to prevent further spread of tuberculosis in the community, this time efficiency is the most significant contribution of Raman spectroscopy.

of progression from LTBI to active tuberculosis, but any disruption of their cellular immunity – suchasinHIVco-infectioncases–canconsiderablyincreasethisrisk[2].Currently,thediagnosis of LTBI is commonly made with the tuberculosis skin test (TST), which is based on the delayed hypersensitivity to purified protein derivative (PPD). Unfortunately, patients sensitized to environmentalnontuberculousmycobacteriaorpatientsvaccinatedwiththebacillusCalmette– Guérin (BCG) vaccine may have a false positive result. On the other hand, a false negative result may occur in immunosuppressed patients and also in children [2]. This immunologic re‐ sponse is often not conclusive as antibodies and delayed type hypersensitivity response persist

Diagnostic Evaluation of Tuberculosis http://dx.doi.org/10.5772/54630 157

Interferon Gamma Release Assays (IGRAs) have been introduced in the clinical setting for the diagnosis of LTBI [19-21]. These more specific whole-blood tests are based on the principle of measuring host interferon-y (IFN-y) released by T-cells specific to *M. tuberculosis* as a marker. IFN-y is stimulated by *early secretory antigen target 6* (ESAT-6) and *culture filtrate protein 10* (CFP-10). These are not present in the BCG or in the most of the NTM [2]. There are two types of IGRAs: The enzyme-linked immunospot assay (ELISpot)-based IGRA, where individual IFN-y producing T-cells responding to *M. tuberculosis* antigens stimulation are counted [22], and the QuantiFERON-TB Gold In-Tube test, an ELISA-based IGRA where the IFN-y produced by those T-cells is measured after stimulation with *M. tuberculosis* antigens [2]. Pai et al. showed that the sensitivity of the ELISpot and ELISA-based approach was around 90% and 70%, respectively, and that the specificity of both was 93% [2, 20]. As there is still no gold standard for the diagnosis of LTBI, these assays potentially may serve as routine diagnosis test other

Cytokine-based detection methods could be useful not only in the detection of LTBI cases but also of active tuberculosis cases. However, considering the high number of LTBI in the community, a single cytokine identification method such as IGRAs is not sufficient to detect active tuberculosis. For this reason the identification of multiple tuberculosis biomarkerscytokines seems to be a promising strategy. Several studies have shown the potential useful‐ ness of TNA-a, IL-2, IP-10, MIG along with IF-g simultaneously [23-26]. Using a multiplex microbead-based assay, Wang et al. showed significant differences in expression of these cytokines/chemokines between active tuberculosis patients and healthy controls. Regarding active pulmonary tuberculosis the sensitivity of IFN-y, IP-10 and MIG was 75.3% and the specificity was 89.7%. They also demonstrated the potential usefulness of this multiplex microbead-based assay for the detection of new tuberculosis cases by documenting a sensi‐

Untill now, smear and culture methods are still the gold standard to detect mycobacteria. Based on our experience, combination of conventional and advanced detection methods would greatly improve the sensitivity and specificity of the assays. Detection of the *mycobacteria* species are quite difficult with culture, therefore we using multiplex PCR as the first confir‐ mation assay to detect the species while it also as confirmation test for negative results from either smear or culture assay. Hence, to overcome the limitation of multiplex PCR in species detection, multiplex PCR- reverse cross blot hybridization assay would further expand the

long after infection or after the diseases has disappeared [12]

than TST to identify people with LTBI [2].

range of *mycobacteria* species detection (Fig. 2).

tivity of 96.3% [23].

**Figure 1.** Multiplex PCR reverse cross blot hybridization assay is able to detect various species of mycobacteria simulta‐ neously. Each column (Col) represents certain species of mycobacteria; Col 1, *M. intracellulare*; Col 2, *M. kansasii* ; Col 3-8, 11, 14, 20, 22, 24, 26, 28, 30-33, *M. tuberculosis*; Col 9, *M. fortuitum*; Col 10, 12, 13, *M. chelonae*; Col 15, 16, 18, 19, 23, 25, 27, 29, *M. avium*; Col 17, *M. genavense*; Col 21, *M. smegmatis* ; 34, pool PCR product of mycobacteria. [7]
