**3. Indirect detection methods**

Even those remarkable molecular detection methods are not yet up to the mark when it comes to in the identification oftuberculosis,particularly latenttuberculosis infection (LTBI).Approx‐ imately 2 milliard people are silent tuberculosis patients, i.e. they have been infected by *M. tuberculosis* but show no tuberculosis symptoms [1, 2]. LTBI has been defined by evidence of a cellular immune response to *M. tuberculosis* derived antigens. It may be the result of incom‐ plete eliminationof*M.tuberculosis*bythehost's adaptive immune system,resultinginasympto‐ maticinfectionwithalmostundetectablebacilli[2].Thus,thediagnosisofLTBIcurrentlydepends ondetectingthehost's immune response tothe infection[2].Affectedindividualshave little risk 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 long after infection or after the diseases has disappeared [12]

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

**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]

Even those remarkable molecular detection methods are not yet up to the mark when it comes to in the identification oftuberculosis,particularly latenttuberculosis infection (LTBI).Approx‐ imately 2 milliard people are silent tuberculosis patients, i.e. they have been infected by *M. tuberculosis* but show no tuberculosis symptoms [1, 2]. LTBI has been defined by evidence of a cellular immune response to *M. tuberculosis* derived antigens. It may be the result of incom‐ plete eliminationof*M.tuberculosis*bythehost's adaptive immune system,resultinginasympto‐ maticinfectionwithalmostundetectablebacilli[2].Thus,thediagnosisofLTBIcurrentlydepends ondetectingthehost's immune response tothe infection[2].Affectedindividualshave little risk

most significant contribution of Raman spectroscopy.

156 Tuberculosis - Current Issues in Diagnosis and Management

**3. Indirect detection methods**

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 than TST to identify people with LTBI [2].

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‐ tivity of 96.3% [23].

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

clinical settings and whether they are economically affordable for developing countries, in

Diagnostic Evaluation of Tuberculosis http://dx.doi.org/10.5772/54630 159

Tuberculosis still remains a major health problem in many developing countries, despite continuous long-standing vaccination and surveillance programs, and worldwide availability of effective anti-tuberculosis drugs. Early detection is of major importance in the control of tuberculosis. The emergence of multidrug resistant *Mycobacterium tuberculosis* and the association of HIV with tuberculosis outbreaks in community both illustrate that rapid diagnosis is essential. Therefore, a fast and reliable diagnosis of tuberculosis would greatly improve the control of the tuberculosis. Regrettably, current conventional laboratory diag‐ nostic methods of tuberculosis are still time-consuming. The rapid development of novel diagnostic methods for the identification of mycobacteria and its species bring new hope, however, for the diagnosis and management this infectious disease. Meanwhile those techni‐

Departments of Medical Microbiology, Molecular Biology and Immunology Laboratory,

[1] Jain, S. K, Lamichhane, G, Nimmagadda, S, Pomper, M. G, & Bishai, W. R. Antibiotic

[2] Connell, D. W, Berry, M, & Cooke, G. Kon OM: Update on tuberculosis: TB in the

[3] Nhlema Simwaka B. Benson T, Salaniponi FM, Theobald SJ, Squire SB, Kemp JR: De‐ veloping a socio-economic measure to monitor access to tuberculosis services in ur‐

[4] Golub, J. E, Mohan, C. I, & Comstock, G. W. Chaisson RE: Active case finding of tu‐ berculosis: historical perspective and future prospects. Int J Tuberc Lung Dis (2005).

[5] Caminero JA: Multidrug-resistant tuberculosis: epidemiologyrisk factors and case

treatment of tuberculosis: old problem, new solution. Microbe (2008).

ques still seem to clash with simplicity and economically affordable issues.

Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

early 21st century. Eur Respir Rev (2011).

finding. Int J Tuberc Lung Dis (2010).

ban Lilongwe, Malawi. Int J Tuberc Lung Dis (2007).

most of which tuberculosis is still rampant [11].

**Summary**

**Author details**

**References**

Mochammad Hatta and A. R. Sultan

**Figure 2.** Microbiologic diagnosis of tuberculosis. Multiplex PCR are used to confirm smears results and negative result of culture assay. Patients with negative multiplex PCR result would be proceed for ELISpot or Tuberculin skin test (TST) to detect latent tuberculosis (LTBI), while specimen from patient with positive culture result would get final confirma‐ tion by Multiplex PCR reverse cross blot hybridization assay to further detect the mycobacterium species
