Diagnosis of extrapulmonary tuberculosis by PCR

Abstract

During the last two decades, the resurgence of tuberculosis (TB) has been documented in both developed and developing nations, and much of this increase in TB burden coincided with human immunodeficiency virus (HIV) epidemics. Since then, the disease pattern has changed with a higher incidence of extrapulmonary tuberculosis (EPTB) as well as disseminated TB. EPTB cases include TB lymphadenitis, pleural TB, TB meningitis, osteoarticular TB, genitourinary TB, abdominal TB, cutaneous TB, ocular TB, TB pericarditis and breast TB, although any organ can be involved. Diagnosis of EPTB can be baffling, compelling a high index of suspicion owing to paucibacillary load in the biological specimens. A negative smear for acid-fast bacilli, lack of granulomas on histopathology and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis of EPTB. Novel diagnostic modalities such as nucleic acid amplification (NAA) can be useful in varied forms of EPTB. This review is primarily focused on the diagnosis of several clinical forms of EPTB by polymerase chain reaction (PCR) using different gene targets.

extrapulmonary tuberculosis, Mycobacterium tuberculosis, nucleic acid amplification test, PCR, GeneXpert assay, Immuno-PCR

Introduction

Tuberculosis (TB) remains one of the leading infectious diseases throughout the world accounting for about 8.8 million incident cases in 2010 (Griffiths et al., 2010; WHO, 2011). India alone accounted for 2.0–2.5 million cases in 2010, thus contributing approximately 26% of all TB cases worldwide (WHO, 2011). According to National Tuberculosis Control Programmes (NTPs), 2.6 million new cases of sputum smear-positive pulmonary TB (PTB), 2.0 million new cases of sputum smear-negative PTB and 0.8 million new cases of extrapulmonary tuberculosis (EPTB) were observed in 2010 worldwide (WHO, 2011). EPTB has become more common since the advent of human immunodeficiency virus (HIV) infection (Cabandugama et al., 2011; WHO, 2011). EPTB constitutes about 15–20% of TB cases and can constitute up to 50% of TB cases in HIV-infected individuals (Noussair et al., 2009; Peto et al., 2009; Cortez et al., 2011). As India has high burden of TB cases, thus proportionately higher number of EPTB cases are also observed in this country (WHO, 2011). The diagnosis of smear-positive PTB has been considerably established, but the diagnosis of smear-negative PTB, TB–HIV co-infection and EPTB poses serious challenges (Golden & Vikram, 2005; Chang, 2007). Diagnosis of EPTB, in particular, is difficult owing to paucibacillary nature of the specimens, lack of adequate clinical sample volumes and nonuniform distribution of bacteria in those specimens as well as the disease localized in sites that are difficult to access (Chakravorty et al., 2005; Cheng et al., 2005; Galimi, 2011).

Various methods are employed for the diagnosis of EPTB such as smear microscopy, culture identification, histopathology, tuberculin skin test (TST), serological assays, interferon-gamma release assays (IGRAs) and nucleic acid amplification (NAA) tests (Katoch, 2004; Lange & Mori, 2010). Smear microscopy is widely used in the diagnosis of EPTB but has drawbacks owing to low and variable sensitivity values (0–40%) and could not differentiate between Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM; Liu et al., 2007; Haldar et al., 2011; Derese et al., 2012). Culture identification for M. tuberculosis also has variable sensitivities (0–80%) in different extrapulmonary specimens (Padmavathy et al., 2003; Sharma & Mohan, 2004; Takahashi et al., 2008; Abbara & Davidson, 2011) with turnaround time of 4–8 weeks and requires skilful technicians (Mehta et al., 2012). Diagnosis of EPTB from tissue samples is usually made by histopathological examination that depends on the presence of granulomatous inflammation and caseous necrosis (Liu et al., 2007; Almadi et al., 2009). However, histology does not distinguish between EPTB and infections from other granulomatous diseases such as NTM, sarcoidosis, leprosy and systemic lupus erythematosus (except for the presence of acid-fast bacilli; AFB; Bravo & Gotuzzo, 2007; Chawla et al., 2009).

The TST is useful for the diagnosis of EPTB; however, false-positive reactions occur as a result of previous Bacille Calmette–Guérin (BCG) vaccination or sensitization to NTM, and false-negative results occur in the immunocompromised patients, elderly persons or overt forms of TB (Lange & Mori, 2010). The in vitro T-cell-based IGRAs have been used for the diagnosis of both latent and active TB, but these assays do not differentiate between latent and active TB infection (Pai & O'Brien, 2008). Therefore, these assays have no utility in disease diagnosis and treatment in highly endemic countries (Dyrhol-Riise et al., 2010). However, these IGRAs have some potential to assist in the diagnosis of active TB in immunocompromised persons, smear-negative PTB and EPTB patients (Pai & O'Brien, 2008). The analysis of cytokine profiles in M. tuberculosis-specific CD4⁺ T cells by polychromatic flow cytometry could differentiate between active and latent TB (Harari et al., 2011). The use of flow cytometry as part of the diagnostic algorithm has been exploited for EPTB infection (e.g. pleural TB); however, owing to high cost, its use as a rapid diagnostic test is limited in the resource-poor settings (Sutherland et al., 2012). The serological antibody detection tests have been widely used, and the tools of genomics and proteomics have led to the use of several antigens for the diagnosis of patients with both PTB and EPTB (Steingart et al., 2011). As a result of inconsistent and imprecise estimates, the World Health Organization (WHO) Expert Group Meeting convened in 2010 has strongly recommended against the use of any of these serological tests for the diagnosis of both PTB and EPTB cases (Morris, 2011). It is believed that the detection of antigens in EPTB patients is relatively more accurate method compared to the antibody detection (Kalra et al., 2010; Steingart et al., 2011).

NAA tests

A major breakthrough in the diagnosis of EPTB especially in health settings with a high prevalence of HIV-EPTB co-infection is achieved by the introduction of NAA tests such as PCR to detect nucleotide sequences unique to M. tuberculosis directly in extrapulmonary specimens which give results within few hours, offering better accuracy than AFB smear microscopy and greater speed than culture (Katoch, 2004; Jacob et al., 2008; Abbara & Davidson, 2011; Haldar et al., 2011). The current review is focused to diagnose several clinical types of EPTB by PCR using different gene targets. Various gene targets such as IS6110, 16S rRNA gene, 65 kDa protein gene (Rv0440), devR (Rv3133c), MPB-64/MPT-64 protein gene (Rv1980c), 38 kDa protein gene (Rv0934), TRC4 (conserved repetitive element) GCRS (guanine-cytosine-rich repetitive sequence), hupB (Rv2986c), dnaJ (Rv0352), MTP-40 protein gene (Rv2351c) and PPE gene (Rv0355) have been employed in these PCR assays (Martins et al., 2000; Bandyopadhyay et al., 2008; Garcia-Elorriaga et al., 2009; Haldar et al., 2011). The reason for widely used IS6110 in PCR tests is the presence of its multiple copies in M. tuberculosis complex genome, which is believed to confer higher sensitivity (Lima et al., 2003; Rafi et al., 2007; Jin et al., 2010). However, a few studies from different geographical regions of the world have reported that some clinical isolates have either a single copy or no copy of IS6110 that leads to false-negative results (Dale et al., 2003; Thangappah et al., 2011). For avoiding the contamination during amplification and increasing the sensitivity, new approaches such as nested PCR (two step amplification; Torrea et al., 2005; Liu et al., 2007; Agashe et al., 2009) and multiplex PCR (amplification of two or more gene targets simultaneously; Okazaki et al., 2005; Colmenero et al., 2010; Sharma et al., 2011a, b) have been exploited for EPTB diagnosis.

The DNA-PCR is unable to differentiate viable and nonviable organisms, while bacterial mRNA with a mean half-life of 3–5 min is more prone to destruction than the genomic DNA; thus, a positive mRNA signal would indicate the presence of viable organisms (Rana et al., 2011). The mRNA-based reverse transcriptase-PCR (RT-PCR) is a rapid method to differentiate viable and nonviable M. tuberculosis and has also been used for the diagnosis of EPTB as well as to monitor drug resistance (Eltringham et al., 1999; Rana et al., 2011). Real-time PCR is a novel and robust assay primarily used to quantify the nucleic acid molecules in EPTB specimens (Baba et al., 2008; Rosso et al., 2011). The main advantages of real-time PCR are shortened turnaround time, quantification of bacterial load and automation of the procedure that reduces hands-on time and decreased risk of cross-contamination (Kalantri et al., 2011; Rosso et al., 2011).

During PCR amplification, several inhibitors such as host proteins, blood and even eukaryotic DNA in extrapulmonary specimens are known to interfere with the sensitivity of PCR and give false-negative results (Gan et al., 2002; Haldar et al., 2011; Sun et al., 2011). A multi-step process is often required to eliminate PCR inhibitors and to obtain highly purified DNA. To achieve this, numerous techniques for DNA sample preparation have been recommended such as freeze-boiling, chelex/proteinase K treatment and sequence capture method (Honore-Bouakline et al., 2003). Chakravorty & Tyagi (2005) introduced a novel multi-purpose universal sample processing (USP) technique using chaotropic property of guanidinium hydrochloride as a principle component and that can be used for inhibitor-free PCR for both PTB and EPTB specimens. The addition of cetyltrimethylammonium bromide or silica membranes in the DNA purification has also been shown to effectively remove the PCR inhibitors and, hence could improve the PCR sensitivity in EPTB specimens (Böddinghaus et al., 2001; Honore-Bouakline et al., 2003; Rafi et al., 2007). However, the additional purification steps could lead to substantial loss of mycobacterial DNA, and to circumvent this problem, a short-culture augmentation step for 2–3 days has been proposed before performing PCR test (Cheng et al., 2005), which could enhance the mycobacterial load, while concomitantly diluting PCR inhibitors. Recently, Santos et al. (2009) compared nine different DNA extraction systems (seven manual and two automatic) in an experimental model of pleural TB for analysis with real-time PCR. Only two methods, that is, extraction using columns (Qiagen) and automated extraction with total nucleic acid isolation (TNAI) system (Roche), were able to detect the presence of M. tuberculosis DNA in all the pleural TB samples, thus demonstrating that the DNA extraction method could affect the performance of real-time PCR. Because of extremely high sensitivity of PCR, the carry-over contamination of amplicon, previous infection or asymptomatic EPTB infection at another site could result into false positivity (Honore-Bouakline et al., 2003; Chakravorty et al., 2005; Sun et al., 2011). The false positivity of PCR reports in the absence of clinical findings poses serious challenges these days in diagnosing EPTB cases (Thangappah et al., 2011).

The lack of proper gold standard remains the major hindrance for evaluating new diagnostics in EPTB-infected individuals (Sun et al., 2011; Vadwai et al., 2011). The true accuracy of PCR tests may actually be different than the reported one when using an imperfect gold/reference standard (Abbara & Davidson, 2011; Tortoli et al., 2012). Culture (on solid and liquid media) is the most widely used gold standard for validating PCR results in diagnosing EPTB specimens although it is suboptimal gold standard with varying sensitivities and leads to inaccurate PCR results (Negi et al., 2005a; Hillemann et al., 2011; Sun et al., 2011). The other gold/reference standards include BACTEC culture, histopathology and response to anti-tubercular therapy (ATT) and also the combination of these methods (Negi et al., 2005b; Kulkarni et al., 2006; Abdalla et al., 2009; Noussair et al., 2009). Chakravorty et al. (2005) as well as Vadwai et al. (2011) have used smear, culture, histology/cytology, clinical findings and response to ATT, all together as the gold/reference standard for validating their PCR results in diagnosing EPTB specimens.

There are several potential commercial kits devised to diagnose TB such as Amplicor M. tuberculosis test (Roche Molecular Systems Branchburg, NJ), Gen-probe Amplified M. tuberculosis Direct Test (AMTD; Gen-Probe, CA), COBAS TaqMan M. tuberculosis (Roche Molecular Systems Branchburg) and LightCycler (Roche Molecular Diagnostics, Mannheim, Germany; Ritis et al., 2005; Causse et al., 2011; Parrish & Carroll, 2011) Among these, Amplicor M. tuberculosis test and AMTD based on 16S rRNA gene have been approved by the US Food and Drug Administration (FDA) for the diagnosis of PTB only (Brodie & Schluger, 2009), and none of these commercial tests have been approved by FDA for the diagnosis of EPTB (Parrish & Carroll, 2011). However, the utility of these commercial tests has been extensively explored in the diagnosis of EPTB (Honore-Bouakline et al., 2003; Causse et al., 2011). Moreover, the meta-analyses of PCR tests have suggested that the commercial tests yielded high specificities but variable sensitivities for the diagnosis of EPTB, while heterogeneous sensitivities and specificities were observed with in-house PCR tests (Pai et al., 2004; Daley et al., 2007). The major drawback of these commercial tests is their high costs, which make them unaffordable in most of the developing countries with high TB burden (Daley et al., 2007). Recently, MTB/RIF GeneXpert (Xpert) assay (Cepheid, Sunnyvale, CA) has been a major breakthrough in the diagnosis of EPTB (Vadwai et al., 2011; Tortoli et al., 2012). Further details of this test are discussed later in this review.

Clinical types of EPTB and diagnosis by PCR tests

EPTB exists in several clinical forms and important research findings related to their diagnosis by PCR are described as follows.

Tuberculous lymphadenitis

Tuberculous (TB) lymphadenitis is the most common presentation of EPTB and has been shown in about 35% of EPTB cases (Mohapatra & Janmeja, 2009; Cortez et al., 2011). Most frequently, this disease involves the cervical lymph nodes followed by mediastinal, axillary, mesenteric, hepatic portal and inguinal lymph nodes (Sharma & Mohan, 2004). Diagnosis of TB lymphadenitis is challenging as it mimics the other pathologic processes (sarcoidosis, leprosy, fungal and NTM infections) and yields inconsistent histopathological findings in the absence of AFB (Osores et al., 2006; Derese et al., 2012). Fine-needle aspiration (FNA) cytology, a less invasive procedure than excision biopsy, has assumed an important role in the diagnosis of TB lymphadenitis (Chakravorty et al., 2005; Derese et al., 2012). However, the amount of material obtained in the FNA is usually so small that it is often inadequate to perform AFB smear and culture examination (Kidane et al., 2002; Mohapatra & Janmeja, 2009). FNA cytology also has difficulty in differentiating TB from other granulomatous or NTM diseases (Baek et al., 2000). Several researchers have performed PCR from the remainders of FNA after cytological examination, and this clinical application of PCR along with FNA cytology could reduce the necessity for open biopsy as the process of biopsy is invasive and leaves unwanted scar tissues in the neck causing aesthetic problems (Baek et al., 2000; Supiyaphun et al., 2010).

Various gene targets such as IS6110, 16S rRNA gene, IS1081, 65 kDa and MPB-64 have been employed to diagnose TB lymphadenitis by PCR from FNA or formalin-fixed paraffin-embedded tissues with varying sensitivities and specificities (Totsch et al., 1996; Pahwa et al., 2005; Osores et al., 2006; Nopvichai et al., 2009; Sharma et al., 2010b; Cortez et al., 2011; Derese et al., 2012; Table 1). Within M. tuberculosis complex, M. tuberculosis and Mycobacterium bovis are the major causative agents of TB lymphadenitis. The rest of FNA after cytological evaluation has been used for PCR based on three gene targets to identify Mycobacterium at the genus (Antigen 85 complex gene), complex (IS6110) and species (pncA gene and allelic variation) levels in patients with TB lymphadenitis. It was found that PCR positivity was 87% at the genus and complex levels, 68.5% at species level for M. tuberculosis and 17% for M. bovis (Kidane et al., 2002). A nested PCR targeting hupB gene has also been documented from FNA specimens to differentiate M. tuberculosis from M. bovis (Verma et al., 2010). PCR test based on individual IS6110 and devR targets showed moderate sensitivity in USP-processed lymph node samples from patients with TB lymphadenitis, and the sensitivity was enhanced by the combination of devR PCR and IS6110 PCR results (by performing separate PCR reaction for each gene target; Chakravorty et al., 2005). The diagnosis of TB lymphadenitis in peripheral blood mononuclear cells has also been examined by the combination of IS6110 PCR and 65 kDa PCR results (Mirza et al., 2003) and that showed better sensitivity than lymph node PCR.

1

Sensitivity and specificity of PCR tests using different gene targets for the diagnosis of EPTB

Reference	Type of EPTB	NAA test	Gene target	Sensitivity (%)	Specificity (%)
Totsch et al. (1996)	TB lymphadenitis	Nested PCR	65 kDa	89	100
Kidane et al. (2002)	TB lymphadenitis	PCR	Ag85 complex/IS6110	87	N.D.
			pncA (M. tb)	69	N.D.
			pncA (M. bovis)	17	N.D.
Mirza et al. (2003)	TB lymphadenitis	LN-PCR	Combined results of IS6110 + 65 kDa	33	87
		PBMC-PCR	Combined results of IS6110 + 65 kDa	63	100
Bruijnesteijn Van Coppenraet et al. (2004)	NTM lymphadenitis	Real-time PCR	Internal transcribed spacer sequence	72	100
Pahwa et al. (2005)	TB lymphadenitis	PCR	MPB-64	90	86
Chakravorty et al. (2005)	TB lymphadenitis	PCR	devR	47	67
			IS6110	60	75
			Combined results of devR + IS6110	67	75
Osores et al. (2006)	TB lymphadenitis	Amplicor MTB	16S rRNA gene	58	94
Nopvichai et al. (2009)	TB lymphadenitis	PCR	IS6110	67	100
Verma et al. (2010)	TB lymphadenitis	Nested PCR	hupB	87	67
Sharma et al. (2010b)	TB lymphadenitis	PCR	IS6110	100	92
Cortez et al. (2011)	TB lymphadenitis	PCR	IS6110	63	87
Derese et al. (2012)	TB lymphadenitis	PCR	IS1081	42	89
Martins et al. (2000)	Pleural TB and TB meningitis	Nested PCR	MPB-64	70	88
Villegas et al. (2000)	Pleural TB	PCR	IS6110	61	90
PCR or IFN-γ	IS6110	81	88
	PCR or ADA	IS6110	90	77
Lima et al. (2003)	Pleural TB	PCR	IS6110	31	96
	PCR or ADA	IS6110	87	72
Moon et al. (2005)	Pleural TB	Amplicor MTB	16S rRNA gene	17	98
Chakravorty et al. (2005)	Pleural TB	PCR (pleural fluid)	devR	76	94
			IS6110	76	94
			Combined results of devR + IS6110	83	94
		PCR (pleural tissue)	devR	63	100
			IS6110	75	100
			Combined results of devR + IS6110	88	100
Srivastava et al. (2006)	Pleural TB	PCR	PPE	78	100
Liu et al. (2007)	Pleural TB	Nested PCR	IS6110	43	95
Baba et al. (2008)	Pleural TB	Real-time PCR	65 kDa	68	73
		Nested PCR	IS6110	64	82
Bandyopadhyay et al. (2008)	Pleural TB	Multiplex PCR	IS6110 + dnaJ + 65 kDa	82	80
Kalantri et al. (2011)	Pleural TB	Real-time PCR	16S rRNA gene	65	98
		Real-time PCR or IFN-γ	16S rRNA gene	97	94
Rosso et al. (2011)	Pleural TB	Real-time PCR	IS6110	43	94
Kulkarni et al. (2005)	TB meningitis	PCR	38 kDa	90	100
Quan et al. (2006)	TB meningitis	PCR	IS6110	75	94
Srivastava et al. (2006)	TB meningitis	PCR	PPE	70	100
Rafi et al. (2007)	TB meningitis	PCR	IS6110	98	100
	Nested PCR	MPB-64	91	91
	Nested PCR	65 kDa	51	92
Bandyopadhyay et al. (2008)	TB meningitis	Multiplex PCR	IS6110 + dnaJ + 65 kDa	73	92
Dora et al. (2008)	TB meningitis	Nested PCR	65 kDa	50	99
Takahashi et al. (2008)	TB meningitis	QNRT-PCR	MPB-64	96	100
	WR-QNRT-PCR	MPB-64	96	100
Haldar et al. (2009)	TB meningitis	Real-time PCR (filtrate)	devR	88	92
		PCR (filtrate)	devR	88	87
		PCR (filtrate)	IS6110	85	84
Real-time PCR (sediment)	devR	53	92
		PCR (sediment)	devR	31	94
		PCR (sediment)	IS6110	40	93
Sharma et al. (2010a)	TB meningitis	PCR	38 kDa	83	100
Sharma et al. (2011a)	TB meningitis	Multiplex PCR	IS6110 + 38 kDa + MPB-64	87	100
Verettas et al. (2003)	Osteoarticular TB	Nested PCR	IS6110	100	N.D.
Yun et al. (2005)	Osteoarticular TB	PCR	rpoB	53	N.D.
Negi et al. (2005b)	Osteoarticular TB	PCR	65 kDa	78	100
Jambhekar et al. (2006)	Osteoarticular TB	PCR	IS6110	85	80
Jain et al. (2008)	Osteoarticular TB	PCR	16S rRNA gene	98	N.D.
Agashe et al. (2009)	Osteoarticular TB	Nested PCR	IS6110	94	63
Pandey et al. (2009)	Osteoarticular TB	Nested PCR	IS6110	73	94
Colmenero et al. (2010)	TVO and BVO	Multiplex real-time PCR	BCSP31 + SenX3-RegX3	93	90
Sharma et al. (2011b)	Osteoarticular TB	Multiplex PCR	IS6110 + MPB-64	82–100	100
Sun et al. (2011)	Osteoarticular TB	PCR/Real-time PCR	IS6110	83	91
Hemal et al. (2000)	Genitourinary TB	PCR	MPB-64	94	N.D.
Moussa et al. (2000)	Genitourinary TB	PCR	IS6110	96	98
			16S rRNA gene	87	99
Bhanu et al. (2005)	Genitourinary TB	PCR	MPB-64	56	97
Garcia-Elorriaga et al. (2009)	Genitourinary TB	Nested PCR	MTP-40	100	82
Sun et al. (2010)	Genitourinary TB	Real-time PCR (CT35)	IS6110	83	86
		Real-time PCR (CT40)	IS6110	93	57
Rana et al. (2011)	Genitourinary TB
	EAs	PCR	MPB-64	45	N.D.
	PF/PW	PCR	MPB-64	10
	CBs	PCR	MPB-64	33
	EAs	RT-PCR	Ag85B	2.8
Thangappah et al. (2011)	Genitourinary TB	PCR	IS6110	25	90
			TRC4	46	100
Gan et al. (2002)	Abdominal TB	PCR	IS6110	64	100
Kulkarni et al. (2006)	Abdominal TB	PCR	38 kDa	77	68
Bandyopadhyay et al. (2008)	Abdominal TB	Multiplex PCR	IS6110 + dnaJ + 65 kDa	79	87
Balamurugan et al. (2010)	Abdominal TB	PCR	IS6110	79	88
Jin et al. (2010)	Abdominal TB	PCR (Kit <A>)	IS6110 + MPB-64	45	88
		PCR (Kit <B>)	IS6110	36	100
		PCR (In-house)	IS6110	5	100
		PCR (Kit <B>) + histopathology	IS6110	58	100
Padmavathy et al. (2003)	Cutaneous TB	PCR	IS6110	64	N.D.
Ogusku et al. (2003)	Cutaneous TB	Nested PCR	IS6110	100	100
			16S rRNA gene	44	90
Negi et al. (2005a)	Cutaneous TB	PCR	65 kDa	95	100
Abdalla et al. (2009)	Cutaneous TB	PCR	65 kDa	88	83
Obieta et al. (2010)	Cutaneous TB	PCR	IS6110	64	92
Madhavan et al. (2000)	Ocular TB	Nested PCR	MPB-64	78	67
Madhavan et al. (2002)	Ocular TB	Nested PCR	MPB-64	21	96
Zamirian et al. (2007)	Pericardial TB	PCR	IS6110	80	80
Rebollo et al. (2006)	Disseminated TB	PCR	IS6110	50	100
Singh et al. (2006)	Disseminated TB	PCR	MPB-64	33	100
Oh et al. (2001)	Combined EPTB	Amplicor MTB	16S rRNA gene	61	99
Ritis et al. (2005)	Combined EPTB	Nested PCR	IS6110	68	100
		Nested PCR (LightCycler)	IS6110	95	100
Torrea et al. (2005)	Combined EPTB (HIV-positive)	Nested PCR	IS6110	67	95
	Combined EPTB (HIV-negative)	Nested PCR	IS6110	46	100
Rebollo et al. (2006)	Combined EPTB	PCR	IS6110	36	100
Noussair et al. (2009)	Combined EPTB	Culture + GTMD	23S rRNA	100	100
Causse et al. (2011)	Combined EPTB	GeneXpert MTB/RIF	rpoB	95	100
		Cobas TaqMan MTB	16S rRNA gene	78	98
Hillemann et al. (2011)	Combined EPTB	GeneXpert MTB/RIF	rpoB	77	98
Miller et al. (2011)	Combined EPTB	GeneXpert MTB/RIF	rpoB	88	87
		Real-time PCR	IS6110	50	87
Vadwai et al. (2011)	Combined EPTB	GeneXpert MTB/RIF	rpoB	81	100
Tortoli et al. (2012)	Combined EPTB	GeneXpert MTB/RIF	rpoB	81	100

LN, lymph node; PBMC, peripheral blood mononuclear cells; TVO, TB vertebral osteomyelitis; BVO, Brucellar vertebral osteomyelitis.

M. tb, M. tuberculosis; QNRT-PCR, quantitative nested real-time PCR; WR-QNRT-PCR, wide-range quantitative nested real-time PCR.

Multiple samples of endometrial aspirates, endometrial biopsies and fluids from the pouch of Douglas; N.D., not done.

MGIT system; GTMD test, GenoType Mycobacteria Direct test; EAs, endometrial aspirates; PF/PW, peritoneal fluid/washing; CBs, cornual biopsies.

NTM lymphadenitis appears to be an emerging disease in children. A real-time PCR has been developed for the rapid diagnosis of this disease on the basis of internal transcribed spacer sequence (between the 16S rRNA and the 23S rRNA genes), hence enabling the identification of the genus Mycobacterium and the species M. avium and M. tuberculosis (Bruijnesteijn Van Coppenraet et al., 2004). The promising results of their assay for the detection of atypical mycobacteria could provide good support for clinical decision-making in children with lymphadenitis.

Pleural tuberculosis

Pleural TB accounts for 3–25% of patients with TB (Light, 2010), and TB pleurisy is the most common aetiology of pleural effusion (Liu et al., 2007; Light, 2010). The conventional diagnosis of pleural TB by identifying tubercle bacilli in pleural fluid and pleural biopsy specimens or by demonstrating granulomas in pleural tissue lack sensitivity and are time-consuming (Chang, 2007). The low yield of microscopy/culture and the invasiveness of pleural biopsy have generated renewed interests in alternative noninvasive diagnostics (Light, 2010). Detection of adenosine deaminase (ADA) and interferon-γ (IFN-γ) in pleural fluid are the useful diagnostic modalities for pleural TB as their levels are elevated in pleural effusion (Villegas et al., 2000; Kalantri et al., 2011). Sharma & Banga (2005) demonstrated the utility of these assays in TB pleural effusion with > 91% sensitivity. Owing to the high cost of IFN-γ assay, ADA assay is preferred over IFN-γ assay in resource-poor countries but ADA assay has been shown to be positive in other diseases such as adenocarcinomas, lymphomas and collagen vascular diseases (Lima et al., 2003; Laniado-Laborin, 2005).

The utility of PCR for the diagnosis of TB pleural effusion has been extensively evaluated using gene targets such as IS6110, GCRS, MPB-64 and devR with varying sensitivities and specificities (Martins et al., 2000; Chakravorty et al., 2005; Haldar et al., 2011; Table 1). Chakravorty et al. (2005) combined the individual results of devR PCR and IS6110 PCR tests together and reported high sensitivity in pleural fluid as well as needle-biopsied pleural tissue using USP method. A new domain of repetitive sequence, that is, CD192, has been identified within a PPE gene of M. tuberculosis genome and its utility has been exploited by PCR to efficiently diagnose both pleural TB and TB meningitis (Srivastava et al., 2006). The presence of CD192 sequences were confirmed in more than 300 clinical isolates of M. tuberculosis including those lacking IS6110 sequences. To further enhance the sensitivity, several researchers have focused on multiplex PCR or real-time PCR assays. Multiplex PCR targeting IS6110, dnaJ and 65 kDa protein genes has been documented for the detection of M. tuberculosis in pleural fluid, CSF as well as peritoneal fluid (Bandyopadhyay et al., 2008). The combination of monoplex/multiplex PCR results with ADA estimation or with histopathologic findings of pleural biopsies could further enhance the sensitivity (Lima et al., 2003; Liu et al., 2007; Bandyopadhyay et al., 2008). A real-time PCR targeting 65 kDa protein gene has been developed for the diagnosis of pleural TB in formalin-fixed paraffin-embedded pleural tissue, and the sensitivity of their assay was comparable with nested PCR targeting IS6110 (Baba et al., 2008). However, Rosso et al. (2011) recently achieved low sensitivity with real-time PCR in patients with pleural TB, although their results were superior to AFB smear and culture.

Based on positivity of either PCR or ADA/IFN-γ results, Villegas et al. (2000) earlier reported good sensitivity and specificity for the rapid diagnosis of pleural TB. Similarly, based on positivity of either real-time PCR or IFN-γ results, Kalantri et al. (2011) recently claimed high sensitivities (96–100%) in the diagnosis of pleural TB.

Tuberculous meningitis

TB meningitis is the most devastating form of meningitis and occurs in 7–12% of TB patients in developing countries (Kulkarni et al., 2005). The fatality rate for untreated TB meningitis is almost 100% and delay in treatment often leads to permanent neurological damage (Takahashi et al., 2008; Sharma et al., 2010a). Hence, the prompt diagnosis of TB meningitis is crucial for an efficient clinical management. The conventional microbiological tests to diagnose TB meningitis almost fail, and therefore, the detection of M. tuberculosis in CSF by PCR has been widely employed using IS6110, 65 kDa, 38 kDa, devR, MPB-64 or PPE gene target with varying sensitivities (Martins et al., 2000; Kulkarni et al., 2005; Quan et al., 2006; Srivastava et al., 2006; Rafi et al., 2007; Dora et al., 2008; Takahashi et al., 2008; Haldar et al., 2009; Table 1). PCR also shows better sensitivity than computed tomography (CT) scan as PCR detects M. tuberculosis DNA in CSF, while CT scan detects only a pathological lesion (Desai et al., 2006). Rafi et al. (2007) compared the relative efficacy of three PCR assays in the same CSF sample, that is, IS6110 PCR and nested PCR based on MPB-64 and 65 kDa protein gene targets. Their study demonstrated that the IS6110 PCR, a single-step assay, had the advantage of being a rapid test for the diagnosis of TB meningitis with better sensitivity and specificity as compared to the nested protocols. Recently, Sharma et al. (2011a) have combined the three gene targets (IS6110, MPB-64 and 38 kDa protein genes) together to devise an efficient multiplex PCR, and their assay showed better results than the monoplex PCR using a single gene target.

The volume of CSF sample is very important to achieve good PCR results, and the difficulty in collecting an adequate volume of CSF sample makes diagnosis of TB meningitis a daunting challenge in the paediatric subjects (Kulkarni et al., 2005; Galimi, 2011). Kulkarni et al. (2005) documented a sensitive PCR test targeting 38 kDa protein gene using small volume of whole CSF for the diagnosis of TB meningitis in children. Their test could detect 10 femtogram (fg) of DNA and that is equivalent to 2–3 tubercle bacilli. Rafi et al. (2007) used ‘whole’ CSF instead of using the ‘sediment’ for their PCR assay, thus proving that the M. tuberculosis DNA could be present as free DNA molecules in CSF samples. The utility of CSF ‘filtrate’ for detecting M. tuberculosis DNA by conventional PCR targeting IS6110 and devR genes as well as by real-time PCR targeting devR has been demonstrated by Haldar et al. (2009). Interestingly, it was found that CSF ‘filtrate’ exhibited better sensitivity and specificity than the ‘sediment’ by both assays.

Takahashi & Nakayama (2006) designed a quantitative nested real-time PCR (QNRT-PCR) assay targeting MPB-64 protein gene to detect M. tuberculosis DNA in CSF samples, and their method was extremely useful for assessing the clinical course of patients with TB meningitis on ATT (Takahashi et al., 2008). To detect M. tuberculosis DNA in CSF samples with a wide detection range (1–10⁵ copy numbers) during the clinical course of disease, a novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay targeting MPB-64 protein gene has been meticulously developed (Takahashi et al., 2008).

Osteoarticular tuberculosis

Osteoarticular TB accounts for about 1–3% of all TB cases and is the major cause of osteomyelitis (Yun et al., 2005; Sun et al., 2011). Any bone, joint or bursa can be infected but the spine, hip and knee are the preferred sites of infection, representing 70–80% of the infections (Pandey et al., 2009). TB of the spine which if not diagnosed properly and treated adequately may develop kyphosis and/or neurological complication (paraplegia; Jain et al., 2008). The accurate diagnosis of osteoarticular TB poses difficulty owing to deep inaccessible lesions and initiation of empirical ATT in majority of the cases (Vardhan & Yanamandra, 2011). Mostly, the diagnosis of osteoarticular TB is based on clinical suspicion and imaging findings, particularly in the endemic regions (Agashe et al., 2009; Sun et al., 2011). PCR tests based on IS6110, 16S rRNA gene and 65 kDa protein gene targets have been widely employed to confirm osteoarticular TB with varying sensitivities (Verettas et al., 2003; Negi et al., 2005b; Jain et al., 2008; Agashe et al., 2009; Sun et al., 2011; Table 1). Using histopathology as the gold standard, application of PCR on formalin-fixed, paraffin-embedded tissue has been documented in the recognition of TB osteomyelitis (Jambhekar et al., 2006), while Chawla et al. (2009) have suggested preserving those tissue samples in normal saline and not in the formalin as the latter is known to cause alterations in DNA for PCR assays. The combined use of nested PCR targeting IS6110 and mycobacterial culture (both automated and conventional) for the diagnosis of osteoarticular TB has also been documented (Agashe et al., 2009). Recently, Sharma et al. (2011b) introduced a highly sensitive and specific multiplex PCR targeting IS6110 and MPB-64 protein genes in the prospective evaluation of synovial fluid and pus samples from 80 cases of osteoarticular TB. The rpoB PCR-plasmid TA cloning-sequencing method to detect M. tuberculosis in the joint tissue, synovial fluid and pus samples from osteoarticular TB has been developed by Yun et al. (2005) and their method could simultaneously determine rifampin (RIF) susceptibility of tubercle bacilli. Fujimoto et al. (2010) also confirmed a case of TB pleuritis with knee-joint involvement by PCR analysis of the synovial fluid.

Interestingly, Colmenero et al. (2010) developed a reliable and sensitive multiplex real-time PCR based on conserved region of the gene coding for an immunogenic membrane protein of 31 kDa of Brucella abortus (BCSP31) and SenX3-RegX3 (intergenic region of M. tuberculosis) gene for the rapid differential diagnosis of TB vertebral osteomyelitis and brucellar vertebral osteomyelitis.

Genitourinary tuberculosis

Genitourinary TB comprising of genital and renal TB is the second most common EPTB and contributes up to 46% cases of EPTB (Jacob et al., 2008). Renal TB occurs up to 20 times more frequently in kidney transplant recipients than in the general population (Wise, 2009). The early diagnosis of renal TB is very important in preventing progressive destruction of the kidney (Wise, 2009). Recently, Sun et al. (2010) described an early and rapid diagnosis of renal TB from renal biopsy specimens by real-time PCR using 35- and 40-cycle threshold (C_T) cut-off values. It was found that the real-time PCR (C_T 40) showed better sensitivity than the real-time PCR (C_T 35).

Genital TB has been involved in the infertility of both men and women, and majority of such cases remain undiagnosed owing to asymptomatic presentation of the disease (Rana et al., 2011). Hence, a high index of suspicion is necessary for the diagnosis of genitourinary TB. To confirm genitourinary TB (both in men and women) in urine samples, PCR targeting MPT-64 protein gene has earlier been demonstrated to be the most sensitive indicator as compared to intravenous urography, bladder biopsy or urine culture (Hemal et al., 2000). The utility of PCR targeting IS6110 or 16S rRNA gene has also been evaluated in urine samples for the diagnosis of genitourinary TB (Moussa et al., 2000; Abbara & Davidson, 2011). High sensitivity up to 100% has been claimed by nested PCR based on MTP-40 protein gene of M. tuberculosis (Garcia-Elorriaga et al., 2009). To determine the tubercular aetiology of female infertility, Bhanu et al. (2005) demonstrated the diagnostic competence of PCR targeting MPT-64 protein gene using multiple samples, namely endometrial aspirates, endometrial biopsies as well as fluids from the pouch of Douglas and also correlated their PCR results with the laparoscopic findings. An mRNA-based RT-PCR assay targeting Antigen 85B protein gene using endometrial aspirate samples as well as DNA-PCR assay targeting MPT-64 protein gene using multiple sampling in 200 subjects has been developed by Rana et al. (2011) to diagnose active female genital TB causing infertility. It was found that DNA-PCR showed much better sensitivity than the RT-PCR and the multiple samples for DNA-PCR included endometrial aspirates, peritoneal fluids/washings and cornual biopsy specimens. Recently, Thangappah et al. (2011) demonstrated better sensitivity with TRC4-based PCR than the IS6110 based PCR with high specificity (91–100%) for the diagnosis of clinically suspected cases of female genitourinary TB in urine samples. Besides diagnosing genitourinary TB as well as the other clinical EPTB forms, the utility of PCR to detect mycobacterial transrenal DNA from urine samples for an early diagnosis of PTB has also been exploited (Torrea et al., 2005; Green et al., 2009).

Abdominal tuberculosis

Abdominal TB contributes up to 10–12% of EPTB cases, and much increase in this disease is because of HIV pandemic (Cabandugama et al., 2011). Abdominal TB comprises TB of gastrointestinal tract, peritoneum, mesentery and other intra-abdominal organs such as liver, spleen and pancreas (Sharma & Mohan, 2004). The use of PCR for the diagnosis of abdominal TB has been exploited as there is a diagnostic dilemma in histopathology, and PCR can further help in ruling out the malignancy in fresh laparoscopic abdominal biopsies (Kulkarni et al., 2011). Taking histopathology as the gold standard, Kulkarni et al. (2006) claimed good sensitivity and specificity by PCR using 38 kDa protein gene to diagnose abdominal TB and their PCR test has also been translated into an Indian commercial kit (Kulkarni et al., 2011). The diagnosis of intestinal TB is challenging owing to its close resemblance to Crohn's disease in clinical and histopathological features (Gan et al., 2002; Pulimood et al., 2008). The ability to distinguish these two diseases is a significant need in TB endemic countries where an increasing incidence of Crohn's disease is set against a background of high prevalence of intestinal TB (Almadi et al., 2009). Gan et al. (2002) recommended that PCR is a valuable test in the differentiation of intestinal TB and Crohn's disease and biopsy is of limited diagnostic value in the differentiation of two diseases. Two commercial PCR kits, that is, kit <A> (targeting MPB-64 and IS6110) and kit <B> (targeting IS6110), widely used in Korea, have been compared with an in-house PCR (targeting IS6110) from endoscopic biopsy specimens (Jin et al., 2010) for differential diagnosis of these two diseases. Among these tests, PCR with kit <B> coupled with histopathologic examination showed promising results in differentiating intestinal TB and Crohn's disease. Recently, faecal-TB PCR test targeting IS6110 has also been documented by Balamurugan et al. (2010) in differentiating these two diseases. However, clinical utility of this PCR test is not validated in large number of patients. One major drawback of conventional PCR is that it requires tissue destruction and nucleic acid extraction making impossible correlation with histological characteristics (Almadi et al., 2009). An in situ PCR has been developed where IS6110 target was amplified within the intact cells and that combined the ability to localize specific DNA within tissues (Pulimood et al., 2008). This method could also differentiate intestinal TB from Crohn's disease in archival mucosal biopsy specimens. However, the sensitivity of in situ PCR needs to be improved and studies should be carried out on large number of patients with Crohn's disease and intestinal TB before its usefulness is confirmed (Pulimood et al., 2008; Almadi et al., 2009).

Cutaneous tuberculosis

Cutaneous TB constitutes about 1.5% of all EPTB cases (Singal & Sonthalia, 2010). However, this disease has re-emerged during the last two decades together with high incidence of PTB and multiple-drug resistant TB (MDR-TB; Abdalla et al., 2009). Differentiation of cutaneous TB from other infectious granulomas of the skin (sarcoidosis, leprosy, fungal or NTM infections) is difficult because of insufficient AFB in the tissue biopsies (Bravo & Gotuzzo, 2007). Of all the clinical types, scrofuloderma is the most commonly encountered variant followed by lupus vulgaris, TB verrucosa cutis and lichen scrofulosorum (Singal & Sonthalia, 2010). These clinical types of cutaneous TB have been confirmed by PCR, while smear microscopy and culture test completely failed (Padmavathy et al., 2003). Interestingly, Okazaki et al. (2005) reported first case of M. bovis BCG-derived cutaneous TB (localized at different area from the vaccination site) without immune deficiency by multiplex PCR assay based on region of difference (RD)1, complement sequence of RD1, RD2, RD8, RD14 and SenX3-RegX3 regions originating from M. bovis BCG Tokyo 172. TB cutis orificialis, a rare manifestation of cutaneous TB (caused by auto-inoculation of M. tuberculosis in patients with advanced internal TB), has been confirmed by PCR (Choi et al., 2009). Using culture/histopathology as the gold standard, IS6110-based conventional PCR/nested PCR has been well documented in diagnosing cutaneous TB and that showed superiority over 16S rRNA gene-based PCR (Ogusku et al., 2003; Obieta et al., 2010). A highly sensitive and specific PCR assay targeting 65 kDa protein gene has also been developed for the diagnosis of cutaneous TB, considering culture/response to ATT as the gold standard (Negi et al., 2005a; Abdalla et al., 2009).

Ocular tuberculosis

Ocular TB represents a rare form of EPTB, which accounts for 0.14–16% of TB cases and arises either from local destruction and inflammation following haematogenous dissemination or as a hypersensitivity reaction following distant TB infection (Alvarez et al., 2009; Cutrufello et al., 2010). PCR has been demonstrated as an extremely useful technique for an early diagnosis of intraocular TB since it can be performed with very small sample sizes obtained from eyes and the clinical improvement with ATT has been observed in most of the patients with positive PCR (Cheng et al., 2004; Gupta et al., 2007). A nested PCR targeting MPB-64 protein gene was earlier demonstrated in formalin-fixed paraffin-embedded tissue of epiretinal membrane (Madhavan et al., 2000). This assay could detect 0.25 fg of DNA, and the quantity is sensitive enough to detect a single bacillus in epiretinal membrane from Eales'disease, however, lesser sensitivity was observed with the same nested PCR assay in vitreous samples (Madhavan et al., 2002; Table 1). Recently, the utility of real-time PCR based on IS6110 or MPT-64 protein gene target has been explored in the diagnosis of ocular TB with promising results (Sharma et al., 2011c; Wroblewski et al., 2011). In addition, M. tuberculosis could be detected in corneas from donors using PCR assay, and such findings may be used to re-evaluate criteria for suitability of donors with active TB, and further studies should be carried out to investigate whether recipients with PCR-positive corneas would eventually lead to disease transmission (Catedral et al., 2010).

Pericardial tuberculosis

Pericardial TB is the most common cause of pericarditis in African and Asian countries (Cherian, 2004). It arises secondary to contiguous spread from mediastinal nodes, lungs or during miliary dissemination (Golden & Vikram, 2005). The elevated levels of ADA and IFN-γ have been documented in pericardial TB (Burgess et al., 2002), but these assays have limitations as detailed earlier in pleural TB. The utility of conventional PCR as well as nested PCR has been described for the diagnosis of acute pleuropericardial TB and chronic constrictive pericarditis (Tzoanopoulos et al., 2001; Zamirian et al., 2007).

Thyroid tuberculosis

The clinical diagnosis of thyroid TB is rarely investigated unless there is multinodular goitre, abscess or chronic sinus in the gland (Bulbuloglu et al., 2006). The diagnosis of primary thyroid TB is mostly dependent on chest X-ray and ultrasonography; however, these methods usually fail (Ghosh et al., 2007). Multiplex PCR targeting IS6110, 65 kDa and dnaJ genes has been established to confirm thyroid TB (Ghosh et al., 2007).

Tuberculous mastitis

TB mastitis or breast TB is a rare presentation of EPTB even in endemic countries. The most common clinical presentation of breast TB is usually a solitary, ill-defined, unilateral hard lump situated in the central or upper outer quadrant of the breasts (Baharoon, 2008). Mycobacterium tuberculosis bacilli can reach breasts through lymphatic, haematogenous or contiguous seeding (Sharma & Mohan, 2004). The utility of PCR in the diagnosis of breast TB has been described (Kao et al., 2010).

Disseminated tuberculosis

Disseminated or miliary TB refers to any progressive and potentially lethal form of TB resulting from widespread haematogenous dissemination of M. tuberculosis bacilli throughout the body (Sharma et al., 2005; Galimi, 2011). Disseminated TB has been observed in 10% of patients who have AIDS + PTB and in 38% of those who have AIDS + EPTB (Golden & Vikram, 2005). The clinical diagnosis of disseminated TB is challenging as it may be confused with other diseases and chest symptoms remain obscure (Escobedo-Jaimes et al., 2003). Isolation of M. tuberculosis from sputum, body fluids or biopsy specimens by PCR is useful for the diagnosis of disseminated TB (Sharma et al., 2005). The utility of PCR targeting MPB-64 protein gene from bone marrow aspirates has been explored for the diagnosis of disseminated TB with 33% positivity, and the clinical improvement with ATT has also been observed in 85% of the patients with positive PCR test (Singh et al., 2006). However, Rebollo et al. (2006) demonstrated 50% PCR positivity targeting IS6110 in urine and/or blood samples of patients with disseminated TB and 36% PCR positivity in other clinical forms of EPTB. The detection of M. tuberculosis in blood and urine samples by PCR is a useful method for the diagnosis of several EPTB forms especially in those patients in which sample extraction is difficult or requires aggressive techniques (e.g. tissue biopsies).

Various researchers have evaluated the performance of PCR in diagnosing together different clinical EPTB forms. Oh et al. (2001) earlier documented a combination of Mycobacteria Growth Indicator Tube (MGIT) method and Cobas Amplicor System in conjunction with duplex PCR (multiplex PCR) targeting 16S rRNA gene and IS6110 for both rapid detection and differentiation of M. tuberculosis and NTM, using ‘extended gold standard’ comprising of gold standard (culture and clinical data) and ‘true DNA positive samples’ originated from EPTB patients with successful ATT. In sub-Saharan African countries like Burkina Faso with high HIV seroprevalence rate, Torrea et al. (2005) developed nested PCR targeting IS6110 for the detection of several EPTB forms in a prospective analysis of urine samples from HIV-infected and noninfected individuals. Differences in PCR sensitivities were observed in the two populations infected and not-infected by HIV. While diagnosing several EPTB forms, two different nested PCR techniques, that is, in-house classic PCR and LightCycler technology targeting IS6110, have been compared (Ritis et al., 2005). It was found that the LightCycler protocol was superior to the in-house system in bone marrow aspirates; however, both methods demonstrated the same reliability when performed in infected tissue samples. A highly sensitive and specific culture-enhanced PCR test has been devised by Noussair et al. (2009) which comprises of a preliminary step of broth culture in BacT/Alert MP bottles with the subsequent detection of M. tuberculosis using GenoType Mycobacteria Direct (GTMD) assay targeting 23S rRNA in several EPTB specimens (tissue biopsies, pleural fluid, CSF, urine, etc.), considering combination of BACTEC culture, histological findings and response to ATT, all together as the gold/reference standard. Various PCR tests employed for the diagnosis of EPTB using different gene targets have been summarized in Table 1. TNF-α inhibitor (e.g. inflixmab and etanercept)-induced EPTB has been established in patients with rheumatoid arthritis and Crohn's disease (Golden & Vikram, 2005; Almadi et al., 2009).

The most notable advantage of PCR tests is their rapid turnaround time and reliability for an early detection of EPTB, which may have important implications for clinical management and TB control; for example, the reliability of PCR to confirm an early diagnosis of TB meningitis and abdominal TB has been well established when smear and culture test are rarely positive (Kulkarni et al., 2011; Galimi et al., 2011). PCR has also been used for an early diagnosis of osteoarticular TB in tissue samples and that can help to start timely ATT (Pandey et al., 2009) and prevent progression to irreversible changes. Cheng et al. (2004) have recommended an early initiation of ATT at least in > 50% cases of their cohort study of 86 patients with EPTB diagnosed by PCR so as to avoid unnecessary mortality and transmission of disease. Similarly, Noussair et al. (2009) have proposed that the PCR results could be used in conjunction with histological findings for the diagnosis of suspected EPTB cases to decide whether presumptive ATT should be continued or discontinued, thereby contributing to decreased costs and decreased potential toxicity related to prolonged unnecessary therapy.

Rifampin resistance and GeneXpert assay

There is a major problem of drug resistance in EPTB individuals and particularly in those individuals co-infected with HIV. MDR-TB and XDR-TB (extensively-drug resistant TB) are two crucial forms of drug resistance (Agashe et al., 2009). The conventional drug susceptibility test takes at least 2 months from the time when the culture is inoculated. RIF resistance is used as a surrogate marker for uncovering MDR as > 90% RIF-resistant isolates are also isoniazid (INH) resistant (Brodie & Schluger, 2009). Eltringham et al. (1999) earlier demonstrated two rapid phenotypic assays for the detection of RIF resistance in M. tuberculosis, that is, the phage-amplified biological assay based on inability of susceptible M. tuberculosis strains to support the replication of bacteriophage D29 in the presence of inhibitory doses of RIF and the RT-PCR assay to demonstrate a reduction in inducible dnaK (Rv0350) mRNA levels in susceptible isolates treated with RIF. The rapid detection of RIF resistance in M. tuberculosis has been meticulously reviewed by Brodie & Schluger (2009) using line probe assays and molecular beacon real-time PCR.

The recently developed Xpert test based on nested real-time PCR and molecular beacon technology targeting rpoB gene of wild-type M. tuberculosis strains has been demonstrated as a rapid test with results for both TB identification and RIF resistance in < 2 h in a single tube (Hillemann et al., 2011; Tortoli et al., 2012). The Xpert test endorsed by WHO for the detection of PTB has been evaluated recently to test its utility in 547 EPTB specimens (Vadwai et al., 2011). The sensitivity and specificity of their Xpert test for TB identification was 81% and 99.6%, respectively, in comparison with a composite reference standard (CRS) made up of smear, culture, clinical findings, ATT follow-up, etc. In addition, their assay correctly identified 98% of phenotypic RIF-resistant cases and 94% of phenotypic RIF-susceptible cases (Vadwai et al., 2011). Considering culture as the gold standard, similar encouraging results have been observed by Hillemann et al. (2011) for TB identification in 512 EPTB specimens. The performance of Xpert assay has also been compared with Cobas TaqMan MTB assay and IS6110 based real-time PCR assay for TB identification in EPTB specimens, and it was found that the Xpert assay exhibited better sensitivity than the other two assays (Causse et al., 2011; Miller et al., 2011). Recently, Tortoli et al. (2012) evaluated the utility of Xpert assay in 1476 EPTB specimens and reported 81.3% sensitivity and 99.8% specificity, considering culture and clinical diagnosis as the gold standard. The high cost of this sophisticated test for the diagnosis of EPTB may be offset in developing countries by the rapid turnaround time similar to that of smear microscopy (< 2 h) with less biohazard risks and minimal training to the technicians (Vadwai et al., 2011; Tortoli et al., 2012).

Immuno-PCR

Immuno-PCR (PCR Amplified Immunoassay; I-PCR) is a novel ultrasensitive assay for detecting protein antigens combining the versatility of ELISA with the sensitivity of NAA by PCR, which leads to at least 10³–10⁴ increase in sensitivity over an analogous ELISA (Malou & Raoult, 2011). PCR tests are restricted to the detection of nucleic acid molecules only. However, most natural processes including EPTB infections involve abundant proteins and other non-nucleic acid molecules in circulation so that the analysis of nucleic acids may be inadequate to fully exploit the biological samples. I-PCR has been used for the detection of proto-oncogenes, cytokines as well as potential viral and bacterial antigens including mycobacterial antigens (Malou & Raoult, 2011; Mehta et al., 2012).

Recently, we developed an ultrasensitive I-PCR assay to detect M. tuberculosis-specific RD1 and RD2 antigens [ESAT-6 (Rv3875), CFP-10 (Rv3874), CFP-21 (Rv1984c) and MPT-64 (Rv1980c)] and antibodies to these antigens in biological specimens of both PTB and EPTB patients (Mehta et al., 2012). With this I-PCR assay, we could detect up to 0.1 fg of RD antigens, which was 10⁷ more sensitive than that detected with an analogous ELISA. The detection of cocktail of RD1 and RD2 antigens in patients with EPTB showed better sensitivity with our I-PCR assay as compared to an analogous ELISA (Mehta et al., 2012).

Merits

PCR tests offer alternative robust approach to detect M. tuberculosis in paucibacillary EPTB specimens that show rapid results with good diagnostic accuracy. Although these tests cannot replace the conventional AFB smear, culture identification or histopathological observations but they contribute significantly for an early diagnosis of EPTB and exert an acceptable impact on the clinical management of disease.

Demerits

Compared to pulmonary specimens, lesser sensitivity of PCR assays observed in some EPTB specimens might result from the use of very small sample volumes available and an irregular dispersion of bacteria in those specimens. PCR assays with EPTB specimens are often associated with false-positive and false-negative results. PCR detects both viable and nonviable M. tuberculosis and could not differentiate between active and latent TB. Furthermore, PCR tests cannot detect non-nucleic acid molecules.

Conclusion

This review has described the utility of PCR for an early diagnosis of EPTB. There is high variation in PCR results owing to different gene targets as well as different gold standards adopted in various laboratories. IS6110 has been shown to be the most widely used gene target followed by 16S rRNA gene or genes encoding MPB-64, 38 kDa and 65 kDa proteins. However, IS6110 has zero or low copy numbers in some M. tuberculosis strains, and the combination of two or more gene targets has been employed in multiplex PCR, for example, IS6110 + MPB-64 or IS6110 + 38 kDa + MPB-64, as an adjunct to the routine battery of laboratory tests for the diagnosis of different clinical types of EPTB. In many suspected EPTB cases, when conventional microbiological tests almost fail, PCR results along with the clinical presentation and/or histopathology may be adequate to initiate ATT. The major drawback of PCR tests is that they do not differentiate between viable and nonviable M. tuberculosis. The mRNA-based RT-PCR can detect viable M. tuberculosis bacilli and is useful for the diagnosis of active disease; however, the sensitivity of the assay is low and it is cumbersome to work with RNA in routine use. Further work is required to devise a simple and cost-effective PCR test for an efficient diagnosis of EPTB that can be used routinely in resource-poor countries.

Acknowledgements

The financial assistance provided (to P.K.M.) by University Grant Commission, New Delhi, is acknowledged. We thank Mahesh Kulharia for critically reading the manuscript.