Diagnostic Performance of Commercially Available Enzyme-Linked Immunosorbent Assay Kit in the Diagnosis of Extrapulmonary Tuberculosis

INTRODUCTION

The World Health Organization (WHO) declared tuberculosis (TB) a global public health emergency in 1993. TB causes ill-health among millions of people each year and ranks as the second leading infectious disease causing death worldwide. Human immunodeficiency virus (HIV) coinfection and the development of multidrug resistant (MDR) and extensively drug resistant (XDR) strains cause a major hurdle in treatment and containment of TB.^[1] The latest estimates of 2011 reports that there are almost 9 million new cases and 1.4 million TB deaths of which 990,000 are among HIV negative people and 430,000 HIV-associated TB deaths.^[2]

TB affects the lung which results in symptoms of respiratory system known as pulmonary TB. However, other organs, such as the pleura, lymph node, kidney, and meninges, may be involved in TB under certain circumstances and this is known as extrapulmonary TB (EPTB). Patients with EPTB present with organ-related symptoms which would develop into serious complications that threaten patients' lives and cause morbidity. Reports of recent years infer that the prevalence of EPTB is getting worse, which has drawn more public attention. In 2011, 19% of new EPTB cases were notified in India.^[2] One critical and early aspect of managing the TB epidemic is early diagnosis, so that the appropriate treatment can be started at the appropriate time. Diagnosis of EPTB is challenging. Routine methods for TB diagnosis such as smear for acid-fast bacilli (AFB) and culture of Mycobacterium tuberculosis on solid media, and X-rays are poorly sensitive worldwide, nucleic acid amplification test, and liquid culture methods, such as BACTEC MGIT 960 are costly and required sophisticated infrastructure.

Due to these diagnostic limitations antibody based immunodiagnostic tests have been widely used which do not require a specimen of the affected organ for microbiological or histological examinations. There are numerous M. tuberculosis antigens, including native, semisynthetic, and recombinant ones that have been used in these diagnostic kits. Some of the antigens such as lipoarabinomannan (LAM) were commonly used and evaluated in areas with low and high burden of TB. These antibody detection methods have evolved into various formats, includes microtiter well enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assays. The latter being the most widely adopted, because these do not require sophisticated instrumentation. Based on these serological methods many studies are carried out on pulmonary TB patients, while only few studies are done on EPTB patients. A meta-analysis conducted in 2007 ^[3] on nine published articles (25 studies) provide glimpse of variability in sensitivity (0-100%) and specificity (59-100%) of these tests. However, only one study was included from India ^[4] in this meta-analysis.

In 2011, World Health Organization (WHO) ^[5] issued strong policy recommendations against the use these antibody-based commercialized serological tests. An expert group ^[3,6] that reviewed the evidence on use of commercial, antibody-based serodiagnostic tests found that these tests provide inconsistent and imprecise results with highly variable values for sensitivity and specificity. No evidence was found that existing commercial serological assays improve outcomes that are important to patients.

We evaluated Pathozyme Myco ^® immunoglobulin (Ig) G, IgA, and IgM (Omega Diagnostic Limited, Scotland, UK) on pulmonary TB patients from Delhi, India ^[7] and the findings were also highly discouraging. Simultaneously we also evaluated Pathozyme Myco ^® IgG, IgA, and IgM in EPTB patients and the results are presented here.

MATERIALS AND METHODS

RESULTS

Performance of PATHOZYME ^® MYCO IgG, IgA, and IgM

ELISAs were performed in all 354 subjects. The performance of all the three ELISAs were analyzed [Table 1]. The individual and overall sensitivity rates of IgM, IgA, and IgG were 29, 24.4, and 34.5%, respectively; while their specificities were 70.8, 77.3, and 68.6%, respectively. When we analyzed these in combination of two or more ELISAs, the sensitivity further decreased and ranged from 7.8 to 11.9%; but their specificities improved significantly and ranged from 83.9 to 96.3% [Table 1]. The PPV for IgM, IgA, and IgG were 61.7, 63.1, and 63.5%, respectively; whereas their NPV were 38.6, 39.2, and 39.8%, respectively. The LRP test for IgM, IgA, and IgG were 0.9, 1, and 1.1; respectively. The mean optical density (OD) values of IgM, IgA, and IgG for confirmed EPTB patients were 2.1 ± 0.7, 1.8 ± 0.8, and 1.6 ± 0.4 OD; respectively and for without TB subjects were 1.78 ± 0.6, 1.8 ± 0.9, and 1.6 ± 0.5 OD; respectively [Figure 1].

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Figure 1:

Optical density values of IgM, IgA, and IgG positive among extrapulmonary tuberculosis (EPTB) patients without EPTB subjects

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Table 1: Performance of Pathozyme Myco® IgM, IgA, and IgG on extrapulmonary tuberculosis cases and non-EPTB subjects

ELISA (n=354)	Type		Sensitivity % (95% CI)	Specificity % (95% CI)	PPV % (95% CI)	NPV % (95% CI)	LPR (95% CI)
	EPTB (n=217)	Without EPTB (n=137)
IgM
Pos	63 (29)	40 (29)	29	70.8	61.7	38.6	0.9
Neg	154 (71)	97 (71)	(23.4-35.4)	(62.7-77.7)	(51.1-70)	(32.8-44.8)	(0.8-1.2)
IgA
Pos	53 (24.4)	31 (22.7)	24.4	77.3	63.1	39.2	1.0
Neg	164 (75.6)	106 (77.3)	(19.1-30.5)	(68.6-83.5)	(54.4-72.6)	(33.6-45.2)	(0.9-1.2)
IgG
Pos	75 (34.6)	43 (32)	34.5	68.6	63.56	39.8	1.1
Neg	142 (65.4)	94 (68.6)	(28.5-41.1)	(60.4-75.7)	(54.5-71.6)	(33.8-46.1)	(1.0-1.2)
IgM, IgA
Pos	26 (12)	12 (8.7)	11.9	91.2	68.4	39.5	1.3
Neg	191 (88)	125 (91.2)	(8.3-16.9)	(85.3-94.9)	(52.2-80.9)	(34.3-45)	(0.6-2.8)
IgM, IgG
Pos	35 (16.1)	22 (16)	16.1	83.9	61.4	38.7	1
Neg	182 (83.8)	115 (84)	(11.8-21.6)	(76.8-89.1)	(48.4-72.9)	(33.3-44.3)	(0.6-1.4)
IgA, IgG
Pos	34 (15.7)	15 (11)	15.6	89	69.3	40	1.4
Neg	183 (84.3)	122 (89)	(11.4-21.1)	(82.7-93.2)	(55.4-80.4)	(34.6-45.5)	(0.9-2.2)
IgM, IgA and IgG
Pos	17 (7.8)	5 (3.6)	7.8	96.3	77.2	39.7	2.1
Neg	200 (92.2)	132 (96.3)	(4.9-12.1)	(91.7-98.4)	(56.5-89.8)	(34.6-45.1)	(0.3-12.3)
Any ELISA*
Pos	113 (52)	70 (51.1)	52	48.9	61.7	39.1	1
Neg	104 (48)	67 (48.9)	(45.4-58.6)	(40.6-57.1)	(54.5-68.4)	(32.1-46.6)	(0.9-1)

ELISA: Enzyme-linked immunosorbent assay, Pos: Positive, Neg: negative, CI: Confidence interval, PPV: Positive predictive value, NPV: Negative predictive value, LRP: Likelihood ratio for positive test, Ig: Immunoglobulin, EPTB: Extrapulmonary tuberculosis; *Any ELISA represents subjects detected positive by at least one of the three (IgM/IgA/IgG) ELISA tests

DISCUSSION

The diagnosis of active TB largely depends upon initial clinical suspicion and radiographic findings, with subsequent laboratory confirmation by bacteriologic studies. Because appropriate specimen might be difficult to obtain from extrapulmonary sites, and the number of bacilli is generally low, bacteriological confirmation of EPTB is often more difficult than pulmonary TB.^[11] Even if the appropriate sample (e.g., CSF in suspected meningitis) is obtained, the sensitivity of conventional bacteriological methods are dismally poor. Because of these limitations, number of investigators relied on serology as a diagnostic tool in all settings.

With the technical advances, sets of M. tuberculosis specific antigens were first identified using either hybridoma or recombinant DNA technologies.^[12] However, the findings that antibody levels are considerably higher and more frequent in the multibacillary than in paucibacillary forms of the disease ^[13] was noted as an obstacle to clinical application of these antigens in many studies, and thus combinations of multiple antigens were used.^[14]

Here, we report poor utility of ELISA test in which the sensitivity of Pathozyme ^® Myco IgM was 29%, IgA was 24.4%, and IgG was 34.5% in our patients of EPTB. These findings were by-and-large similar to our findings in pulmonary TB patients, where the sensitivity rates of the same kit were 48.7, 25.7, and 24.4%; for IgM, IgA, and IgG, respectively.^[7] However, the specificity of serology was better in EPTB than pulmonary TB [Table 1]. But, the sensitivity and specificity of these kits varied among various disease forms, for example, among bone/joint TB and lymph node TB cases IgG had the highest OR of 0.83 (95% CI: 0.11-6.01), and 3.69 (95% CI: 1.02-13.34); IgA had OR of 1.6 (95% CI: 0.18-14.13), 1.25 (95% CI: 0.24-6.25), and 2.55 (95% CI: 0.71-9.08) for disseminated, gastrointestinal and pleural TB respectively, while CNS TB had the maximum OR of 2.67 (95% CI: 0.69-11.06) for IgM.

Not many authentic studies are published, but reported sensitivity of ANDA IgG detection kit used for the diagnosis of EPTB was 64% (95% CI: 28-92) for lymph node TB and 46% (95% CI: 29-63) for pleural TB, and its specificity was reported to be 90% and 87%, respectively for these patients.^[3] The alternative serology method MycoDot™ is reported to have higher sensitivity of 80% and 66.7% in TB lymphadenitis and in pleural TB patients, respectively.^[15] However, all studies have reported highly variable results, mainly because of difference in patient selection, criteria of diagnosis and cut-offs used. In one study, sensitivity of microwell ELISA based Pathozyme ^® Myco IgM in EPTB has been reported as low as 9% ^[16] but in another study based on 38 kDa antigen immunochromatographic-TB test the sensitivity of 46% and specificity of 59.6% were reported in pleural TB patients.^[17] Senol et al., ^[18] also reported sensitivity of 22.2% and 25% in pleural TB and lymph node TB, respectively; and had similar specificity of 93.3% using Pathozyme-TB Complex Plus test.

This explains the fact that not all TB patients produce antibodies against all antigenic epitopes in the cell walls of the tubercle bacilli which infers on the inconsistency in specificity of antibody based assays among different patient groups like gender, age, ethnicity, and geographical distribution. It is also well known ^[19] that person-to-person variation in antigen recognition is the key feature of human humoral immunity against TB.^[20]

Despite the inconsistency and imprecise sensitivity and specificity of the commercial serological tests, these were being marketed liberally and used widely in major endemic regions including India until recently when WHO banned commercialization of these kits.^[5] As a result of this WHO guideline, in June 2012 the Government of India stopped the import, manufacture, distribution, and sale of commercial serodiagnostic tests for TB. This bold action is expected to greatly reduce the frequency of false diagnoses of TB and facilitate the introduction of WHO approved diagnostics into the market. Therefore, in conclusion even though the specificity of the Pathozyme Myco IgA, IgM, and IgG was significantly better and acceptable as compared to pulmonary TB, the sensitivity of these kits was dismally poor and their use cannot be recommended for diagnosis of EPTB.