Introduction

Generally the diagnosis of TB is made on the basis of clinical, radiological and bacteriological examinations. For the later case smear and culture examination for the presence of AFB in clinical samples is done. The limitation of  smear examination for AFB is that it may give false negative results and require a high degree of bacillary load of at least 50,000 bacilli/ ml and subject to inherent errors like contamination.¹ Another demerit is that it is positive in open cases only², thus it is not helpful in extra pulmonary form of TB, particularly in TBM/ childhood TB where sputum is not available. The limitations of bacterial culture are that the results are available only after 4 – 6 weeks and though reliable for pulmonary TB but not so for extra pulmonary form.³ X-ray is not suitable for field studies in developing countries like India and also the reports are non specific. Despite an enormous amount of research we still do not have simple, sensitive and  specific test for the diagnosis of TB in active form.⁴

The diagnosis of extra pulmonary TB (EPTB) is difficult, delayed and frequently made on circumstantial evidence alone and delay in diagnosis might have threatening consequences.

Attempts have been made to improve the sensitivity and the speed of detection of TB bacilli of components thereof by other methods such as radiometric determination of bacterial growth (BACTEC system), gas chromatography/ mass spectrometry and DNA hybridization. All of these have meet  with problems of either sensitivity or prohibitive cost. Thus, when the current criteria for diagnosis such as clinical and radiological signs and bacteriological and biochemical methods that aid in diagnosis are unreliable, there is a search for alternative test which can stand

with its merits in vigorous clinical trials. Serodiagnostic tests have attracted considerable attention of the investigators.

The history of serodiagnosis starts with Robert Koch’s unsuccessful attempt to diagnose the disease by direct aggulutination  of TB bacilli. Subsequently several workers tried several ways of  immunoassays like agglutination of RBC, complement fixation, preparation in fluid and gels and radio immunoassays, but hardly any one is accepted for routine clinical use.

ELISA is having the added advantage as it is sensitive, specific, reproducible and useful in field studies to detect mycobacterial antigens^5-9 or antibodies^10,11.

The choice of antigen is the major determinant for the success of any serological test. A wide variety of antigens have been applied ranging from crude antigens such as culture filtrate antigen, whole cell sonicate antigen, mycobacterium saline extract antigen to purified antigens such as antigen 5, glycolipid antigen, Lipo arabinomannan etc. Grange suggested that the study of antibody response to those antigens that are actively secreted by living mycobacteria rather than cytoplasmic antigens liberated by sonication or other mechanical means are more fruitful for TB diagnosis.¹² The most pronounced specific antibody, which correlated with active disease was IgG.^13,14

In our laboratory studies using different antigen preparations (whole protein to purified proteins) have been employed for exploring their potential in detecting tubercular IgG antibodies in tuberculosis.^15,16 On exploring use of ES –31 & ES- 41 antigens of M. tbH₃₇Ra in stick penicillinase ELISA for the detection of seroreactivity in                                                                pulmonary TB and EPTB, ES-31 showed good potential in detecting IgG antibody in TB Lymphnode, Tubercular meningitis and pulmonary TB¹⁷ while ES –41 in abdominal TB and Bone and Joint TB. Hence, the present study was undertaken to evaluate ES –31 and ES –41 antigen in immunodiagnosis of pulmonary and extra pulmonary tuberculosis.

Experimental

The study subjects comprised of  patients with tuberculosis, non- tuberculous disease control and healthy controls, who attended Kastruba Hospital, Sevagram during 1999 – 2001. Tuberculosis groups were showing group for pulmonary tuberculosis (having progressive tuberculosis proven bacteriologically), lymphnode tuberculosis (confirmed by histopathologically), Central nervous system (CNS) tuberculosis (confirmed by CSF studies or response to anti- tubercular therapy), Genitourinary tuberculosis (confirmed by histopathologically, radiology , CT and clinical study), Abdominal tuberculosis (confirmed by biopsy, radiology, ascitic fluid examination and clinical study) and Osteoarticular tuberculosis (confirmed by clinico- radiological measures).

Sera were collected before starting of antitubercular and antibiotic treatment. Approximately 3 cc of  blood was collected in sterile vials. Sera was separated and stored at

-20⁰C after addition of 0.1% sodium azide as preservative. Pure laboratory strain of Mycobacterium tuberculosis H₃₇Ra from Tuberculosis Research Centre, Chennai was used in this study. Lowentstein – Jensen (L-J) medium was used for seed culture and modified synthetic Sautan medium was used as subculture medium. Mycobacterium tuberculosis ES antigen was isolated and labelled as M.tb ES antigen and stored in aliquots at – 20⁰C with sodium azide (0.1%) until use. ES-31 & 41 antigen was isolated by affinity chromatography. Polyclonal antibodies to ES- 31 & 41 antigen were raised in goat and specific antibodies against ES –31 & 41 antigen isolated from immune sera using ES-31 & 41 antigen coupled CN Br- activated sepharose B column.¹⁸

Stick indirect penicillinase ELISA for tuberculous IgG antibody detection was carried out. Stick sandwich penicillinase ELISA  was carried out for detection of circulating TB antigen and CIC – antigen in sera samples with some modification. The concentration of ES-31 & 41 antigen was 1ng/stick while that of affinity purified goat anti ES – 31 & 41 antibody was 1 mg per stick. Optimum sera dilution in case of antibody detection was 1:600, while in case of antigen detection was 1:300. In the detection of CIC antigen sera samples were pre-treated with glycine  HCl buffer (0.1 M pH 2.8) followed by heating at 65⁰C for 15 minutes.¹⁹ Combined sensitivity were also tested.  The results obtained are depicted in Table 1-2.

Table 1: Comparative analysis of M. tb ES-31 and ES-41 antigen in the detection of tubercular,  IgG antibodies by indirect ELISA pulmonary and extra pulmonary TB.

Figure in parenthesis denotes percentage positivity

^*Sera showing positive reaction at 1: 600dilution.

 Table 2: Combine sensitivity of antibody, antigen or CIC –Ag screened with ES –31, ES-41 antigens and affinity purified anti ES – 31 antibody.

Figures in parenthesis indicate percentage positivity

* Screened with ES –41 antigen

# Sera showing positive reaction at 1: 600dilution

Results and Discussion

In a preliminary study, a total of 52 sera samples belonging to different tubercular groups viz. pulmonary TB, abdominal TB, lymphnode TB, CNS TB, osteoarticular TB, genitourinary TB along with disease controls and healthy controls were analysed for the detection of tubercular IgG antibody using ES-31 and ES- 41 antigens. Serological analysis of tuberculosis cases on ATT by penicillinase ELISA showed presence of antibody in 11 cases out of 22 cases; out of 11 cases which were negative for antibody, 10 each were  positive for antigen  and CIC antigen with a combined positively of 95%.

The study has shown that  M. tb ES –31 antigen is more sensitive in detecting tubercular IgG antibody in pulmonary TB cases (80%), in some systemic groups of extra pulmonary TB like, Lymphnode TB (80%), Central Nervous System (CNS) TB (80%) and genitourinary TB(60%) compared to M. tb ES –41 antigen which showed less sensitivity ( 40%, 40%, 20% respectively), whereas ES-41 antigen is more sensitive in some groups of extra pulmonary TB, like, abdominal TB(80%) and osteoarticular TB(80%) compared to ES-31 antigen which showed less sensitivity in these two forms of extra pulmonary TB (20% each). Percentage positivity in combined antibody / antigen / CIC – antigen sensitivity was found more as depicted in Table – 2.

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