Tubercle 67 (1986) 5560 0 Longman Group Ltd.

SERODIAGNOSIS OF INTESTINAL TUBERCULOSIS BY ENZYME IMMUNOASSAY AND SOLUBLE ANTIGEN FLUORESCENT ANTIBODY TESTS USING A

SALINE EXTRACTED ANTIGEN

T. C. Chawla, Anjana Sharma, Usha Kiran, D. K. Bhargava* Shriniwas, and B. N. Tandon

All India Institute of Medical Sciences, New Delhi - 110 029, India

Summary The efficacy of enzyme-linked immunosorbent assay (ELISA) and soluble antigen fluorescent antibody JSAFA) tests in the diagnosis of intestinal tuberculosis was compared using saline extracted surface antigen of M. tuberculosis H3zRa. Of the 24 proved cases of intestinal tuberculosis ELISA detected positivity in 92 % compared with 83 % detected by the SAFA test. The 2 tests showed a strong correlation with each other and yielded a clear cut difference between patients and controls, although there was some degree of overlap.

R&urn6 Utilisant un antigene de surface de M. tuberculosis H37Ra, on a compare I’efficacite du test ELISA et celle des tests aux anticorps a I’antigene fluorescent soluble dans le diagnostic de la tuberculose intestinale.

Sur les 24 cas prouves de tuberculose intestinale, le test ELISA a decele la maladie dans 92 % des cas, contre 83 % par le test SAFA. Les deux tests ont montre une forte correlation entre eux et ont donne des differences bien nettes entre malades en temoins, en depit d’un peu de chevauchement entre ces deux categories.

Resumen Se compare la eficacia de la prueba ELISA y la de las pruebas de 10s anticuerpos al antigen0 soluble fluorescente (SAFA) en el diagnostic0 de la tuberculosis intestinal, utilizando un antigen0 de superficie de M. tuberculosis H3zRa. En 24 cases comprobados de tuberculosis intestinal la prueba ELISA detect6 la enfermedad en 92 % de 10s cases, comparados con 83 % para la prueba SAFA. Las dos pruebas mostraron una correlation importante entre ellas y dieron diferencias netas entre enfermos y controles, aunque se observe cierto grado de superposition entre estas dos categorias.

Introduction The diagnosis of intestinal tuberculosis (ITB) is often difficult. Most commonly used methods are detection of tubercle bacilli and histopathological demonstration of typical granulomas in biopsy material, which can only be obtained by surgery or colonoscopy [I, 21. Since these methods are cumbersome and time consuming, reliable and simple serodiagnostic tests have long been sought.

*Address for correspondence: Dr D. K. Bhargava, Department of Gastroenterology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India.

56 Chawla and others

A bewildering array of serological tests have been employed using different mycobacte- rial antigens in attempts to detect and distinguish acute, subacute and past cases of tuberculosis 13-71. The soluble-antigen fluorescent-antibody (SAFA) test and the enzyme- linked immunosorbent assay (ELISA) have been introduced relatively recently and were found to be simple and sensitive methods for the immunodiagnosis of pulmonary tuberculosis [8-141. The ELISA test has not been evaluated in cases of intestinal tuberculosis (ITB) and there are scanty reports of its use in extra-pulmonary and disseminated tuberculosis j151. We intended, therefore, to evaluate the usefulness of this test in the serodiagnosis of intestinal tuberculosis by using saline extracted antigen. Our recent experience with saline extract, sonicate and PPD antigens has revealed that saline extract (SE) antigen yielded the most consistent results both with ELISA and SAFA tests [Ill.

Serum Materials and methods

A total of 24 samples of serum were obtained from clinically, bacteriologically and/or histopathologically proven cases of ITB and another 24 controls sera were taken for subjects with other gastrointestinal disorders like ulcerative colitis, eosinophilic entritis, jejunum carcinoma, carcinoma ileum, were taken. In addition, 11 sera from normal healthy individuals were included as controls.

All samples were stored at -20 “C until use.

Antigen

Saline extracted protein antigen was prepared by the method of Bennedsen et a/. [16]. In brief, M. tuberculosis (H3,Ra) strain was grown on Sauton’s liquid medium for 7 weeks, cells were killed by adding phenol to a concentration of 0.5 % and filtered. The cells were washed twice with phosphate-buffer saline (PBS), pH 7.3 (0.01 M phosphate; 0.15 M Nacl) and finally suspended in 6 volumes of 0.15 M saline. Cells were incubated at 37 “C for 72 hours, centrifuged for 1 hour at 4 “C and supernatant collected. The proteins were precipated with ammonium sulfate (560 g/litre), dissolved and reprecipitated twice. The final precipitate was dissolved in normal saline and dialysed against 0.01 M PBS, pH 7.3 and then ultracentri- fuged at 1 05000 x g for 1 hour at 4 “C. The clear supernatant was filtered through millipore membrane (0.45 u size) and stored in small aliquots which contained mycobacterial surface antigens at protein concentration of 5 mg/ml.

ELISA test

The detailed protocol has been described elsewhere [I I-141. The antigen (5 ug/ml in 0.06 M carbonate-bicarbonate buffer, pH 9.6) was coated in polystyrene micro-ELISA plates (Dynatech) by incubating overnight at 4°C. The plates were then washed 3 times with PBS, pH 7.2 containing 0.05 % Tween 20 (PBS/T). All sera were diluted to I:100 in PBS/T containing 0.5 % of bovine serum albumin (BSA) and 200 PI of each was added to the microwells in duplicate. After incubating for 1 hour at 37 “C, the plates were again washed similarly and 200 PI of I:800 diluted anti-human IgG-peroxidase conjugate, as used routinely in our laboratory [I71 was added to each well. The plates were again incubated at 37 “C for 1 hour. After further washings, 0-phyenylenediamine substrate (40 mg/lOO ml) of phosphate citrate Buffer, pH 5.0 containing 40 ul of 30 % H202, was added in equal amounts of 200-ul to each well and the reaction was allowed to proceed at room temperature for 20+5 min. The reaction was terminated by adding 50 1.11 of 5 N H2S04 to each well and the readings were taken on an ELISA reader (Dynatech) at 492 nm.

Serodiagnosis of intestinal tuberculosis 57

SAFA test

A standard method was followed [3, 8, 91. For each test serum, 2 cellulose-acetate paper discs (0.45 mu pore size) were taken. One was sensitized with optimally diluted antigen (50 ug/ml), whereas the other disc without antigen served as a control. The discs were treated with fixative (ethanol-HCI), washed and dried. Both discs were moistened with 5 yl of 0.01 M tris-buffered saline (TBS) pH 8.0 and then reacted with 40 $ of test serum diluted to 1:3. After incubation in a humid chamber for 45 minutes the discs were thoroughly washed in TBS and treated with suitably diluted rabbit-antihuman IgG for 45 minutes and washed. Finally, the discs were incubated with optimal dilution of anti-rabbit IgG (goat) tagged with FITC, washed and dried. The fluorescence was measured on Aminco-Bowman Spectrophoto-fluorometer at an incident excitation of 495 nm and emission at 550 nm.

Results interpretation of ELISA & SAFA readings

All sera were tested in duplicate by both the tests. A set of reference positive and negative sera along with buffer blanks were included for each assay. ELISA results were expressed in terms of absorbance (OD) values 1141 while SAFA results by calculating fluorescent coefficient (FC) of each serum by the following equation 18, 9) -

FC = (Reading of test serum disc with antigen)-(reading of test serum disc without antigen

(Reading of standard serum disc with antigen)-(reading of standard serum disc without antigen)

.

1 t I

2.0 2.4

OPTICAL DENSITY (00) IN ELISA

Figure 1. Scatterogram showing correlation between values of FC and OD obtained in SAFA and ELISA, respectively for intestinal tuberculosis patients.

58 Chawla and others

I I I

INTESTINAL CONTROL TUBERCULOSIS

Figure 2. Scatterogram showing optical density values in Intestinal tuberculosis (ITB) and control subjects by ELISA test.

.

.

: . . . . . ??*w

cut off

. : :. :’

I I INTESTINAL CONTROLS TUBERCULOSIS

Figure 3. Scatterogram showing fluorescent coefficient (FC) values in ITB and control subjects by SAFA test.

Serodiagnosis of intestinal tuberculosis 59

ELISA and SAFA values of 0.81 and 2.56 were taken as cut off points on the basis recent reports [I 1, 141. Incidentally the values remained unaffected by using control mean + 2 SD estimates or applying an arbitrary selection criteria. For comparison of test and control results student ‘t’ test was done.

Correlation between ELlSA and SAFA

A total of 22 and 3 out of 24 sera each from test (ITB cases) and control groups were found to be positive by ELISA: the corresponding figures by SAFA test were 20 and 2 respectively (p<O.Ol). Clustering of antibody levels revealed a strong correlation between the two tests (r=0.82) (see Fig. 1).

Two ITB samples negative in SAFA test were positive by ELISA whereas none was negative in ELISA but positive by SAFA test. Individual optical density and fluorescent coefficient values of patient and control serum samples are shown in Figures 2 and 3. In general, the 2 tests gave (45/48) concordant results.

Discussion The present study demonstrated a good concordance of results by ELISA and SAFA tests. Only 3 out of 48 samples gave discordant results and all such sera had low levels of antibodies. In an earlier study, Nicholls 141 using an agglutination assay with phenol killed M. tuberculosis Hz7Ra cells, reported positivity in 87 % cases with active tuberculosis, though he could not detect antibodies in any of the 40 healthy subjects. By contrast, in our study both ELISA and SAFA detected positivity in 12 % and 8 % of apparently healthy individuals also. Our ELISA results are in good agreement with the results obtained by Nassau et a/. [12, 141, where more than 80 % positivity has been reported among patients with only 8 % among the healthy controls. Similar results have been reported by Karr and Coworkers [18] where 67 % and 31 % were positive in active tuberculosis cases and culture negative subjects, respectively. In contrast, Winters and Cox [61, using a sensitive radioimmuno-assay could detect positivity only in 56 % (with whole cell antigen), 45 % (with a cell wall antigen) and 50 % (with PPD antigen) patients. In a recent report however, ELISA has shown to yield excellent results in the serological diagnosis of extra pulmonary (bone and joint) tuberculosis 1151, where geometric mean titre of active tuberculosis cases was as high as I:179 as compared to only I:19 in the control subjects and I:19 in active tuberculosis cases. The detection rate among advanced cases of joint tuberculosis was 100 % indicating that ELISA may be of high diagnostic value 1151.

Although the degrees of sensitivity and specificity of the serologic tests have been acceptable in the present and preceding studies [5, 151, the overall picture indicates that a significant number of culture positive tuberculosis patients too, do not manifest circulating antibody against mycobacterial antigens and the disease remission is not essentially followed by a decrease in antibody level [61. Furthermore, the serology has failed to detect antibodies in many proven cases of tuberculosis and that a significant number of healthy subjects had demonstrable mycobacterial antibodies [3-151. There are sufficient reasons to conclude that none of the tests is fool proof and therefore, it is unlikely that these assays alone will prove useful in the serodiagnosis of individual cases of ITB. At this stage they should only be used as additional methods to confirm a previous diagnosis.

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