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  IN THIS Article
 ::  Introduction
 ::  Material and methods
 ::  Results
 ::  Discussion
 ::  References

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Year : 1986  |  Volume : 32  |  Issue : 2  |  Page : 77-81

Dinitrochlorobenzene contact sensitization in patients with pulmonary tuberculosis.

How to cite this article:
Sharma T N, Jain N K, Sharma R C, Purohit S D, Sarkar S K. Dinitrochlorobenzene contact sensitization in patients with pulmonary tuberculosis. J Postgrad Med 1986;32:77-81

How to cite this URL:
Sharma T N, Jain N K, Sharma R C, Purohit S D, Sarkar S K. Dinitrochlorobenzene contact sensitization in patients with pulmonary tuberculosis. J Postgrad Med [serial online] 1986 [cited 2023 Mar 25];32:77-81. Available from:

  ::   Introduction Top

A functional impairment of the cell mediated immune response as manifested by the inability to elicit sensitization to 1-chloro 2, 4-dinitrochlorobenze (DNCB) has recently been reported in pulmonary tuberculosis.[1], [11] In the present study, cell mediated immune response in pulmonary tuberculosis has been assessed by DNCB skin sensitization in healthy controls, freshly diagnosed patients of pulmonary tuberculosis, patients who have received a treatment for one to three months and in patients who had completed chemotherapy, so as to assess the effect of chemotherapy on cell mediated immunity in pulmonary tuberculosis. A simultaneous comparison has also been made with PPD reactivity.

  ::   Material and methods Top

The study included a group of 40 healthy persons (Gr. I), 40 freshly diagnosed sputum positive patients of pulmonary tuberculosis (Gr. II 'untreated'), 40 tubercular patients who have received anti-tuberculosis chemotherapy for one to three months (Gr. III 'on-treatment') and 40 patients who had completed chemotherapy of one year (Gr. IV 'fully-treated').
DNCB skin sensitization was done by the method of Catalona et al[3] Briefly, after cleaning the skin of the volar surface of both the forearms, 2,000 mcg and 50 mcg of freshly prepared DNCB solution in 0.1 ml acetone was applied over a 3 centimeter square area through a metal ring on the left and right forearms respectively.
The sites were covered with adhesive plaster for three days and washing was avoided for 24 hours. The site with 2,000 mcg. of DNCB application was observed for the signs of non-specific inflammatory response (NIR), which was recorded as positive or negative. Between 7 and 14 days both the sites of DNCB application were observed for any spontaneous flare. Occurrence of spontaneous flare represented the development of cell mediated immunity (CMI) to DNCB. If no response was visible after 14 days at either DNCB application site, a challenge dose of 50 mcg. was given and the response was observed after 48 hours for a delayed cutaneous hypersensitivity (DCH) reaction. The response was graded as 4+ if spontaneous flare was observed at both the DNCB application sites within first 2 weeks, 3+ if the reaction occurred only at the 2,000 mcg site, 2+ if spontaneous flare did not occur at either of sites upto 14 days but second challenge dose site showed unequivocal response within 72 hours. The reaction was labelled as 1 + if the second challenge dose gave an equivocal response. If needed the site was biopsied for the confirmation of contact dermatitis. The reaction was recorded as negative if there was neither any reaction to sensitization dose nor to challenge dose.
Tuberculin sensitivity was tested by 0.1 ml of 10 TU PPD-RT 23 with Tween 80 applied on the anterior surface of the left forearm and reading taken after 72 hours. An induration of 10 mm was taken as positive.

  ::   Results Top

[Table - 1] shows age and sex distribution of 40 healthy controls and 120 patients studied. The age and sex distribution of patients were comparable to those with controls.
[Table - 2] shows the results of DNCB response and its correlation with PPD reactivity and NIR in four groups of cases. All the healthy controls (Gr. I) could be sensitized to DNCB, majority of them were 4+ responders, 55 per cent of them had PPD positivity and all had a normal NIR. The proportion of subjects developing contact sensitivity to DNCB in Gr. II and III were significantly less than that of the control group. A positive DNCB response was observed in 35 per cent of Gr. II patients and in 70 per cent of the patients who had received treatment for 1 to 3 months. With continuation of treatment, there was further improvement in cell mediated immunity and the percentage of positive responders increased to 95 per cent in Gr. IV patients. Most of the patients had normal NIR and those having negative NIR were also consistently negative for DNCB contact sensitization.
[Table - 3] shows correlation of Mantoux positivity with DNCB response. Out of 120 patients, 59 (49%) were reactive to both DNCB and PPD, 33 (28% ) reacted to the recall antigen PPD but were negative to DNCB, 21 (17%) were DNCB positive but PPD negative and 7 (6%) were anergic to both DNCB as well as PPD.
[Table - 4]shows correlation of radiological extent of disease with DNCB response in Group II (untreated) patients. Out of 14 patients who could be sensitized to DNCB, 6 (43% ) had far-advanced disease. In contrast, out of 26 patients who were anergic to DNCB, 17 (65 % ) had far-advanced disease.

  ::   Discussion Top

Of the 40 freshly diagnosed sputum positive cases, only 14 (35%) could be sensitized to DNCB. Anergy to DNCB has also been reported by others.[1], [2], [9], [11], [12], [15] Thus, Agnihotri et al,[1] Gross[9] and Malaviya et al[11] reported anergy to DNCB in more than one-half of their patients while in the present study DNCB anergy was observed in 65 per cent of the patient. Malaviya et al[12] have also observed DNCB anergy in 60 per cent of their patients. Bhatnagar et al[2] have reported a DNCB anergy in upto 75 per cent of cases of active pulmonary tuberculosis. Our results are, therefore, not very different from those of previous workers. On the other hand Sharma[15] could not observe any evidence of depression of delayed hypersensitivity in patients with tuberculosis who were tuberculin positive. He used much higher doses for sensitization as well as challenge. DNCB response is concentration dependent[8] and higher doses can convert 'nonresponders' to 'responders'.[14] Moreover, it has been Dostulated that there may be a defect in the affarent limb of immune response leading to its partial suppression which could be overcome by strong stimulation.
In the present study, 65 per cent of DNCB anergic patients had far advanced pulmonary tuberculosis. In comparison to this, only 43 per cent of DNCB positive patients had far advanced disease. In the study of Agnihotri et al[1] also, more of the DNCB negative patients had far advanced disease in comparison to those who mounted a normal cell mediated immune response to DNCB.
With treatment, the ability to be sensitized with DNCB increased. The patients who had received treatment for more than one month (Gr. III) had positive skin sensitization to DNCB in 70 per cent cases and the same procedure, done in 'fully treated' patients (Gr. IV) resulted in DNCB sensitization (95%) almost similar to that of the control group (100%). These observations indicate that the DNCB responsiveness improved with treatment and reached normal proportion simultaneously. with clinical improvement. A similar finding has also been reported by Malaviya et al.[12] However, the degree of reactivity as measured by Catalona et al[3] method remained low. Majority of the control group were 4+ responders while most of those 'on treatment' had a 2+ response and those 'fully treated' had a response of 2+ to 3 + in majority. Whether this subtle degree of immunodeficiency contributes towards the development of post-primary tuberculosis in these individuals remains to be seen. Alternatively, they might not have as yet fully recovered from the immune deficiency induced by the infection.12 The improvement in the proportion of DNCB responders among the 'fully treated' patients indicates that the defect was a secondary immunodeficiency related to the disease. Such a defect has been documented in leprosys and in patients with miliary tuberculosis.[13], [17], [19]
Correlation of the results of DNCB sensitization and PPD reactivity brought out 4 groups of patients. The largest group was that of 59 patients who were reactive to both DNCB and PPD. The second largest group was of 33 patients who could not be sensitized 'to DNCB (a new antigen) while they did respond to PPD (an antigen encountered previously). Most of the patients had normal NIR which indicates that poor inflammatory reaction of the body was not responsible for DNCB anergy. Furthermore, factors known to cause suppression of CMI[5], [16] e.g., malnutrition, debility, advanced age, immunosuppressive drugs and others were not present in these patients. Also, the presence of such factors would have equally affected PPD reactivity. The inability to respond to an antigen not encountered previously while showing a positive response to an antigen encountered earlier is indicative of a defect in the afferent limb of immune response.[10] Patients with Wiskott Aldrich Syndrome' and sonic patients with lepromatous leprosy[18] and sarcoidosis[8], [10] I show such a defect and it is likely that a similar defect is also present in tuberculosis. Two other minor groups of patients were detected in this study. One was that of 21 patients who were PPD negative but DNCB positive. Normal DNCB response indicates that their CMI was normal, probably those patients had false negative PPD response due to any of the various factors known to cause such negativity,[5] despite the best efforts to exclude these. The last group was that of 7 patients who showed non-reactivity to both DNCB as well as PPD. This small group probably represented those patients who had more generalised depression of CMI.

  ::   References Top

1.Agnihotri, M.S., Chaturvedi, U.C. and Pande, S.K.: Immunological classification of pulmonary tuberculosis, Ind. J Pub., 25: 65-76, 1978.  Back to cited text no. 1    
2.Bhatnagar, R., Malaviya, A.N. and Narayanan, S., Rajgopalan, P., Kumar, R. and Bhardwaj: Spectrum of immune response abnormalities in different clinical forms of tuberculosis. Amer. Rev. Respir. Dis., 115: 207-212, 1977.  Back to cited text no. 2    
3.Catalona, W.J.: Taylor, P.T., Rabson, A.S. and Chretein, P.B.: A method for dinitrochlorobenzene contact sensitization, a clinico-pathological study. New Engl. J. Med., 286: 399-402, 1972.  Back to cited text no. 3    
4.Cooper, M.D., Chase, H.P., Lowman, J.T., Krivit, W. and Good, R.A.: Wiskott-Aldrich Syndrome. An immunologic deficiency disease involving the afferent limb of immunity. Amer. J. Med., 44: 499-513, 1968.  Back to cited text no. 4    
5.Edwards, P. Q.: Tuberculin negative?. New Engl. J. Med., 286: 373-374, 1972.  Back to cited text no. 5    
6.Godal, T.: The role of immune response to mycobacterium leprae in host defence and tissue damage in leprosy. "Progress in immunology II, Volume 4, editor L. Brent and J. Holborow, North Holland, Amsterdum, 1974, p. 161.  Back to cited text no. 6    
7.Greenwood, R., Smellie, H., Barr, M. and Cunliffe, A.C.: Circulating antibodies in sarcoidosis, Brit. Med. J., 1: 1388-1391, 1958.  Back to cited text no. 7    
8.Grolnick, M.: Studies in contact dermatitis, spontaneous flare up of negative test sites in experimental sensitization in man. J. Immunol., 41: 127-142, 1941.  Back to cited text no. 8    
9.Gross, L.: Immunological defect in aged population and its relationship to cancer. Cancer, 18: 201-204, 1965.  Back to cited text no. 9    
10.Jones, J.V.: Development of sensitivity to DNCB in patients in sarcoidosis. Clin. Exp. Immunol., 2: 477-487, 1967.  Back to cited text no. 10    
11.Malaviya, A.N., Kumar, R. and Dingley, H.B.: DNCB skin sensitization and its relationship with PPD reactivity in pulmonary tuberculosis. Ind. J. Med. Res., 61: 885-890, 1973.  Back to cited text no. 11    
12.Malaviya, A.N., Sehgal, K.L., Kumar, R. and Dingley, H.B.: DNCB contact sensitization in pulmonary tuberculosis. Clin. Esp. Immunol., 22: 399-403, 1975.   Back to cited text no. 12    
13.Mc Murray, D. N. and Echeverri, A.: Cell mediated immunity in anergic patients with pulmonary tuberculosis. Amer. Rev. Respir. Dis., 118: 827-834, 1978.  Back to cited text no. 13    
14.Saha, K. and Mittal, M.M.: A study of cell mediated immunity in leprosy: changing trends in immunological spectrum of disease, Clin. Exp. Immunol., 8: 901909, 1971.  Back to cited text no. 14    
15.Sharma, O.P.: Immunological relationship between sarcoidosis and tuberculosis Ind. J. Med. Res., 58: 1551-1559, 1970.   Back to cited text no. 15    
16.Sharma, O.P. and Evans, P.A.: Negative tuberculin test in active pulmonary tuberculosis. New Engl. J. Med., 286: 374-378, 1972.  Back to cited text no. 16    
17.Uberoi, S., Malaviya, A.N., Chattopadhyaya, C., Kumar, R. and Shrinivas: Secondary immunodeficiency in miliary tuberculosis. Clin. Exp. Immunol., 22: 404408, 1975.  Back to cited text no. 17    
18.Waldorf, D.S., Sheagren, J.N., Trautman, J.R. and Block, J.B.: Impaired delayed hypersensitivity in patients with lepromatous leprosy. Lancet, ii: 773-775, 1966.  Back to cited text no. 18    
19.Waxman, J. and Lockshin, M.: In vivo and In vitro cellular immunity in anergic miliary tuberculosis. Amer. Rev. Respir Dis., 107: 661-664, 1973.  Back to cited text no. 19    

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Online since 12th February '04
2004 - Journal of Postgraduate Medicine
Official Publication of the Staff Society of the Seth GS Medical College and KEM Hospital, Mumbai, India
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