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Year : 1960  |  Volume : 8  |  Issue : 1  |  Page : 11-15

Complement fixation test for diagnosis of trachoma in India

1 Department of Ophthalmology, All India Institute of Medical Sciences, India
2 United States Naval Medical Research Unit No. 2, Taipei, Taiwan
3 Department of Medicine, University of Chicago, School of Medicine, Chicago, Illinois, USA

Date of Web Publication6-May-2008

Correspondence Address:
L P Agarwal
Department of Ophthalmology, All India Institute of Medical Sciences
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Source of Support: None, Conflict of Interest: None

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How to cite this article:
Agarwal L P, Grayston J T, Woolridge R L. Complement fixation test for diagnosis of trachoma in India. Indian J Ophthalmol 1960;8:11-5

How to cite this URL:
Agarwal L P, Grayston J T, Woolridge R L. Complement fixation test for diagnosis of trachoma in India. Indian J Ophthalmol [serial online] 1960 [cited 2020 Aug 15];8:11-5. Available from: http://www.ijo.in/text.asp?1960/8/1/11/40690

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Table 2

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Table 1

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Table 1

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The recent isolation of trachoma virus and its growth in series and quan­tity iii the yolk sac of chick embryos -Tang. et al (1957), Collier and Sowa (1958), Snyder et al (1959) Bernkopf et al (1959), Hanna et al (1959), Grayston, Wang and Johns­ton (1960) and at AIIMS - Laboratories (1960) - has provided an opportu­nity for studies of laboratory diagnos­tic methods for trachoma. Virus isola­tion has not proved to be a practical method of assistance in diagnosis of the questionable and borderline clinical cases of trachoma, since it can be ac­complished most readily only from the more active cases that are inclusion body positive. Snyder et al (1959) Grayston et al (1960) Collier (1959) and AIIMS Laboratories (1960).

Serological tests are valuable me­thods of diagnosis in a large number of infectious diseases. Previously, studies have been made of complement fixing antibodies in serums from patients with trachoma utilizing group antigens of the psittacosis-lymphogranuloma ven­ereum group. Rake and co-workers (1942) and Kornblueth and co-workers (1954) reported the presence of com­plement fixing antibodies in the serum of some trachoma patients with lym­phogranuloma venereum virus antigen. Other workers employing similar anti­gens or trachoma antigens prepared from scrapings of conjunctival epithe­lium of patients with active infection have reported complement fixing anti­bodies in the serum of patients with trachoma, reviewed by Thygeson (1958). None of these tests have pro­ven to be of general diagnostic useful­ness,

The use of trachoma virus as group antigen in the complement fixation test with trachoma patient serums has given. disappointing results. A relatively small percentage of cases show antibody with the group antigen and then in low serum titer. However, when the tra­choma virus is purified, a specific antigen can be prepared which mea­sures antibody in a significant portion. of those patients with trachoma Grays- ton. Wang Woolridge et al (1960) This present report concerns the use of this specific "purified" elementary body (PEB) antigen with serum from. patients with trachoma in India.

  Materials and Methods Top

Serum : Blood specimens were . obtained at the same time as ophthal­mological examination for diagnosis of eye condition from two groups of per­sons. One group was patients and: staff personnel of the All India Insti­tute of Medical Sciences, and the other was persons at three health sta­tions (Kotla Mubarakpur, Masjid Moth and Palam centre) in the greater Delhi area. The blood specimens were obtained with sterile syringes and tubes. The blood was first allowed to clot and then the tubes centrifuged and the serums removed aseptically.. Serums were stored in the frozen state.

Preparation of antigens : Six to eight day old chick embryos were inoculated into the yolk sac with a predetermined virus inoculum which would produce a maximum yield of virus. Yolk sacs from all embryos surviving for three days or longer were harvested just be­fore or after death, smeared, cultured and stored at -65° C. Only sterile membranes, found to be heavily infect­ed with virus by microscopic examina­tion using Macchiavello's stain, were used for antigen preparation. The TW­29 trachoma virus strain - Grayston et al (1960 a), was used throughout this study.

PEB antigens were prepared according to the technique developed by Grayston et al (1960). Twenty per cent buffered saline sus­pensions of yolk sac tissue adjusted to pH .8.0 with 1 per cent sodium carbonate were incubated -,with 0.1 ml of 0.1 per cent tryp­sin per ml of suspension at 37°C for 3 hours. Trypsin was added and the pH adjusted at the end of the first and second hours of di­gestion. Only the pH was adjusted at the end of the third hour of incubation. Then following two cycles of low speed centri­fugation to remove the floating fat and large particles, the virus was sedimented three times at 10,000 rpm for 30 minutes in the No. 40 head of the spinco model L centrifuge. The resuspended elementary bodies were further purified by precipita­tion of foreign material with polymyxin B, so gamma per nil, and elution of any ac­companying virus from the precipitate. For­maldehyde USP was added to a 0.02 per cent concentration. The PEB antigens were adjusted in volume to a 40 per cent original yolk sac suspension. "These pre­parations had complement fixing antigen titers of 1:12 to 1: 24 in the presence of 2 units of homologous antiserum.

Boiled phenolized antigens were prepared after the method of Nigg, Hilleman and Bowser (X3,16) . Phenol to a 0.5 per cent concentration was added to to per cent suspensions of infected yolk sacs, This material was incubated at 37°C for four weeks and then placed in boiling water for 2o minutes. After centrifugation at 15oo rpm for ten minutes the straw colored supernatant was removed and used as group antigen.

Complement Fixation Test Procedure:The test used in the current work - Rosenbaum Max and Woolridge (1956) was a modified Kohlmer (1954) technique employing the optional proportions method of Friedewald (1943). In all tests two units of antigen (in 0.25 ml.) and two full units of complement (in 0.5 ml) were mix­ed with serial dilutions of serum (in 0.25 ml.) and incubated overnight at 5°C. The following day after the tubes were warmed at 37°C for ten minutes, two units of hemolysin and 2 per cent sheep red blood cells (in 0.5 ml.) were added. The materials were again incubated at 37°C for thirty minutes after which the tests were read. The highest serum dilution in which three plus fixation occurred (75%) was taken as the endpoint and reported as the reci­procal of the original serum dilution.

  Results Top

A total of 123 serum specimens were successfully tested in the complement fixation test for trachoma. Of the per­sons from whom these serum specimens were taken, 109 had the clinical diag­nosis of trachoma, while 14 were thought not to have trachoma on eye examination. [Table - 1] shows the results of the complement fixation test by area from which the patients were obtained and by eye diagnosis. Sixty and 67 per cent of the patients from the first two areas of field collection (Kotla Mubarakpur and Masjid Moth) showed complement fixing antibody titers in their serum. It should be noted that in these two areas the people were informed that an eye doctor was com­ing to examine those with problems, and that the people who came to the clinic undoubtedly had more severe eye disease than in the third area of field collection (Palam centre) where most of the persons examined con­sisted of middle school students who were brought to the clinic. In Palam centre only 35 per cent of the persons with trachoma had complement fixing .antibodies and then in low titer. Of the patients and personnel of the All India Institute who were tested, 42 per cent. of those with the diagnosis of trachoma had complement fixing antibodies. Two of 13 persons without clinical evidence of active trachoma showed complement fixing antibodies at a 1:8 serum dilution.

Of the total of 109 persons with trachoma, 47 per cent showed anti­bodies with the specific trachoma an­tigen. Patients with the definite diag­nosis of active trachoma, but without yet showing scarification (Trachoma II) had the highest percentage of com­plement fixing antibodies and also showed the highest antibody titer.

[Table - 2] shows the complement fixa­tion test results by age for the persons with trachoma. There was an increase in the percentage of persons with anti­body with increasing age until the 40 to 49 year age group. Eight of the 14 persons (57%) age 40-49 had tra­choma antibodies. In persons over 50 years of age there was a fall off in the frequency of antibodies.

In [Table - 3] the results of testing 18 serums from trachoma patients with boiled, phenolized group antigen in addition to PEB antigen are shown. Only 5 of the serums with the highest antibody titer against PEB antigen showed any reaction with the group antigen.

  Discussion Top

Previous studies with the purified elementary body trachoma virus anti­gen have shown that this antigen measures complement fixing anti­bodies only in persons with trachoma. It fails to measure antibodies deve­loped by persons who are infected with psittacosis or lymphogranuloma vene­reum - Woolridge, Jackson and Grayston (1960) and Grayston et al (1960 a). On the other hand, the group antigen made from the tra­choma virus has been shown to effect­ively measure antibodies in patients with psittacosis and lymphogranuloma venereum - Grayston et al (1960). The results reported in [Table - 3] demon­strate that the antibodies measured in the trachoma patients are due to tra­choma infection and not psittacosis or lymphogranuloma venereum and also again demonstrate that the PEB antigen is greatly superior to the group antigen for showing antibodies in trachoma infection.

We have tested serum from 229 young American service men without evidence of trachoma. None of their serums reacted at a titer of 1:8 in the complement fixation test with the PEB antigen-Woolridge & Grayston (1960). This is the best evidence of specificity of the test since most Americans are never exposed to trachoma. The occa­sional person in the endemic area who shows trachoma antibodies but has no clinical evidence of trachoma (such as the 2 of 14 in this study) may have been infected in the past.

Sera from persons on Taiwan with the diagnosis of trachoma have shown complement fixing antibodies with the purified arnigen in from 32 to 61 per cent of those tested, depending upon the severity of the disease, and the age of the individual - Grayston, Wang Woolridge et al (196o a). In this study the increase with age in percent­age of those with antibody was not great, but since all persons still had active disease it is presumed that those with longer standing disease had a greater chance of developing comple­ment fixing antibodies. The frequency and titer of antibody was related to severity of disease. In the field collec­tions the somewhat older persons with more severe disease in Kotla Mubarak­pur and Masjid Moth showed much more frequent and higher titer anti­body than middle school boys in Palam centre. Also, there was a dif­ference in reaction among those per­sons bled at the All India Institute of Medical Science. The physicians and other staff members, although show­ing signs of trachoma infections, rare­ly had antibody. The patients and other personnel of the Institute had antibody more frequently.

Finding that about 50 per cent of the persons in India with the diagnosis of trachoma possess antibody in their -serum against the specific trachoma virus antigen, suggests that the com­plement fixation test may find useful­ness in the diagnosis of this disease. Further improvements in the antigen may allow a higher percentage of the patients to be diagnosed. The test has proven to be adequately sensitive for some research purposes. We have de­monstrated on Taiwan that most per­sons with the clinical diagnosis of -chronic follicular conjunctivitis have trachoma - Grayston, Wang Wool­ridge et al (1960 a).

Bernkoff and associates (1960) have devised an agglutination test for tra­choma in which they report 6o to 88 per cent of patients tested to show antibody, although II per cent of con­trols were also positive. Now that the trachoma virus can be cultivated in the laboratory, it is reasonable to suppose that there will be continued improve­ment in laboratory diagnostic methods.[19]

  Summary Top

A newly devised complement fixa­tion test for trachoma in which the antigen is a purified trachoma virus was used to test serum from persons in India. Forty-seven per cent of Tog persons with trachoma showed comple­ment fixing antibodies in serum titer from 1:4 to 1:256 in this test. It was concluded that serologic tests offer hope for more simple laboratory diag­nosis of trachoma.

  References Top

A.I.I. Med. Sciences Laboratories (1960) - Personal Communications.   Back to cited text no. 1
Bernkopf, H., Nishmi, M., Maythar B.; and Feitelberg, 1, (1959) . A. M. A. Arch. Ophth., 62: 33-34.  Back to cited text no. 2
Bernkopf, H., Nishmi, M., Maythar. B., and Feitelberg, I. (1960). J. Inf. Dis., 106: 83-86.  Back to cited text no. 3
Collier, L. H. and Sowa, J. (1958). Lancet I : 993-996.  Back to cited text no. 4
Collier, L. H. (1959). Brit. Med. Bull., 15 : 231-23.1.  Back to cited text no. 5
Friedewald, W. F. (1943). J. Exper. Med., 78 : 347-366.  Back to cited text no. 6
Grayston, J.T., Wang, S.P. and Johnston, P. B. (196o April). Proc. Soc. Exper. Biol. & Med., V. 103.  Back to cited text no. 7
Grayston, J.T., Wang, S.P., Woolridge, R.L., Yang, T.F, and Jonston, P.B. (1960 a). J.A.M.A., V. 172, April 9.  Back to cited text no. 8
Hanna, L., Thygeson, P., Jawetz, E. and Dawson, C. (1959). Science, 130:1339-1340.  Back to cited text no. 9
Kolmer, J.A. and Boerner, F. (1954) Approved Laboratory Technique, 4th ed., D. Appleton Century Company, New York.  Back to cited text no. 10
Kornblueth, W. Feigenbaum A. and Bernkopf, H. (1954). Acta Cone. Ophth. XVII : 1506-1511­  Back to cited text no. 11
Nigg, C., Hilleman M.R. and Bowser, B.M. (1946) J. Immunol., 53: 259-268.  Back to cited text no. 12
Rake, G., Shaffer, M.F. and Thygeson, P. (1942). Proc. Soc. Exper. Biol. and Med., 49 : 545 - 547.  Back to cited text no. 13
Rosenbaum, Max J. and Woolridge, R. L. (1956). J. Infect. Dis., 99: 275-281.  Back to cited text no. 14
Snyder, J.C., Page, R. C. Murray, E. S., Daggy, R.H., Bell, S.D. Jr., Nicols, R.L., Haddad, N.A., Hanna, A.T. and McComb, D. (1959) Am. J. Ophth., 48: 325-329.  Back to cited text no. 15
Tang, F.F., Chand, H.L., Huang, Y. T. and Wang, K. C. (1957). Chinese Med. J., 75: 429 - 447.  Back to cited text no. 16
Thygeson, P. (1958). Bull. Wld. Hlth. Org., 19 : 129-152.  Back to cited text no. 17
Woolridge, R.L., Jackson, E.B. and Grayston, J.T. (1960). Proc. Soc. Exper. Biol. & Med., V. 103 No. 3.  Back to cited text no. 18
Woolridge, R.L, and Grayston, J. T., Unpublished data.  Back to cited text no. 19


  [Table - 1], [Table - 2], [Table - 3]


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