|Year : 1960 | Volume
| Issue : 1 | Page : 11-15
Complement fixation test for diagnosis of trachoma in India
LP Agarwal1, JT Grayston2, RL Woolridge3
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 Publication||6-May-2008|
L P Agarwal
Department of Ophthalmology, All India Institute of Medical Sciences
Source of Support: None, Conflict of Interest: None
|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 May 30];8:11-5. Available from: http://www.ijo.in/text.asp?1960/8/1/11/40690
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|| |
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|| |
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|| |
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.
| Summary|| |
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.
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[Table - 1], [Table - 2], [Table - 3]