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   Table of Contents      
ARTICLE
Year : 1958  |  Volume : 6  |  Issue : 2  |  Page : 21-29

The serological diagnosis of trachoma and its application to the mass examination of trachoma


Kyoto Prefectural Medical University, Kyoto, Japan

Date of Web Publication8-May-2008

Correspondence Address:
Hiromu Ueno
Kyoto Prefectural Medical University, Kyoto
Japan
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Source of Support: None, Conflict of Interest: None


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How to cite this article:
Ueno H, Kashii T, Shimizu E. The serological diagnosis of trachoma and its application to the mass examination of trachoma. Indian J Ophthalmol 1958;6:21-9

How to cite this URL:
Ueno H, Kashii T, Shimizu E. The serological diagnosis of trachoma and its application to the mass examination of trachoma. Indian J Ophthalmol [serial online] 1958 [cited 2019 Sep 23];6:21-9. Available from: http://www.ijo.in/text.asp?1958/6/2/21/40717

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Table 1

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Table 1

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From the knowledge we have got to-day regarding trachoma, we can well say that the correct diagnosis of trachoma is rather difficult. Proving the presence of Prowazek-Halber­staedter's inclusion bodies that are re­garded as infectious agents of trachoma is thought to have the most scientific diagnostic value, but, to-day, in Japan, the rate of proving the same is too low and it has little practical diagnostic value. Since ten years, we have been observing trachoma in Japan clinically, for a long time, and as a result we have been insisting that it is hard to catch trachoma from only the finding in the conjunctiva. So at the same time it is necessary to examine the trachomatous changes in the cornea and specially diagnose trachoma on the basis of the finding of pannus. It is true, however, that some ophthal­mologists are opposed to it and we can­not deny the fact that some trachoma has no pannus. But without adoption of such a criterion for the diagnosis of trachoma it is impossible to find more trachoma scientifically. Even to-day I believe that it is a correct diagnostic criterion.

Fortunately, as the co-worker of Assistant Professor Arakawa of the Institute of Infectious Diseases of Tokyo University, I have been engaged in the study of Arakawa's mouse-fixed trachoma virus (this will be abbreviated MFTV) for the last more than eight years, and have published several reports. Goto, Takahashi, Shimizu and myself have proved by histo-pathological examinations, that the changes produced in mice inocula­ted by this virus were of viral nature; and Takahashi, Kashii and I have de­monstrated the hematological changes and antibody formation in infected mice. These have led us to presume the existence of sero-immunological re­action among trachoma patients.

Yuge, Professor of our Ophthalmo­logical Department, Kashii, one of my co-workers and I by way of serological tests, have tried the neutralization test, skin and complement fixation test (this will be abbreviated CFT) using this virus, and among them we have found the special excellency of CFT and carried out several fundamental experi­ments which have been published.

Such experiments are carried out not only by us but also by the Arakawa Group, that is, Inoue, Murakami and Sugiura, Assistant Professor of Tokyo University, and Tsutsui of Okayama University, and so on. But all of them, using this virus, do not go beyond the field of fundamental experiment. Last year, Professor Yuge, from the results of our experiments, considered CFT as a reliable reaction for trachoma and thought this to be of practical value for clinical application, and worked out on this, the mass examination method of trachoma which has been published in japan. With all the might of our de­partment, in two heavily infected tra­choma areas, we have used this method practically and have got good results. Of course, we do not think that this method is the most ideal, though we think this to be the most scientific one considered on the basis of our knowledge.


  I. Fundamental Experi­ments Concerning CFI Top


The Test Sera

The test sera were obtained from patients with typical trachoma in vari­ous stages, inactivated by heating to 60°C, for 20 minutes and then diluting with normal saline by double dilution method. Sera of non-trachomatous persons were used as controls.

The Antigen

The brains of the mice, which show­ed the characteristic signs by the inoculation with the MFTV were made into an emulsion of 10% in normal saline solution with pH 7.6, left in an ice-box for 24 hours and centrifuged for 30 minutes at 3,000 r.p.m. The supernatant fluid thus obtained was frozen and thawed 3 times, and centri­fuged again for 1 hour at 15,000 r.p.m. To the sediment thus obtained was added normal saline solution to make as large a volume as the original one which was centrifuged for 30 minutes at 3,000 r.p.m. and then the superna­tant fluid we used as the antigen, after addition of Merthiolate to give a final concentration of 0.01%.

Normal mouse brain, treated with similar procedures as above mentioned was used as the normal (control) anti­gen. The anticomplementary action of the antigen was estimated, and for the proper test, the antigen of 2 fold dilu­tion was used.

The Complement

The complement was prepared from pooled blood of 3 normal guinea pigs. The dilution of the complement was, such that 0.5 ml of the solution con­tained 2 full units of 30 fold solution.

Sheep Cells and Antisheep Hemolysin

A 3% suspension of washed sheep cells was made and mixed with an equal volume of the 3 units antihemo­lysin solution.

The scheme of the test is shown in [Table - 1]. The Wassermann test was carried out in all cases.

Experiment 1. [Table - 2] shows the results of Experiment 1. Of 81 cases of typical trachoma 46 were test posi­tive (57%), 2 doubtful and 33 nega­tive. The highest antigen titer with positive test was 1.64, but in general the titer was moderate. The Wasser­mann test in 2 cases was positive. In control subjects, in which one case of Behcet's disease and four cases of kerato-conjunctivities epidemica were involved, all except one doubtful case, were negative. The case of doubtful result in the control group is one of an oculist, treating trachoma patients every day. As the table shows, the rate of positive cases varies according to the stage of trachoma but the differences between one another are not signifi­cant.

Experiment 2. This experiment was carried out in a heavily infected area of trachoma, and 219 cases were examined in all and CFT was carried out in all of them. Of 219 cases in 148 we were able to judge the test; the re­sults of which are shown in [Table - 3].

Notwithstanding the findings in the conjunctiva, when we observed the relationship of CFT positive rates among cases with and without pannus, making 0.3mm the limit of elognation of endcapillary loops as pannus, we could not recognize a statistically significant difference between the two,-59.2% and 39.1% respectively.

Experiment 3. In a certain heavily infected area we have tried CFT of conjunctivitis follicularis and follicul­ous conjunctivae. Of course, we did not find pannus in those cases by the slit lamp examination. Among folliculosis 10 cases and conjunctivitis follicularis 4 cases, we have found positive tests in 5 cases.

Experiment 4. We have tried for trachoma patients, the treatment of sulphonamide systemic administration and that of local application of Aureo­mycin ointment, and examined and compared CFT respectively before the treatment, 21 days later,66 days later, in the group treated by systemic administration and 135 days later in the group treated by local application only, but there was no change.


  II. Practical Experiments on Mass Examinations for Trachoma Applying CFT For Diagnosis Top


We have tried in 2 trachoma sections the mass examinations, using methods for mass examination applying CFT introduced by Professor Yuge.

Methods of the Experiment

The technique of CFT that we have tried is just the same that we had described before, but the antigen that we have used is a little different from that we had used before and which we have prepared as follows

The brains of the infected mice with the MFTV (Shiratori strain) were made into an emulsion of 10% in the normal saline solution and centrifuged. The supernatant fluid thus obtained was inoculated in the chorio-allantoic membrane of the chicken eggs which had already been incubated for to days at 38°C, and then these eggs were in­cubated furthermore for 5 days at 35°C. After this incubation the chicken embryos and the membranes were re­moved and together made into an emulsion of 10% in normal saline solu­tion. This emulsion was frozen and thawed 5 times repeatedly and centri­fuged for 30 minutes at 3,000 r.p.m. The supernatant fluid thus obtained was ultracentrifuged in Sherpless centri­fugal machine, and then to the sedi­ment obtained was added normal saline at the rate of 1/25 of the original volume which was made homogeneous and centrifuged for 30 minutes at 3,000 r.p.m. This supernatant fluid was used as the antigen, after addition of Mer­thiolate to give a fluid concentration of 0.01% By the way, before the material is removed out of the eggs, if it is left overnight in an ice-box, good antigen will be obtained unmixed with blood. The antigenicity of this refined antigen was measured by the immune serum and the standard serum.2 units were used in this proper test.

Experiment 1.(Scheme 1). This experiment was tried on 1,798 pupils of a middle school and a primary school on the seashore of a trachoma area. At this time, according to Yuge's first method, we removed the blood for the test and then carried out CFT. We made a mistake to keep a group of the blood too long and could not carry out this test because of hemolysis, etc., and thus we could judge this test in 1,226 cases. From the results of the CFT which was carried out first, a clinical examination with the help of a hand slit lamp was carried out, of the con­junctiva and the cornea in the CFI positive cases, and those who needed treatment were thus caught. On the other hand, from the CFT-negative cases, similarly those who were suffer­ing from trachoma and in need of treat­ment were picked out. Besides these, as a control group, we tried CFT in 49 persons who were normal and living in .a place where trachoma was not pre­vailing.

The results are shown in [Table - 4]. In the trachoma group, the rate of positiveness of CFT was 57.5% (60.0%>p>55.4%) ; in the non-tracho­matous conjunctivitis group, 39.5%; in the group of normal cornea and conjunctiva, I8.1% but in the control group all 49 cases were negative.

Experiment 2. (Scheme 2). This experiment was tried for the inhabitants in a certain trachoma section in Hyoga Prefecture. This time, according to the second method of Yuge's mass exami­nation of trachoma, we first used the hand slit lamp for all and examined carefully the cornea and the conjunc­tiva, and then decided the diagnosis. Then, we tried CFT for all non-tracho­matous conjunctivitis and doubtful trachoma except the typical trachoma.

Furthermore, as control, we tried CFT for 50 cases which were normal and were living in a place where trachoma was not prevailing. Technique of CFT was the same as I described in Experi­ment I, and the preparatory method of the antigen used was also the same.

[Table - 5] shows the distribution of doubtful or healed trachoma and other conditions in 186 clinically non-tracho­matous cases, The results concerning the groups in which we tried CFT are shown in the Table. 30 cases show CFT positive, out of which 18 were confirm­ed as trachomatous, which shows that about 10% had escaped diagnosis by only a clinical examination.

Besides the control group, we carried cut this test for each stage of trachoma and a part of those in whom we recog­nised no pathological findings of the conjunctiva and the cornea and which we considered normal.


  Comment Top


The above mentioned experiments consisted of primarily fundamental ex­periments on immuno-serological reac­tion of CFT and the experiments were then applied practically to mass exami­nation of trachoma. Regarding such fundamental studies in Japan, besides ours, recent reports tell us that the following experimenters have tried CFT of the sera of trachoma patients; Arakawa, Tsutsui, etc. and Murakami, with the MFTV; Terayama, Ikeda and Kondo etc. with the developing chiken egg-fixed trachoma virus; Fujiyama, with the rabbit testicle-fixed trachoma virus. Since Roemer's trial of CFT in 1908, there have been many studies at home and abroad, and yet we cannot recognise a decisively diagnostic value. Of course, the antigenicity of the anti­gen used, I think matters considerably, but in the case of trachoma, clinically speaking a local disease, antibody against the virus is formed in sera, and therefore its relation matters not a little.

Recent reports in Japan are shown in [Table - 7] and [Table - 8]. It shows the highest 90%, the lowest 53% positive results (arranged from 53% to 90%). The results of Inoue and Murakami who also used the mouse-fixed tra­choma virus show higher value than ours. Arakawa and Murakami describ­ed, "the specificity of antigenicity of the MFTV was confirmed which is of great value in the diagnosis of tra­choma." Yuge and Kashii described, "as our studies suggest, we should use CFT when the source of the antigen and its preparation are proper. Al­though the rate of our positive tests is not so high (57%) that we cannot decide the negative cases to be non-trachomatous this does not mean that the tests are of no significance, for the control tests are all negative."

Thus our examinations generally speaking are in accord with a little less than 60% positive rate in trachoma patients, as a whole. The strongest rea­son why we think this test specific for trachoma, lies in the fact that all nor­mal persons in non-trachoma popula­tions are CFT negative.

It is an interesting result that in a trachoma section we have often found some cases diagnosed as non-trachomat­ous conjunctivitis or normal, showed the positive reactions; thus we find a difference between trachoma in tra­choma section and that in non-trachoma section. We can see here such problems as the natural cure of trachoma, the cure without any trace -- the carrier state in trachoma, etc. This is nothing but my presumption, and I hope future researches will prove it. Many tra­chomatologists have not recognised the carrier state in trachoma. No valid evidence has been advanced to indicate that an individual who has never had clinical trachoma can be a carrier. There is Bodian's claim for the exis­tence of natural cure of trachoma since long ago. This belief becomes stronger when I consider it on the basis of CFT. Cannot we presume the possibility of the existence of the carrier state in trachoma ?

Well, it is true that all trachoma cannot be caught by only CFT nor by only pannus. But, last year, Yuge brought forward the view that the positive rate of CFT in all the trachoma cases from Experiment I was supposed to be 65% and that in pannus cases 70% and then they were calculated mathematically. From the examina­tion, about 10% trachoma cases might be escaping by either method and thus Yuge introduced a new mass examina­tion method. The principles are as follows

(1) Try CFT for all who are exa­mined, and then examine those who are negative with the objective of pannus.

(2) Examine all who are examin­ed with the objective of pannus and then try CFT for those whose pannus are negative.

So I, according to this method, carri­ed out the mass examination for tra­choma, combined with CFT as I men­tioned above. The first experiment got the figure - about 10% trachoma cases that escaped from the examina­tion as Yuge had pointed out. At any rate according to his examination methods, trachoma cases that escape by the corneo-conjunctival examination, can be caught by CFT; and then those that escape by the CFT are caught by the corneo-conjunctival examination.

Consequently, it is natural that this method is superior to the past methods. Again, CFT is profitable because be­sides the oculist, the technician at the health centre can try it; and in addi­tion to it, we can spare time for the physical examination. It is needless to say that this method is not yet com­plete. In future, I do hope that fur­ther researches will get antigen of higher antigenicity, by clarification and purification of the antigen, and will surely catch all trachoma by only CFT. Recently, some have expressed their doubts against the MFTV; some have made it public, to oppose it, a certain virologist has denied it, saying that no virus can be cultured in the mouse brain. But, recently I have heard that at Adams in the United States of America, they have succeeded in culturing the virus in the mouse brain.

We thus think that we have medi­cally improved, more or less the diffi­culties that the diagnosis of trachoma presents and have been successful to catch more trachoma. Chiefly in Asian countries, where trachoma is rampant, it is significant to improve the trachoma diagnosis in order to stamp out tra­choma. In that sense we think this experiment has a certain value.


  Summary Top


Complement fixation test (CFT) with an antigen prepared from mouse-fixed trachoma virus (MFTV) of Arakawa as a serological diagnostic test for tra­choma is described and evaluated. Method of preparation of the antigen in described.

For a quicker and a more exact diagnosis the test has been applied in mass examinations for trachoma in two ways. (1) CFT first and then picking out from CFT + yes, cases which require treatment, (2) clinical examina­tion first and then CFT for clinically negative cases.

Controls have been carried out in non-trachomatous populations to prove the specificity of the test, and evalua­tion of the results in mass examinations have been commented upon.


    Figures

  [Figure - 1]
 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7], [Table - 8]



 

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