|Year : 1958 | Volume
| Issue : 2 | Page : 21-29
The serological diagnosis of trachoma and its application to the mass examination of trachoma
Hiromu Ueno, Tadao Kashii, Eiji Shimizu
Kyoto Prefectural Medical University, Kyoto, Japan
|Date of Web Publication||8-May-2008|
Kyoto Prefectural Medical University, Kyoto
Source of Support: None, Conflict of Interest: None
|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
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|| |
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 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 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|| |
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|| |
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|| |
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.
[Figure - 1]
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7], [Table - 8]