Methyl green-pyronin stain for the diagnosis of trachoma.

The clinical diagnosis of TRIC-agent infection of the conjunctiva is not difficult for ophthalmologists in endemic areas such as Egypt, but the similarity of the disease to other types of follicular conjunctivitis makes laboratory investigations especially important (Thygeson, I960; Duke-Elder, I965). These include the staining of conjunctival scrapings with Lugol's iodine or Giemsa stain for demonstration of the inclusion bodies of Halberstaedter and von Prowazek (1907) in the cytoplasm of infected cells (Rice, 1936; Gilkes, Smith, and Sowa, I 958; Dunlop, Jones, and Al-Hussaini, I 964; Al Hussaini, Jones and Dunlop, I 964) , and the use of the fluorescent antibody technique has recently attracted interest (Nichols, Bobb, Haddad, and McComb, I967; Surman, Hardy, and Howarth, I967). Isolating the TRIC-agent or finding the inclusions are the most conclusive means of diagnosis; complement-fixation tests give variable results (Barwell, Dunlop, and Race, I967), and intradermal tests are usually negative (Barwell and others, 1967). All these methods require an experienced investigator familiar with virological methods and very well equipped laboratories, which are not always available in developing countries. The demonstration of the inclusions by Giemsa stain is time-consuming and does not differentiate the colours. To isolate the TRIC-agent two or three egg passages are sometimes required, and this may take about a month. Moreover, Giemsa-stained smears from infected yolk sacs sometimes present artefacts of fine and coarse deposits which make it difficult to read the slides. It is obvious, therefore, that easier and quicker methods of laboratory diagnosis are needed. Histochemical stains have been successfully used to detect viral particles in other conditions, such as rabies (Shokeir and Elbagoury, I 966). Pollard and Tanami (i 962) have used acridine orange to study the replication of TRIC-agent in tissue cultures. Since TRIC-agent contains both desoxyribonucleic acid and ribonucleic acid (Jawetz, i964), infected cells containing inclusion bodies at any stage of development may be expected to react with stains specific for nucleic acids. For this reason we selected methyl green-pyronin stain as described by Brachet (1942, I953) and Gurr (I958) to demonstrate TRIC-agent in conjunctival scrapings and yolk-sac smears.