The use of cycloheximide-treated cells for isolating trachoma agents under field conditions*

Bulletin of the World Health Organization, 56 (4): 629-632 (1978) The use of cycloheximide-treated cells for isolating trachoma agents under field conditions* J. SCHACHTER,1 C. R. DAWSON,2 I. HOSHIWARA,3 T. DAGHFOUS,4 & J. BANKS5 Standard procedures for the isolation of Chlamydia trachomatis require pretreatment of the tissue culture cells. We evaluated the use of cycloheximide, an antimetabolite that can be added to the cells with the inoculum. Cycloheximide-treated cells provided a sensitive system for isolating trachoma agents. This system was applicable to field studies as require- ments for equipment were minimal and the cells were 2 weeks old when inoculated. Chlamydia trachomatis is a significant pathogen of man, being the agent of trachoma, which is still a major cause of blindness in some parts of the world (6, 7). Chlamydiae are also recognized as common agents of sexually transmitted disease (6, 9). Much of the recent information on human chlamy- dial infections has been due to the development of a tissue culture technique for isolating these organ- isms (5). The commonly used isolation procedures require pretreatment of the host cells with radiation or an antimetabolite (2, 11). This pretreatment requirement poses potential difficulties to diagnostic studies and large-scale surveys. Cycloheximide suppresses the metabolism and reproduction of eukaryotic cells while not affecting prokaryotic cells (1), and this substance therefore provides an alternative method of treating cells for chlamydial isolation because it can be added to host cells after infection (8). * This work was supported in part by National Institutes of Health grants EY 00427 and CA 14427; by PL 83-480 grant 07-075 from the Health Services Administration, US Public Health Service, and by the World Health Organi- zation. 1 Co-Director, WHO Collaborating Centre for Reference and Research on Trachoma and Other Chlamydial Infections, and George W. Hooper Foundation, University of California, San Francisco, CA 94143, USA. ' Co-Director, WHO Collaborating Centre for Reference and Research on Trachoma and Other Chlamydial Infections, and Francis I. Proctor Foundation, University of California, San Francisco, CA 94143, USA. 3 Medical Director, Phoenix Indian Medical Center, Phoenix, AZ 85016, USA. 'Directeur, Institut d'Ophtalmologie, Tunis, Tunisia. 'George W. Hooper Foundation, University of California San Francisco, CA 94143, USA. In previous studies on trachoma in Tunisia, we used Giemsa- and immunofluorescent-stained con- junctival smears to determine the prevalence of chla- mydial infections (3, 4, 12). C. trachomatis was isolated from only 5% of specimens from selected patients with active trachoma, although up to 60% of matched conjunctival smears had inclusions. It appeared that technical problems were preventing chlamydial recovery in the cell culture systems. In previous studies, frozen clinical specimens were shipped in liquid nitrogen and took 3-5 weeks in transit from Tunisia to San Francisco. When Ripa & Mirdh (8) described the use of cycloheximide in the treatment of cells for C. trachomatis isolation, we decided to employ this technique in our studies. An attractive feature of this method is that the cells need no pretreatment. Because untreated cell cultures are stable for 2-3 weeks at moderate temperatures (20°C), it would be feasible to send normal cells from San Francisco to Tunisia and process the specimens there. MATERIALS AND METHODS The collection medium consisted of Eagle's mini- mal essential medium with 10% fetal calf serum and contained 1 ,g cycloheximide, 10 ,g gentamicin, 100 ,ug vancomycin, and 4 ,ug amphotericin B per ml. Approximately 75 000 McCoy cells were planted on 12-mm cover slips in 15-mm-diameter shell vials (10), sealed with silicone rubber stoppers, and incubated at 37°C for 48 h. The vials and other tissue culture supplies were then sent by air freight from San Francisco to Tunis in a suitcase measu'ring 3727 - 629 - 630 J. SCHACHTER ET AL. approximately 760 x 460 x 230 mm., Before inocu- lation, the cells were incubated in a standard bac- teriological incubator at 37°C for about 36 h. Clinical specimens were collected from the conjunc- tiva of the lower lid of patients with active trachoma by means of a wire stem calginate nasopharyngeal swab and placed into 3 ml of collection medium in 15-ml screw-cap tubes. The specimens were held slightly below ambient temperature for 10 h after collection and then refrigerated overnight (another 12 h) prior to inoculation into the cells. One ml of each specimen was placed on each of three cover slips and centrifuged for 1 h at 1850 g. The vials were then incubated at 37°C for 48 h without chang- ing the medium. The cell monolayers from one vial/specimen were then fixed with absolute metha- nol, stained with 5% iodine, and examined for the presence of inclusions. After 72 h of incubation, the second inoculated tissue culture vial was frozen in liquid nitrogen and the third was taken by hand back to San Francisco (about a further 40 h) at ambient temperature. The material was passed into new cyclohe;imide-treated cells immediately on ar- rival at the Collaborating Centre. A duplicate clinical specimen had been frozen in liquid nitrogen in the field and both the frozen cell passage material and duplicate clinical material were sent by air freight to San Francisco, arriving approximately 5 weeks after collection. These frozen specimens were thawed and spun into cycloheximide-treated cells using the same media, the only modification in the procedure being that they were centrifuged at 2700 g. For cytological studies, scrapings collected from the tarsal conjunctiva were spread on glass slides, air dried, and fixed in methanol. They were then stained by Giemsa's technique. RESULTS Of 31 specimens collected in the field and directly inoculated into the cells, 24 were examined at 48 h (7 tubes were lost owing to various handling mishaps). Twelve of these 24 (50%) were inclusion-positive. All 31 inoculated cell monolayers were carried back to San Francisco and 19 (62%) yielded iodine- staining inclusions on subsequent passage there. Two of the positive specimens had been inclusion- a Details of this portable tissue culture system are available on request from the WHO Collaborating Centre for Refer- ence and Research on Trachoma and Other Chlamydial Infections, University of California, San Francisco, CA 94143, USA. negative in the first passage. Of the 31 matched Giemsa-stained scrapings, 12 (39%) were inclusion- positive. The isolation attempts on all 12 of these patients were positive. Only 2 (6%) of the 31 frozen duplicate clinical specimens yielded isolates in San Francisco. The cell cultures that had been inoculated in Tunis and then frozen in liquid nitrogen were also thawed and passaged in San Francisco and 3 of the 31 (10%) yielded chlamydial isolates. DISCUSSION The conditions and logistic problems in this study were not unique. Relatively sophisticated laboratory procedures are often not available in a trachoma- endemic area. Indeed, the transportation problems, lack of dry ice, and the high ambient temperatures (these specimens were taken in the northern Sahara, where daytime temperatures are frequently above 30°C, even in November) are common in trachoma- endemic areas. Although our previous studies had employed a variety of different holding media for freezing specimens, none had successfully preserved the trachoma agents during transit. We do not know why specimens lose infectivity during their 3- to 5-week shipment period from North Africa to San Francisco, since specimens stored in liquid nitrogen in our laboratory lose very little infectivity during this time. However, the tissue culture pro- cedure we used in this study proved to be a sensitive method for isolating trachoma agents. It was approxi- mately 60% more sensitive than the Giemsa stain (62% positive versus 39% positive). We used the iodine stain to detect inclusions in the infected tissue culture monolayers after 48 h of incubation. If there is urgency in establishing a diag- nosis, or there are technical restrictions, the mono- layers can be incubated for 24 h and examined for inclusions by immunofluorescent techniques. Our goal was not early diagnosis but rather the establish- ment of isolates for further laboratory studies. The use of cycloheximide-treated cells for the isolation of trachoma agents requires only a centri- fuge and a bacteriological incubator as permanent laboratory equipment. We have often transported this equipment and an electrical generator to the sites of our field studies. The rest of the supplies needed for the isolation attempts are easily carried and may be disposable. The major advantage of cycloheximide is that it can be added with the inoculum, eliminating the need to pretreat cells with radiation or 5-iodo-2-deoxyuridine, the previously ISOLATING TRACHOMA AGENTS UNDER FIELD CONDITIONS 631 employed techniques (2, 11). Untreated, light mono- layers of McCoy cells maintain viability for several weeks at moderate temperatures. The cells used in this study had been planted 17 days before the time of infection; it is therefore possible to send material from a tissue culture laboratory for use in the field 2 weeks later. The 48 h in transit from Tunis to San Francisco resulted in no loss of isolates, although infectivity (titre) may have decreased. Thus, the use of McCoy cell monolayers prepared in the base laboratory, shipped with the materials necessary to process the clinical specimens and the subsequent cycloheximide cell treatment, provides a useful tech- nique for the isolation of trachoma agents in the field. RESUME EMPLOI DE CELLULES TRAITEES PAR LE CYCLOHEXIMIDE POUR ISOLER LES AGENTS DU TRACHOME DANS LES SPE'CIMENS RECUEILLIS SUR LE TERRAIN Dans les 6tudes men6es precedemment sur le trachome en Tunisie, onaeu recours aux m6thodes cytologiques pour d6terminer la prevalence des infections a Chlamydiae. Mais seules 5% des tentatives faites pour isoler Chlamy- dia trachomatis dans des prelevements conjonctivaux ont abouti, alors qu'un examen sur lame avait permis de deceler des inclusions dans 60% des echantillons appair6s. Les tentatives d'isolement avaient et6 effectuees sur des specimens cliniques congel6s dans l'azote liquide et expedies a San Francisco. La communication de Ripa & March au sujet de l'em- ploi du cycloheximide pour traiter les cellules en vue de l'isolement de C. trachomatis a determin6 les auteurs de la pr6sente etude a utiliser cette technique pour leurs travaux. Le cycloheximide stoppe le m6tabolisme et la ,reproduction des cellules eucaryotes sans affecter les cellules acaryotes, et l'avantage de cette substance est de pouvoir etre ajoutee au milieu de culture tissulaire apres l'inoculation. Ce sont donc des couches mono- cellulaires non traitees de cellules McCoy (dont la viabilit6 est toujours d'au moins 2 semaines) qui ont ete expedi6es de San Francisco en Tunisie. La collecte de materiel conjonctival a e faite chez des enfants atteints de trachome. Les echantillons ont ete places dans un milieu appropri6 et centrifuges dans les 24 heures sur des couches monocellulaires de cellules McCoy. Apres une incubation a 37°C pendant 48 heures sans changer le milieu, les cellules avec l'inoculum ont 6t6 transport6es directement a San Francisco (comme bagage & main). L'examen des cellules, trait6es par le cycloheximide pour un second passage des I'arrivee a San Francisco, a permis d'isoler des Chlamydiae sur 19 des 31 echantillons (soit 62 %). On avait deceI6 des inclusions dans 12 (39%) des prelevements conjonctivaux appaires colores au Giemsa, et les tentatives d'isolement int6ressant ces 12 sujets ont toutes ete positives. En revanche, les difficultes rencontrees prec6demment avec les echantillons expedies dans l'azote liquide se sont repetees, puisque seuls 2 des 31 sp&cimens cliniques correspondant aux inoculums (soit 6%) qui ont ete congel6s pour leur expedition ont permis d'isoler des Chlamydiae. Les cellules traitees par le cycloheximide constituent par consequent un systeme favorable a l'isolement de l'agent du trachome, et leur emploi ne necessite qu'un incubateur et une centrifugeuse comme dquipement de laboratoire permanent. Quant aux couches monocellu- laires de cellules McCoy initiales, celles-ci peuvent etre envoy6es sur le terrain par le laboratoire de base, puisque leur viabilite est de plusieurs semaines & tempe- rature mod6ree. REFERENCES 1. ALEXANDER, J. J. 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