Treatment of hydatid cysts (Echinococcus granulosus) by Cetrimide (R)

447 TRANSACTIONS OF TH!Z ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 75, No. 3, 1981

Treatment of hydatid’ cysts (Echinococcus

granulosus)

by Cetrimide

(R)

GEORGE J. FRAYHA~, KAMAL J. BIKHAZI~ AND TALAL A. KACHACHP IDept. of Microbiology, School of Medicine, American University of Beirut, Beirut, Lebanon 2Dept. of Surgery, School of Medicine, American University of Beirut, Beirut, Lebanon 3Service d’Anesth&iologie, Hbpital de I’Hotel Dieu de France, Beirut, Lebanon

Summary Cetrimide (a) has been efficiently assessed against hydatid protoscolices as a scolicidal adjunct to hydatid surgery. It was tested in four concentrations (0.05. O-1. 0.5 and 1%) against the orotoscolices of hydatid-cysts in sheip and cattle >ndin 0.1% concentration against the protoscolices of hydatid cysts in humans. For each concentration, three incubations of one, five and 10 min exposure were tested in vitro and in vivo on sheep and cattle cysts, and two successive incubations each of five min exposure on human cysts. All experiments resulted in rapid mortality of the protoscolices, as judged by their loss of motility, readiness to take vital stains and inability to develop into secondary hydatid cysts when inoculated into albino mice. The low toxicity of Cetrimide (a), its rapid action against the protoscolices, its scolicidal effect at a low concentration, and its low degree of absorption by the host tissues make it the most promising drug in hydatid surgery, displaying no side effects on the host. Introduction Cetrimide (a) or cetyl-trimethyl-ammonium bromide and other chemicals have been evaluated in vitro for their scolicidal activities as adjuncts to hvdatid surgerv (MEYMARIAN et al., 1963; FRAYHA, 1970, 1971;1975). Many Lebanese surgeons have used Cetrimide (a) since 1970 and nrefer it to other chemicals in hydatid surgery because of its high scolicidal action, low toxicity, low absorption and lack of side effects. These qualities make this compound unique among all drugs and chemicals so far tested, including the most recent systemic compounds such as mebendazole (AFRICAN MEDICAL & RESEARCH FOUNDATION. 1978: HEATH & LAWRENCE,1978; KERN et al., 1979). &formation on the systematic in vivo application of Cetrimide (a) to animals and human hydatid cysts is fragmentary (ESLAMI et al., 1978; AHRARI, 1978; AFRICAN MEDICAL & RESEARCH FOUNDATION, 1979), and its use in surgery is becoming a common practice in midde-eastern (ESLAMI et a{., 1978; AHRARI, 1978) and, probably, other countries of the world (AFRICAN MEDXAL & RESEARCH FOUNDATION, 1979). It was, therefore, deemed necessary to conduct more comprehensive research leading to its proper aoolication and usage. AThis work reports a systematic study of the in vitro and in vivo evaluation of Cetrimide (a) in the treatment of hydatid cysts of animals and man.

Materials

and Methods

In vitro and in vivo application of Cetrimide (R) to animal hydatid cysts Fertile hydatid cysts of Echinococcus granulosus were obtained from the lungs and livers of cattle and sheep slaughtered a few hours previously in local abattoirs. The protoscolices of some of the cysts were removed and prepared according to the techniques of MEYMARIAN et al. (1963) for in vitro scolicidal evaluation of Cetrimide (a). The protoscolices were collected and washed three times in physiological saline by suspension and sedimentation. Two ml of each of the four Cetrimide (a) testing solutions (O-05%, O-l%, 0.5% and 1%) were temperature -equilibrated in a test-tube in a 37°C water-bath. One drOD of orotoscolex suspension containing about 500 p;otoscolices was added to each Cetrimide (a) solution and thoroughly Control samples contained physiological mixed. salines only. After 10, 5 and 1 min incubation, the protoscolices were washed three times in physiblogical saline by suspension and sedimentation and keot at 37°C for about 30 min. One or two drops of each suspension were placed on a glass slide and the protoscolices examined microscopically for motility. Similarly, one or two drops of each suspension were placed on a glass slide and a 0.1 y0 solution of either eosin or janus green was added to Only dead protoscolices were darkly the slide. The remaining protoscolices from each stained. Cetrimide (a) solution were divided into batches of 500 protoscolices each and then inoculated i.p. into at least five albino mice aged 25-30 days (NAMRU-3 strain, Cairo, Egypt). The mice were killed after six to seven months and their peritoneal cavities and organs examined for hydatid cysts. The remaining fertile hydatid cysts were prepared for in vivo
448

TREATMENT

OF HYDATID

CYSTS

BY

CETRIMIDER

treated with physiological saline. The cysts in the beakers were placed in a shaking water bath at 37°C and each group, including the controls, was incubated for three exposure times, namely one, 5 and 10 min. At the end of each exposure time the cysts of each group were removed from beakers and transferred to conical flasks. Their protoscolices were collected, washed three times in physiological saline and samples examined for viability using the motility and staining techniques as described above. About 500 protoscolices from each treated cyst group were inoculated i.p. into immature albino mice as described above for further confirmation of death of the protoscolices. Similarly, about 500 protoscolices from control cysts were inoculated into albino mice.

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Cetrirnide (R) application in human hydatid surgery

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Before the removal of the hydatid cyst, abdominal pads soaked in 0.1 y0 Cetrimide (n) were placed in the cyst region. In addition, whenever possible the cyst was covered with a small pool of 0.1% Cetrimide (a). The cyst was then punctured with a large needle, GE, and its contents emptied away from the operating field by suction through one limb of a 3-way stopcock system. A 0.1 y0 Cetrimide (a) solution was injected by a syringe through the other limb of a 3-way stopcock into the cyst via the same inserted needle. In this system, the closure of one of the three limbs allow the other two to connect, thus displaying the advantage of (i) avoiding the use of many instruments during surgery, and (ii) decreasing the chances of spilling hydatid fluid into the operating field. The volume of Cetrimide (a) injected should be a little less than the volume of hydatid fluid aspirated in order to prevent any seepageof the cyst contents. Cetrimide (R) was kept in the cyst for five min, then withdrawn through the three-way stopcock and a fresh volume of 0 - 1% Cetrimide (a) solution injected into the same cyst. After another five min exposure the cyst contents were again withdrawn. In some instances, samples from the first untreated batch, as well as from the second and third treated batches of protoscolices, were examined immediately in the operating room to test their viability using the motility and staining techniques. The rest of the untreated and treated protoscolices of each batch were pooled separately in three conical flasks, washed three times with physiological saline and preserved at 4% for later examination. The treated protoscolices were inoculated into albino mice to confirm their death (see above), while the untreated protoscolices were used for in vitro testing of Cetrimide (a) as described above. The surgical area was repadded with fresh abdominal pads soaked in O+l% Cetrimide (a). The cyst was then opened, usually with a cautery, and carefully removed from the organ. Where multiple daughter cysts were found inside a mother cyst, these were ruptured inside the mother cyst, thus emptying their contents into the Cetrimide (a) which already filled the mother-cyst. Following cyst removal (endocyst plus ectocyst), the pericyst was then rubbed with a sponge stick soaked in Cetrimide (a). If any debris of germinal (endocyst) or laminated (ectocyst) membranes remained inside the pericyst, this was peeled off

G. J. FRAYHA et al.

and treated within the pericyst with 0.1 y0 Cetrimide (a) before removal. The pericyst was then filled with physiological saline and closed completely. The region from which the cyst had been removed (peritoneal, thoracic or other cavities), was then thoroughly irrigated with 0.1% Cetrimide (a) for 5 to 10 min. Cetrimide (n) solutions were used fresh and were prepared and assayed in the American University I-Iospital Pharmacy, Beirut, Lebanon. The LD, of Cetrimide (R) was determined on adult albino mice, average 26 gm weight over 96 hours, according to the method of REED & MUENCH (1938). .

I

Results The in vitro and in viva results of all incubations at 10, 5 and 1 min exposure times with the four concentrations of Cetrimide (a) (1, 0.5, O-1 and O*OS%), showed all the protoscolices in cattle and sheep hydatid cysts (Table I) to be completely dead, as examined by the motility and staining techniques and confirmed by their inability to form secondary hvdatid cysts in mice. When the in vitro Cetrimide (i) treated protoscolices were washed with physiological saline and incubated in it for 30 min at 37°C they showed no sign of activity or motility. In addition, they rapidly took up the vital stain which is another indication of their death (MEYMARIAN et al., 1963). Moreover, they did not develop into secondary hydatid cysts six to seven months after being inoculated i.p. into albino mice. In some cases, the protoscolices treated with the most dilute Cetrimide (n) solution (O-05%), did not display an unequivocal mortality at 1 min exposure time when tested by motility and staining techniques. However, this uncertainty was eliminated and death confirmed when the same protoscolices failed to develop into secondary hydatid cysts in mice (see Table I). In contrast, the non-treated control protoscolices, i.e. those incubated in physiological saline, showed obvious motility, did not stain with the vital stain and grew in albino mice into secondary hydatid cysts or masses varying in size between 3 and 12 mm diameter. The sheep and cattle hydatid cysts which were treated in vivo in livers and lungs were selected on size, because those between 3 and 5 cm in diameter had been shown to be the most fertile. Protoscolices treated in vivo were all dead. Their mortality was again detected by loss of motility, readiness to take up vital stain and inability to develop into secondary hydatid cysts after six to seven months in albino mice (Table I). On the contrary, the control cysts which were treated with physiological saline gave viable protoscolices as judged by the same criteria. In human hydatidosis, Cetrimide (n) treatment gave good results. Out of 175 patients surgically treated during the last 10 years at the American University Hospital, 142 patients at the Hopital de 1’Hotel Dieu de France in Beirut, and 61 patients at the Hopital de Bhannes in Metn, none returned for hydatid recurrence, whereas the pre-Cetrimide (a) recurrence rate was significant amounting to 10% of the surgical cases per year. In addition, all samples of protoscohces that were collected from Cetrimide (a) treated cysts and examined in the

449

operating rooms were judged dead because they lost their motility and were easily stained by vital.-_ stains. They were also unable to produce secondary hvdatid infections when inoculated in. into albino nice (Table I) The untreated protoscolices that were collected from human hydatid cysts before Cetrimide (n) application, also displayed the same pattern of results when treated in vitro with 0.1% Cetrimide (n) solution, i.e. rapid death as judged by motility, staining and mouse inoculation techniques (Table I). Discussion Hydatid disease is a public health problem in Lebanon (FRAYHA, 1970). Before 1970, it was common pratice for the surgeon, during removal of the cyst, to aspirate a certain volume of the cyst fluid and replace it with formalin which at that time was presumably considered the best scolicidal chemical. However, long experience with formalin as an adjunct to hydatid surgery gave some undesirable results. Some cases died from an overdose of chemical and in others, when the dose was ineffectively low, the disease recurred. Attempts to find more efficient chemicals (MEYMARIAN et al., 1963; FRAYHA et al., 1971) resulted in the discovery of about 13 promising drugs and chemicals including Cetrimide (a). These were selected on the criteria of being scolicidal during a short exposure time and at a low and stable concentration and exhibiting low toxicity to the host. Cetrimide (n) has since been used as the drug of choice in Lebanon and, more recently, in Iran (ISLAMI et al., 1978; AHRARI, 1978) although no systematic study had been made of its proper application and use. The four concentrations of Cetrimide (R) used in our in vitro and animal in vivo experiments were arbitrarily selected. However, two of these namely 0.1 andOm5%,areactually in use in human hydatid surgery in .Lebanon and Iran respectively (for Iran see ESLAMI et al. 1978;

AHRARI,

1978).

The efficacy of Cetrimide (R) as assessed in this study is probably due to its high scolicidal effect as compared with formalin and other chemicals tested against hydatid cysts. This implied a low degree of toxicity to the host, as measured by its LD,, (76mg/kg body-weight/96 hours), and the short time needed to kill the parasite at high dilution. Its mode of action is probably due to its affinity for the thiol group, which enables it to alter structurally the essential thiol-containing compounds of the protoscolex or germinal membrane of the cyst, such as dehydrogenases, co-enzyme A, lipoic acid, etc. The mechanism would be presumably accomplished by the easy oxidation of the thiol groups. Cetrimide (R) may also act as an electrophylic compound or redox inhibitor interfering with oxidative phosphorylation and/or the electron transfer system, hence culminating in impaired cellular respiration (FRAYHA, 1970; FRAYHA et aE., 1971), thus probably blocking essential metabolic processes, some of which have been already demonstrated in E. granulosus protoscolices (FRAYHA, 1968, 1971, 1974). Apparently the low degree of absorption of Cetrimide (a) by the host tissues prevented the occurrence of such drastic reactions in the host cells and permitted us to introduce it safely into delicate organs and

450

TREATMENT

OF

HYDATID

tissues of the peritoneal cavity, into the bile and hepatic ducts and into thoracic and other infected regions. Consequently, and during the last 10 years of using Cetrimide (n) as adjunct to hydatid surgery, no recurrence of the disease had been observed in our patients and no side effects have su far been detected. Acknowledgements

This study was financed by the National Council for Scientific Research in Lebanon, Grant No. 385704. The authors record their esteem and gratitude to the late Dr. Joseph Naffah, the former Secretary General of the Council, for his help and encouragement. They are also grateful to Drs. Il. Saadeh and A. Ghossein, Professors of Surgery, for supplying human hydatid records from Hopital de l’Hote1 Dieu de France in Beirut and H6pital Bharmes in Metn. The final copy was prepared during the sabbatical of George J. Frayha as a Wellcome Trust Research Fellow (Reg. 9410/1.4) at the Department of Zoology and Applied Entomology, Imperial College of Science and Technology, London. References

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the Period

African Medical & Research Foundation. (1979). Studies on the epidemiology and treatment of hydatid disease in the Turkana District of Kenya. Third Annual Progress Report for the Period Ju& 1978-June 1979, pp. 1-5 with Appendices

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de Pathologic Eiotique,

71, 90-94.

Eslami, A., Ahrari, H. & Saadatzedeh, H. (1978). Scolicidal effects of Cetrimide (R) on hydatid cyst

CYSTS

BY

CETRIMIDER

Frayha, G. J. (1968). A study on the synthesis and absorption of cholesterol in hydatid cysts (Echinococcus granulosus). Comparative Physiology, 27, 875-878.

Biochemistry

Frayha, G. J. (1970). Studies on hydatid disease in Lebanon. Lebanese Medical Journal, 23, 135-150. Frayha, G. J. (1971). Comparative metabolism of acetate in the taeniid tapeworms Echinococcus granulosus, E. multilocularis and Taenia hydatigena. Comparative Biochemistry and Physiology, 39B, 167-170. Frayha, G. J. (1972). The mode of action of certain drugs against helminth parasites of man. Lebanese Medical Journal,

25, 507-520.

Frayha, G. J. (1974). Synthesis of certain cholesterol precursors by hydatid protoscolices of Echinococcus granulosus and cysticerci of Taenia hydatiygn;. gCon$zrative Biochemistry and Physiology, Frayh;, G.-J.,‘Saheb, S. E. & Dajani, R. M. (1971). Systematic search for a systemic hydatid scolicide. I. In vitro screening of chemicals against the scolices of hydatid cysts (Echinococcus granulosus). Chemotherapy, 16, 371-379. Heath, D. D. & Lawrence, S. B. (1978). The effect of Mebendazole and Praziquantel on the cysts of Echinococcus gramdosus, Taenia hydatigena and T. ovis in sheen New Zealand Veterinary_- ‘fournal ‘J 26, 11-15. * Kern, P., Dietrich, M. & Volkmar, K. J. (1979). Chemotherapy of Echinococcosis with Mebendazole. Clinical observations of 7 patients. Tropenmedizin

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Meymarian, E., Luttermoser, G. W., Frayha, G. J., Schwabe, C. W. & Prescott, B. (1963). Hostparasite relationships in echinococcosis : X. Laboratory evaluation of chemical scolicides as adjuncts to hydatid surgery. Annals of Surgery, 158, 211-215. Reed, L. J. & Muench, H. (1938). A simple method of estimating 50 per cent endpoints. American Journal of Hygiene, 27, 493-497.

(Echinococcus granulosus). Transactions of the Royal Society of Tropical Medicine and Hygiene,

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and

Accepted for publication

1st September, 1980.