DIFLUOROMETHYLORNITHINE FOR ARSENO-RESISTANT TRYPANOSOMA BRUCEI GAMBIENSE SLEEPING SICKNESS

1431

placed in EDTA bottles labelled fetal (F) or maternal (M) and stored at 2°C until tested. All samples were tested within 24 h by one of us (K. P. J.). The same samples were then randomised and numbered sequentially such that the F and M indicators could not be seen. The samples were then tested in batches of 10-20 by A. W. W. and by another colleague within 48 h of collection. Each sample was therefore tested once in the usual way and twice by a were

single-blind

random method. Thus

fifty pairs

of

samples

were

.7 obtained and tested with a simple modification of the Apt test Nine drops of a 1 % aqueous solution (0 1 mol/1) NaOH were placed in each of two small tubes. One drop of blood was added and the tubes were shaken briefly, the colour being noted after 1 min. Fetal red cells are resistant to alkali and therefore the mixture remains red, whereas maternal (adult) red cells are denatured by the alkali and the mixture turns brown/green. The colour change should be observed within 2 min since the reaction is not stable after a few minutes.

Results

Of the 100 samples tested all were correctly identified as maternal or fetal and there were no equivocal results. The test is therefore 100% sensitive and 100% specific. Maternal samples were contaminated with liquor and vemix but the contamination did not affect the results since tests of a few samples of clear liquor did not show any colour change. Furthermore, the test is not affected if the vaginal blood is partly clotted since enough red blood cells remain in suspension for a correct result. 50/50 (volume/volume) mixtures of maternal and fetal blood were tested and produced a red colour change which is easily distinguished from "pure" maternal blood.

Preliminary Communication DIFLUOROMETHYLORNITHINE FOR ARSENO-RESISTANT TRYPANOSOMA BRUCEI GAMBIENSE SLEEPING SICKNESS

J. PEPIN1,4 C. GUERN1,2

F. MILORD1,2

P. J. SCHECHTER3

University of Sherbrooke, Sherbrooke, Canada;1 Zone de Santé Rurale, Nioki, Zaire;2 Merrell Dow Research Institute, Strasbourg, France;3 and University of Manitoba, Winnipeg, Canada4 with arseno-resistant brucei gambiense trypanosomiasis were treated with difluoromethylornithine (eflornithine), an inhibitor of ornithine decarboxylase, given intravenously, then orally. There was rapid disappearance of trypanosomes in the cerebrospinal fluid (CSF), gradual decrease of CSF lymphocytosis, and parallel improvement in central nervous system status. Side-effects, including diarrhoea, anaemia, and hair loss, were common but tolerable and reversible. 5 patients died during or shortly after treatment. None of the 21 patients who completed therapy has had a relapse during the 6-30 month follow-up.

Summary

26

patients Trypanosoma

INTRODUCTION

DESPITE considerable efforts at active screening and early by mobile teams, the incidence of Trypanosoma bruceigambiense trypmosomiasis (sleeping sickness) in West and Central Africa has increased in the past few years,1 with over 10 500 cases being diagnosed in Zaire in 1986. The case-fatality rate of untreated trypanosomiasis is 100%. The treatment of trypanosomiasis has remained unchanged for 40 years-suramin and pentamidine when cerebrospinal fluid (CSF) is normal and melarsoprol when the central nervous system is affected.1.5 5-8% of melarsoprol-treated patients die of a poorly understood but probably autoimmune encephalopathy.6 Moreover, in approximately 5% of patients relapses occur after melarsoprol therapy;7B subsequent courses of melarsoprol 9 are ineffective and death supervenes.9 rz-Difluoromethylornithine (eflornithine; DFMO) is a specific and irreversible inhibitor ofomithine decarboxylase and thereby blocks polyamine biosynthesis.1O Polyamines play a prominent part in the cellular metabolism of trypanosomes."DFMO was effective against Tb brucel* ...... T b gambiense, 12 and T b rhodesiense14 infections in laboratory animals. In a limited trial in man DFMO given orally was effective against Tb gambiense trypanosomiasis in the Sudan.1s However, 4 of 15 patients with late-stage treatment

Discussion

Rhesus-negative women are routinely tested for fetal haemoglobin after delivery with commercial kits based on the method described by Kleihauer et al.8 However, when fetal haemorrhage occurs on the labour ward, the time the test takes (at least 30 min) is too long for there to be much chance of saving the baby. Torrey1 quotes Williamson (1912) who recommended a routine blood smear to detect fetal blood elements, but the best hope lies in biochemical means. The alkali denaturation test for fetal haemoglobin was first described by Apt and Downey7 with simplified techniques offered subsequently by Gelsthorpe,9 Israel6 and Ogita et al. 10 The simplicity of our test makes it attractive for use on the labour ward, and we have shown that it is rapid and reliable. At present the test does not work in cases where there is heavy contamination with maternal blood as in a "show", when only the Kleihauer test is diagnostic, but we are working on a quantitative colorimetric assessment of the colour change which may make it usable in these circumstances. We recommend that this test should be part of the routine management of antepartum haemorrhage and that the detection of fetal haemoglobin in the vagina should be an indication for immediate caesarean section with the same priority as cord prolapse, unless the fetal heart is already inaudible. Furthermore, a- paediatrician equipped with umbilical catheter and group 0 rhesus negative blood should be standing by to transfuse the infant directly after delivery. Thus we hope that the high infant mortality of fetal haemorrhage from vasa praevia may be reduced. REFERENCES 1. 2

Torrey WE Jr. Vasa praevia Am J Obstet Gynecol 1952; 63: 146-52 Kouyoumdjian A. Velamentous insertion of the umbilical cord. Obstet Gynecol 1980; 56: 737-41.

3. 4

Vago T, Caspi E Antepartum bleeding due to injury of velamentous placental vessels Obstet Gynecol 1962; 20: 671-74. Curl CW, Johnson WL. Vasa praevia, antepartum diagnosis Obstet Gynecol 1968; 31:

328-30. 5. Barham KA. The

diagnosis of a vas praevium by amnioscopy Med J Aust 1968; 2: 398-400. 6. Israel R. Vasa Previa in binovular twins. Obstet Gynecol 1961, 17: 691-94 7. Apt L, Downey WS Melaena neonatorum The swallowed blood syndrome. J Paediatr 1955; 47: 6-12. 8 Kleihauer E, Braun H, Bethe K Demonstration von Fetalem Haemoglobin in den Erythrocyten eines Blutausstrichs Klin Wochenschr 1957, 35: 637-38. 9 Gelsthorpe K. A rapid method for differentiating fetal from maternal blood. Vox Sang 10.

1960; 5: 172-75. Ogita S, Ishiko O, Matsumoto M, Hasegawa H, Sugawa T. A simplified method of measuring fetal haemoglobin Obstet Gynecol 1976, 48: 237-39

-

1432 PAT rENT CHARACTERISTICS

CSF

lymphocytes

count

before, during, and after DFMO

treatment.

Means

=

height of box; SD = horizontal bar.

diarrhoea and then resumed at the same dose. Initially, the goal was to give DFMO orally for 30 days. In 2 patients, oral DFMO was discontinued after 22 and 24 days because of severe anaemia. For the last 11 patients the oral course was reduced to 21 days. All patients were reviewed daily by a doctor while in hospital. The CSF was examined for trypanosomes and white blood cell (WBC) count and the peripheral blood for haemoglobin (Hb) concentration and WBC count before start of DFMO, weekly during treatment, and 1, 3, 6, 12, 18, 24, 30, and 36 months after the end of the treatment. RESULTS

trypanosomiasis relapsed shortly after completing a course of oral DFMO. Intravenous administration of DFMO approximately doubles oral bioavailability16 without increasing gastrointestinal toxicity. Two small studies (4 patients,17 and 14 patients18) of a combination of intravenous and oral DFMO have yielded encouraging results. We have assessed, in an open clinical trial, the efficacy and toxicity of combined intravenous and oral DFMO therapy in 26 patients with late-stage T b gambÙnse trypanosomiasis who had relapsed after arsenical therapy. Because of very limited laboratory facilities and because transportation of specimens to reference laboratories was unreliable only clinically obvious toxicity was assessed. METHODS

The study was conducted at Nioki Hospital (Zaire), a 110-bed rural hospital 500 km northeast of Kinshasa, between September, 1984, and January, 1987. 410 patients with trypanosomiasis were treated there in 1984, 252 in 1985, and 288 in 1986. The hospital laboratory does common parasitological tests including examinations for trypanosomes in lymph node aspirates, CSF, fresh blood, and Giemsa-stained thick smears. A card agglutination test (’Testryp’, Smith Kline RIT, Rixensart, Belgium) and Woo’s test are available for confirmation of doubtful cases. The subjects (see accompanying table) were inpatients. All had had a relapse after treatment with one or more courses of either melarsoprol or trimelarsan according to a modified Neujean schedule.19 A relapse was defined as the presence of trypanosomes in the CSF and/or a CSF lymphocytosis of more than 40/1 and it was usually diagnosed at follow-up, done every 6 months for 3 years after arsenical treatment.

DL-K-dinuoromethylomithine hydrochloride monohydrate, mg/kg every 6 h (400 mg/kg/day), was infused intravenously over 1 h, for 14 days in 23 patients, 17 and 19 days in 2 patients because of impaired consciousness, and only 10 days in 1 because of 100

behavioural abnormalities. Parenteral DFMO was followed by a course of oral therapy, 75 mg/kg every 6 h (300 mg/kg/day) provided in sachets whose contents were to be dissolved in water. When diarrhoea developed during oral therapy, DFMO was withheld until resolution of the severe

Efficacy 12 patients had trypanosomes in the CSF before DFMO, but none had trypanosomes in CSF examined during or after therapy. In every patient the first CSF sample obtained 1 week after start of DFMO showed a decrease in lymphocyte count (mean CSF lymphocyte count pre-DFMO 193/ul versus 56/pl after a week of DFMO) (figure). The count dropped further in subsequent samples, with a mean CSF count of 11/1 after 5 and 6 weeks of DFMO. Only 1 patient had more than 30

lymphocytes/ul

in the CSF

at

the end of

treatment.

The clinical response paralleled the disappearance of trypanosomes and the fall in CSF lymphocyte counts. Patients usually noted improvement in symptoms after the first 2 weeks of therapy and most of them were symptomless when discharged from the hospital. The 21 patients who survived the course of DFMO were followed-up for 6-30 months (mean 16). 17 patients have been followed up for 12 months or more and 5 for at least 24 months. No relapses occurred after DFMO therapy.

Toxicity 5 patients died during treatment. These patients were severely cachectic at the start of DFMO therapy. A 7-year-old girl (weight 10-5 kg) died of measles contracted in hospital. A 15-year-old boy, comatose on admission, had generalised seizures 5 h after the first dose of DFMO. The seizures were difficult to control despite aggressive treatment (phenytoin, phenobarbitone, diazepam, hydrocortisone). 6 weeks later (while on oral DFMO) he died during an attack of status epilepticus. 2 other patients (14 and 20 years old) initially improved with DFMO but died after a progressively deepening comatose state without seizures. A 53-year-old woman had severe anaemia (Hb 6 g/dl, initial Hb 12.5 g/dl) after 35 days of DFMO. She died at home a month after discharge, following a subacute deterioration of her level of consciousness. All these patients had nevertheless shown considerable improvement in CSF lymphocyte counts and trypanosomes had disappeared from the CSF of the 4 patients in whom they had been found

1433

of the 5 deaths 1 (of the boy with to DFMO, 1 (of the girl with related seizures) probably unrelated to DFMO, and the other 3 was measles) probably could have been related either to DFMO, the underlying disorder, or both. Another patient, a 53-year-old man, died of septic shock 9 months after completing DFMO treatment. 1 day before death the CSF contained no parasites and 1 lymphocyte/ /11; examination of peripheral blood was also negative for trypanosomes. Side-effects associated with DFMO were common, but usually tolerable. 13 (50%) patients had diarrhoea, which occurred only during oral therapy and lasted 1-4 days. Diarrhoea was controlled merely by withdrawing DFMO and usually did not recur on resumption of DFMO. Anaemia (defined as an Hb drop >3 g/dl) occurred in 9 (35%) patients. 2 (Hb 6 and 6-5 g/dl) required transfusions. In the remaining 7 patients spontaneous correction of the anaemia occurred gradually over a few months after treatment. In 7 (27%) patients leucopenia (WBC < 3000/[tl) developed without any obvious clinical consequence. 14 patients (54%) had hair loss affecting mainly the scalp. This occurred either at the end of treatment or soon after discharge and was reversible. Apart from the previously described patient, 1 other had seizures (on day 7), which were readily controlled and did not recur when DFMO was resumed. 2 patients had jaundice, the cause of which remained undefined; in both the jaundice disappeared when DFMO was stopped and did not recur on resumption of DFMO. 2 patients (53 and 60 years old) had some loss of hearing. before DFMO. Thus,

out

was

DISCUSSION

This study shows the remarkable efficacy of DFMO in patients with arseno-resistant late-stage trypanosomiasis. In every patient who completed the course of treatment trypanosomes disappeared rapidly from CSF, CSF lymphocyte count fell to or towards normal, and CNS status

improved progressively. Most patients who relapse after melarsoprol treatment do so within the first 12 months. The absence of relapses in our DFMO-treated patients is extremely encouraging, especially since 17 have been followed up for a year or more. Giving DFMO intravenously for 2 weeks before an oral course seems to result in a lower relapse rate than those16 reported for DFMO given only orally. However, there are serious logistic difficulties with intravenous infusions every 6 h for 14 days in short-staffed rural hospitals. The high mortality rate (5/26, 19%) was not unexpected in view of the patients’ extremely poor condition at start of therapy, and it is acceptable since arseno-resistant trypanosomiasis is otherwise uniformly fatal. Use of DFMO as a first-line treatment in patients newly diagnosed as having late-stage trypanosomiasis-ie, before the disease is too advanced and the patient’s overall condition too poor-would undoubtedly result in fewer deaths during treatment.

Side-effects were common but usually tolerable and reversible. Reducing the oral phase from 30 to 21 days may lower the frequency of side-effects but our study is too small for us to draw conclusions about this point. We believe that DFMO represents the only form of treatment for T bgambiense trypanosomiasis relapsing after a course of melarsoprol, and it is likely to be curative in these patients. A prospective randomised trial comparing DFMO (intravenously plus orally) against melarsoprol as a first-line

treatment

should

for late-stage Tb be conducted.

gambiense trypanosomiasis

now

This study was partly supported by the Canadian International Development Agency. We thank Dr Mpia and the nursing staff of Nioki Hospital for their collaboration; the Bureau Central de la Trypanosomiase (Kinshasa, Zaire) for its logistical help; Dr R. Brunham, Dr F. Plummer, Dr P. Piot, and Dr J. M. Pepin for critical review of the paper; and Mrs C. Reimer, Mrs G. Bilan, and Mrs C. Mamont for secretarial assistance.

Correspondence should be addressed to J. P., Infectious Diseases Section, University of Manitoba, Room 530-730, William Avenue, Winnipeg, Manitoba, Canada R3E OW3. REFERENCES 1 World Health

Organisation. Epidemiology and control of African trypanosomiasis. Geneva: Report of a WHO Expert Committee. WHO Tech Rep Ser 1986 (no 739). 2 Greenwood BM, Whittle HC. The pathogenesis of sleeping sickness Trans R Soc Trop Med Hyg 1980; 74: 716-25. 3. Ellis CJ. The trypanosomiases. Br Med J 1985; 291: 1524. 4. Van Miervenne N, LeRay D. Diagnosis of African and American trypanosomiasis Br Med Bull 1985, 41: 156-61 5. Dumas M, Breton JC, Pestre Alexandre M, Girard DL, Giordano C Etat actuel de la thérapeutique de la trypanosomiase humaine africaine. La Presse Médicale 1985, 14: 252-56. 6 Haller L, Adams H, Merou ze F, Dago A. Clinical and pathological aspects of human Afncan trypanosomiasis (T.B. gamiense) with particular reference to reactive arsenical encephalopathy. Am J Trop Med Hyg 1986; 35: 94-99. 7. Kazyumba GL L’endemie sommeilleuse en Republique du Zaire au cours des 25 dernières années (1952-1976) Med Afr Noire 1979; 26: 47-52. 8. Gmoux PY, Lancien P, Frezil JL, Bissadidi N. Les échecs du traitement de la trypanosomiase à T gambiense au Congo Med Trop 1984; 44: 149-54 9 Molyneux DH. African trypanosomiasis. Clin Trop Med Commun Dis 1986; 1: 535-55. 10 Sjoerdsma A, Schechter PJ. Chemotherapeutic implications of polyamine biosynthesis inhibition. Clin Pharm Ther 1984; 35: 287-300 11. Bacchi CJ, Nathan HC, Hutner SH, McCann PP, Sjoerdsma A Polyamine metabolism: A potential therapeutic target in trypanosomes. Science 1980; 210: 332-34. 12. McCann PP, Bacchi CJ, Clarkson AB Jr, et al Further studies on difluoromethylomithine in African trypanosomes. Med Biol 1981; 59: 434-40 13. Clarkson AB Jr, Bienen EJ, Bacchi CJ, et al. New drug combination for experimental late-stage African trypanosomiasis. DL-&agr;-difluoromethylomithine (DFMO) with suramin Am J Trop Med Hyg 1984; 33: 1073-77. 14. McCann PP, Bacchi CJ, Hanson WL, et al. Effect on parasitic protozoa of &agr;-difluoromethylomithine—an inhibitor of omithine decarboxylase In. Caldarera CM, Zappia V, Bachrach U, eds. Advances in polyamine research. New York Raven Press, 1981; 3: 97-110. 15. Van Nieuwenhove S, Schechter PJ, Declercq J, Boné G, Burke J, Sjoerdsma A. Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL-&agr;-difluoromethylomithine), an inhibitor of omithine decarboxylase; first field trial. Trans Roy S Trop Med Hyg 1985, 79: 692-98 16. Haegele KD, Alken RG, Grove J, Schechter PJ, Koch-Weser J. Kinetics of &agr;-difluoromethylomithine. An irreversible inhibitor of omithine decarboxylase. Clin Pharmacol Ther 1981; 30: 210-17 17. Taelman H, Schechter PJ, Marcelis L, et al Difluoromethylomithine, an effective new treatment of gambian trypanosomiasis. Am J Med 1987, 82: 607-14. 18. Doua F, Boa FY, Schechler PJ, et al. Treatment of human late stage gambiense trypanosomiasis with &agr;-difluoromethylomithine (eflornithine) Efficacy and tolerance in 14 cases of Cote d’Ivoire. Am J Trop Med Hyg (in press). 19. Neujean G Ghimiothérapie et chimoprophylaxie de la maladie du sommeil à Tb gambiense Revue Méd Liège 1959; 14: 5-13

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