One group of BALB/c mice infected with a highly virulent strain of Trypanosoma brucei gambiense were treated intraperitoneally with three series of three injections (each injection of 10 mg/kg) of Mel-B separated by seven days of rest, while a second group was treated once by a single injection. All the Mel-B treated mice in both experiments were negative for parasites when examined using either the wet blood film or buffy coat methods, but were intermittently PCR positive during the sampling period. We encourage the use of a repeat negative PCR test over a one month period in combination with corroborative clinical and parasitological investigation to be suggestive of cure in experimental animals previously infected with trypanosomosis. In view of the exorbitant costs of Mel-B and its extreme toxicity, it is recommended that Mel-B be given as one course of two injections (each equivalent to 10 mg/kg) separated by 2 d of rest in experimentally infected rodent models.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Japan International Cooporation Agency (JICA) through the Ministry of Research.
Technical Training of the Government of Kenya.mn201

Hirumi, H.

Hirumi, K.

Inoue, N.

Mbati, P.A.

Situakibanza, N.H.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.One group of BALB/c mice infected with a highly virulent strain of Trypanosoma brucei gambiense were treated intraperitoneally with three series of three injections (each injection of 10 mg/kg) of Mel-B separated by seven days of rest, while a second group was treated once by a single injection. All the Mel-B treated mice in both experiments were negative for parasites when examined using either the wet blood film or buffy coat methods, but were intermittently PCR positive during the sampling period. We encourage the use of a repeat negative PCR test over a one month period in combination with corroborative clinical and parasitological investigation to be suggestive of cure in experimental animals previously infected with trypanosomosis. In view of the exorbitant costs of Mel-B and its extreme toxicity, it is recommended that Mel-B be given as one course of two injections (each equivalent to 10 mg/kg) separated by 2 d of rest in experimentally infected rodent models.Japan International Cooporation Agency (JICA) through the Ministry of Research.
Technical Training of the Government of Kenya.mn201

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Onderstepoort Journal of Veterinary Research, 67:71-74 (2000) RESEARCH COMMUNICATION Suggested dosage rates of melarsoprol in the treatment of mice experimentally infected with Trypanosoma brucei gambiense p.A. MBATI1 *, K. HIRUMI2, N . INOUE2, N .H. SITUAKIBANZA3 and H. HIRUMI2 ABSTRACT MBATI, P.A., HIRUMI, K., INOUE, N., SITUAKIBANZA, N.H. & HIRUMI , H. 2000. Suggested dosage rates of melarsoprol in the treatment of mice experimentally infected with Trypanosoma brucei gam-biense. Onderstepoort Journal of Veterinary Research, 67:71-74 One group of BALB/c mice infected with a highly virulent strain of Trypanosoma brucei gambiense were treated intraperitoneally with three series of three injections (each injection of 10 mg/kg) of Mel-B separated by seven days of rest, while a second group was treated once by a single injection All the Mel-B treated mice in both experiments were negative for parasites when examined using either the wet blood film or buffy coat methods, but were intermittently PCR positive during the sampling period. We encourage the use of a repeat negative PCR test over a one month period in combination with corroborative clinical and parasitological investigation to be suggestive of cure in experimental animals previously infected with trypanosomosis. In view of the exorbitant costs of Mel-B and its ex-treme toxicity, we recommend that Mel-B be given as one course of two injections (each equivalent to 10 mg/kg) separated by 2 d of rest in experimentally infected rodent models. Keywords: Mel-B, mice, PCR, Trypanosoma brucei gambiense INTRODUCTION Only four drugs are currently in use for the treatment of human sleeping sickness caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhode-siense infections. Suramin and Pentamidine are used to treat the early stages of the disease, the former being used against T.b. rhodesiense and the latter against T.b. gambiense infections. Only two drugs are available to treat the late stage of the dis-ease when trypanosomes have invaded the central nervous system: melarsoprol (Mel-B) and DL-alpha-* Author to whom correspondence is to be directed 1 Qwa-Qwa Campus, University of the North, Parasitology Re-search Programme, Private Bag X13, Phuthaditjhaba, 9866, Republic of South Africa 2 The Research Centre for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Obi-hiro, Hokkaido 080, Japan 3 Cliniques Universitaires de Kinshasa, Medicine Interne, B.P. 123 Kinshasa XI , Democratic Republic of Congo Accepted for publication 6 December 1999-Editor difluoromethyornithine (DFMO). Melarsoprol is the drug of choice in the treatment of both T.b. gambien-se and T.b. rhodesiense infections. It is an arsenical compound, and is associated with severe side effects in 20% of treated patients (Pepin, Milord, Guerin, Mpia, Ethier & Mansinsa 1989). In addition, relapse cases have been reported following Mel-B treatment, though the reasons for these are unknown. Melarso-prol is relatively toxic and produces encephalopathic reactions which may be fatal (WHO, 1998). Never-theless, with attention to detail , these toxic effects can be minimized. The drug is issued in ampoules, ready for use, as a 3,6% solution in propylene gly-col. Melarsoprol must never be used prior to a pre-liminary course of suramin which is administered to clear the patient's blood of trypanosomes in order to avoid a Jarisch-Herxheimer reaction which occurs when large numbers of trypanosomes are killed (Bryceson 1976). The usual dose of melarsoprol re-commended for each administration is 3,6 mg/kg body weight, the maximum single dose being 5 mQ. One such administration is given on each of 3 or 4 consecutive days and, after a rest period of one 71 Melarsoprol in treatment of mice experimentally infected with Trypanosoma brucei gambiense week, the course is repeated. A third course of three or four injections may be given after a further rest period of 1 week. In th is study, the Mel-B dose re-quired to clear an infection in mice experimentally in-fected with a virulent strain of T.b. gambiense was examined, and a possible yardstick of assessment of cure is given. MATERIALS AND METHODS Two experiments were devised. In the first, the treat-ment schedule of mice experimentally infected with trypanosomes closely mimicked that given to hu-mans suffering from sleeping sickness (i.e . three series of three injections separated by 7 d of rest). In brief, ten 5-week-old male inbred BALB/c mice each were individually infected with 5 x 103 blood stream-forms of T.b. gambiense IL3707 intraperi-toneally (ip) in 0,5 m~ of PSG(+) (phosphate, sodium, glucose) buffer. This parasite is a highly virulent strain , as has been described by Inoue, Narumi, Mbati , Hirumi, Situakibanza & Hirumi (1998). The mice were then separated into two equal groups which were labeled as experimental and control groups, respectively. At 4 d post-infection (DPI) , the mice in the experimental group were started on a treatment regimen which consisted of Mel-B (ob-tained from WHO, Geneva) administered ip at the dosage rate of 10 mg/kg/day (Poltera, Hochmann & Lambert 1981) and comprised three series of three injections each (one injection per day on 3 consecu-tive days) separated by 7 d of rest. In the control group, PSG( +) buffer was administered using a simi-lar protocol. In the second experiment, ten 5-week-old male BALB/c mice were similarly infected and divided into two equal groups: experimental and con-trol. At 4 DPI , the mice in the experimental group were treated once with a single ip injection of Mel-B at the dose rate of 10 mg/kg. The mice in both ex-periments were examined periodically (see below) for the appearance of parasites in peripheral blood by wet blood film and buffy coat scrutiny, measure-ment of packed cell volume (PCV) , and PCR. Parasitological diagnosis The diagnostic examinations of the mice in the ex-perimental group in the first experiment were per-formed as follows: 4 DPI (just before the start of the first schedule of treatment) ; 7 DPI (one day after the first schedule of treatment) ; 14 DPI (just before the second schedule of treatment); 17 DPI (a day after the second schedule of treatment); 24 DPI (just be-fore the third and last schedule of treatment); and 27 DPI .(a day after the last schedule of treatment). Thereafter, the diagnostic examinations were con-ducted at random time intervals up to 104 DPI. The mice in the experimental group in the second experi-ment were observed at random time intervals for 72 evidence of infection 1 d after treatment, and for up to 61 DPI. Diagnostic examination of the control groups of mice in both experiments were performed at the same intervals as the experimental groups until they died. DNA extraction from small blood samples About 1 0 IJ ~ of blood from the tail of each mouse on each occasion that diagnostic tests were performed, was pi petted into 100 IJQ of lysis buffer containing a final concentration of 10 mM Tris HCI (pH 8,0) , 10 mM EDTA (pH 8,0),100 mM NaCL, 0,5% SOS and 300 IJg/mQ of proteinase K. After incubation for 2 h S at 55 °C (Eyla, DryThermobath MG-11 00) , 100 IJQ of a phenol :chloroform (1 : 1) solution was added and thoroughly mixed with a Vortex machine. The sam-ples were then centrifuged at 4 000 g for 5 min at room temperature and the supernatant (containing DNA) transferred to a new tube. This phenol :chloro-form extraction procedure was repeated twice. Ten IJQ of 3 M sodium acetate (pH 5,2) and 250 IJQ of 100% ethanol was added to the supernatant and frozen at -80°C for 30 min. These samples were subsequently thawed at room temperature. After centrifugation at 4 000 g for 15 min at 4 °C, the supernatant was re-moved, and 500 IJQ of 70% ethanol was added to the DNA pellet. The samples were again centrifuged at 4 000 g for 5 min at 4 °C and the supernatant re-moved. The DNA samples in the micro-tubes were then air-dried (Eyela, Centrifugal Vaporizer). Each DNA sample was dissolved in 10 IJQ of Tris-EDTA buffer and kept frozen at 30 °C until used. peR of VSG 117 genes DNA preparations were amplified in a final volume of 30 IJQ in a Perkin Elmer PCR machine (Gene Amp 2400). In preparation of the master mix, the follow-ing volumes were used: 20,4 IJQ DDW; 3 IJQ of 10 x PCR buffer (Perkin Elmer); 3 IJQ of 2 mM dNTPs (Perkin Elmer), 0,3 IJQ of VSG 117 primer (50 pmol/ IJQ each of sense and antisense) (Bromidge, Gibson, Hudson & Dukes 1993), and 0,3 IJQ Taq (Thermus aquaticus) DNA polymerase (Perkin Elmer). Three microlitres of DNA sample was used as template per reaction programme. In the PCR reaction pro-gramme, samples were incubated at 94 °C for 10 min in an initial denaturation step, followed by 40 cycles at 94 °C (30 s) , 51 °C (1 min) and 72 °C (2 min). The PCR reaction products were analysed by agarose gel electrophoresis and detected visually by UV transil-lumination (Atta, Bioinstrument) of the ethidium bro-mide-stained gel. RESULTS Summaries of results of the diagnostic tests of the mice in the experimental group in the first and sec-ond experiments are shown in Tables 1 and 2, P.A. MBATI et al. TABLE 1 Results obtained in Experiment 1 of various diagnostic tests used to detect Trypanosoma brucei gambiense (IL3707) in BALB/c treated with either Mel-B or PSG(+) Days post-infection Test Group 4 7 14 17 24 27 41 53 67 74 80 88 104 Tail blood PSG(+) 4/5a 1/1 b Mel-B 4/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Buffy coat PSG(+) 4/5 1/1 b Mel-B 5/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 PCR PSG(+) 4/5 1/1 b Mel-B 5/5 1/5 0/5 0/5 0/5 0/5 ND 2/5 3/5 1/5 3/5 0/5 ND a Results are shown as the proportion: Number of mice in which the presence of parasites was detected Number of mice tested All mice dead ND: Not done TABLE 2 Results obtained in Experiment 2 of various diagnostic tests used to detect Trypanosoma brucei gambiense (IL3707) in BALB/c treated with either Mel-B or PSG(+) Days post-infection Test Group 4 5 26 41 47 61 Tail blood PSG(+) 4/5a 5/5 b - - -Mel-B 5/5 0/5 0/5 0/5 0/5 0/5 Buffy coat PSG(+) 5/5 5/5 b - - -Mel-B 5/5 0/5 0/5 0/5 0/5 0/5 PCR PSG(+) 5/5 5/5 b - - -Mel-B 5/5 0/5 0/5 2/5 0/5 0/5 a Results are shown as the proportion: Number of mice in which the presence of parasites was detected Number of mice tested b All mice dead respectively. In both treatment schedules, the mice revealed no trace of parasites 24 h after treatment when examined by parasitological methods and PCR. Untreated mice from both experiments were dead by 10 DPI, all revealing heavy parasite bur-dens. Mice treated with either a single dose or three courses of Mel-B were negative for parasites when examined using both the wet blood film and buffy coat techniques upto the end of the experiment at 61 or 104 DPI respectively, but were intermittently PCR positive during the sampling period. No toxic effects resulting from the Mel-B therapy was observed in any of the treated mice which were physically healthy with normal PCV's of around 56 % from approximately the second week of treatment and throughout the rest of the experimental period. No relapses to the treat-ment were observed. DISCUSSION Trypanosoma b. gambiense IL3707 is a highly viru-lent parasite in BALB/c (Inoue et at. 1998). Most of control (PSG-treated) mice had died by about the 7th DPI. There was an excellent correlation between the PCR and parasitological methods of diagnosis at both 4 DPI and 5 DPI in the groups of experimental animals where all parasitologically positive mice were also PCR positive. The most significant observation 73 Melarsoprol in treatment of mice experimentally infected with Trypanosoma brucei gambiense is that at about 24 h after a single administration comprising one injection of Mel-B at a dosage rate of 10 mg/kg, all the mice were both parasitologically and PCR negative, demonstrating the efficacy of S Mel-B. Parasitological methods of diagnosis of whole blood and buffy coat of all the Mel-B treated mice were negative until the end of the observation peri-ods. Various techniques used in the diagnosis of trypano-somosis, such as the parasitological methods and PCV, and the highly sensitive and specific PCR which were used in this study are all useful tests. We con-sider that a repeated negative PCR test during a 1 month period, in combination with corroborative clini-cal and parasitological methods of diagnosis, is sug-gestive of cure. In view of the exorbitant costs of Mel-B and its extreme toxicity, we recommend that Mel-B, given as one course of two injections (each equiva-lent to 10 mg/kg) separated by 2 d of rest is adequate to cure rodents suffering from Tb. gambiense in labo-ratory experimental studies. ACKNOWLEDGEMENT We are grateful to the advice received from Prof. em. Naoyoshi Suzuki and Prof. Yutaka Toyoda, both of The Research Centre for Protozoan Molecular Im-munology, Obih iro, Japan . This study was possible 74 in part through a training fellowship awarded to Dr P.A. Mbati by the Japan International Coorporation Agency (JICA) through the Ministry of Research and Technical Training of the Government of Kenya. This paper is published with the approval of the Director, KEMRI. REFERENCES BRYCESON, AD.M. 1976. Clinical pathology of the Jarisch-Herx-heimer reaction. Journal of infectious Diseases, 133:696-704. BROMIDGE, T. , GIBSON, W., HUDSON, K., & DUKES, P 1993. Identification of Trypanosoma brucei gambiense by PCR am-plification of variant surface glycoprotein genes. Acta Tropica, 53:107-119. INOUE, N., NARUMI , D. , MBATI, PA., HIRUMI, K., SITUAKI -BANZA, N.H. & HIRUMI , H. 1998. Susceptibility of severe com-bined immuno-deficient (SCI D) mice to Trypanosoma brucei gambiense and T.b. rhodesiense. Tropical Medicine and In-ternatioanl Health, 3:408-412. PEPIN , J., MILORD, F., GUERIN , C., MPIA, B., ETHIER, L. & MANSINSA, D. 1989. Trial of prednisolone for prevention of melarsoprol-induced encephalopathy in gambiense sleeping sickness. Lancet, 1 :1246-1250. POLTERA, A.A., HOCHMANN, A. & LAMBERT, P.H. 1981 . Trypanosoma brucei brucei: the response to Melarsoprol in mice with cerebral trypanosomiasis. An immunopathological study. Clinical and Experimental Immunology, 46:363-374. WORLD HEALTH ORGANIZATION. 1998. Control and survei l-lance of African trypanosomiasis. Report of a WHO Expert Committee (Technical Report Series, no. 881) . 

Suggested dosage rates of melarsoprol in the treatment of mice experimentally infected with Trypanosoma brucei gambiense

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Suggested dosage rates of melarsoprol in the treatment of mice experimentally infected with Trypanosoma brucei gambiense

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