TRYPANOSOMA BRUCEI GAMBIENSE FROM SLEEPING SICKNESS PATIENTS IN SOUTH SUDAN: ISOLATION AND PROPAGATION IN RODENTS 

DETECTION DE TRYPANOSOMA BRUCEI GAMBIENSE CHEZ DES MALADES SOMMEILLEUX AU SUD-SOUDAN: ISOLEMENT ET PROPAGATION CHEZ LES RONGEURS

Naomi W. N. Maina^1,2, Michael Oberle², Charles Otieno¹, Christina Kunz², Joseph M. Ndung’u¹ & Reto Brun²

¹Trypanosomiasis Research Centre  (TRC), P. O. Box 362, Kikuyu, Kenya
²Department of Medical Parasitology and Infectious Biology, Swiss Tropical Institute (STI), P. O. Box CH-4002, Basel, Switzerland

Résumé

            A Ibba, dans le comté de Maridi, dans la région de l’Equatoria de l’Ouest, au Sud-Soudan, les taux d’échec du traitement au mélarsoprol étaient, d’après les estimations, élevés (>20 %). Le rôle des trypanosomes résistants dans les échecs de traitement n’est cependant pas connu, et il est urgent de procéder à une caractérisation de T. b. gambiense isolé dans cette région. Cette étude vise à isoler le parasite chez des malades sommeilleux à l’hôpital de MSF-F. Cinquante-trois malades sommeilleux identifiés lors d’une surveillance active ont été sélectionnés. La plupart d’entre eux (51 sur 53) étaient au deuxième stade de la maladie, dont sept avaient été traités auparavant : quatre à l’eflornithine et trois à la pentamidine. Les échantillons de sang et de liquide céphalo-rachidien prélevés à des patients ont été cryoconservés à l’aide du Triladyl® utilisé comme cryomédium. Sur les 42 isolats de parasites testés positifs, 18 (soit 43 %) pouvaient être transmis à des rongeurs de laboratoire (des souris Mastomys natalensis immunodéprimées et des souris SCID). La parasitémie était plus forte chez les SCID que chez les Mastomys. La viabilité in vivo dépendait de la parasitémie du patient – elle est plus élevée chez les patients à forte parasitémie. Les stabilats de ces isolats ont été effectués et conservés dans deux cryothèques au TRC et au STI. Tous les 18 isolats prélevés à des souris Mastomys ou SCID étaient infectieux pour les souris blanches suisses immunodéprimées, et 6/18 isolats provoquaient une parasitémie de plus de 106/ml. Une comparaison des souris blanches suisses, des Mastomys C57/bl et des BALB/C, toutes immunodéprimées, a montré que ces races de rongeurs étaient sensibles après la seconde sous-inoculation et qu’elles développaient une parasitémie plus forte que 106/ml au cinquième jour après l’infection. La plus forte parasitémie a été obtenue chez les C57/bl et les BALB/C, ce qui indique que la propagation des isolats de T. b. gambiense après l’isolement initial chez les souris Mastomis ou SCID immunodéprimées pourrait se réaliser chez un grand nombre de rongeurs de laboratoire immunodéprimés. Il est recommandé d’effectuer la première propagation et les premières sous-inoculations en utilisant des souris Mastomys ou SCID immunodéprimées. Pour les transimissions ultérieures, les souris C57/bl ou BALB/C immunodéprimées constituent l’alternative la plus économique par rapport aux souris Mastomys ou SCID.

            In Ibba, Maridi county, western Equatorial, South Sudan, the rate of melarsoprol treatment failure was earlier reported to be high (>20%). The role of resistant trypanosomes in treatment failures is however not known and characterisation of T b gambiense isolated from this area is of high priority. This study aimed at isolating the parasite from Human African Trypanosomiasis (HAT) patients attending the MSF-F hospital. Fifty-three HAT patients identified during active surveillance were recruited. Most (51/53) were in second stage disease with seven having been previously treated: four with eflornithine and three with pentamidine. Blood and cerebrospinal fluid samples from the patients were cryopreserved with Triladyl® as cryomedium. Of the 42 parasite positive isolates, 18 (43%) could be propagated in laboratory rodents (immunosuppressed Mastomys natalensis and SCID mice). Parasitaemia was higher in SCID than in Mastomys. In vivo viability was dependent on the parasitaemia in the patient - being higher in patients with high parasitaemia. Stabilates of these isolates were made and stored in two cryobanks at the TRC and STI. All 18 isolates recovered from Mastomys or SCID mice were infective to immunosuppressed Swiss White mice, with 6/18 of them causing a parasitaemia of over 106/ml. A comparison of Swiss White mice, Mastomys C57/bl and BALB/C, all immunosuppressed, demonstrated that these rodent breeds were susceptible after the second sub-passage, developing a parasitaemia higher than 106/ml by day 5 post-infection. The highest parasitaemia were achieved in C57/bl and BALB/C. This indicated that the propagation of T b gambiense isolates after initial isolation in immunosuppressed Mastomys or SCID mice could be done in a range of immunosuppressed laboratory rodents. It is recommended that primary propagation and first sub-passages be done in immunosuppressed Mastomys or SCID mice. For subsequent passages, immunosuppressed C57/bl or BALB/c mice are the more economical alternative to Mastomys or SCID mice.

Introduction

            In Sudan sleeping sickness is highly endemic with an estimated 1 to 2 million people at risk of infection, and more than 1000 people are infected annually. Control of the disease depends mainly on chemotherapy and, to a limited extent, vector control. In recent years, high rates of melarsoprol treatment failure (>20%) have been reported in T. b. gambiense endemic areas including Sudan (Legros et al., 1999 and Van Niewenhove et al., 1985).

            In Ibba, Maridi county, Western Equatoria, South Sudan the situation was even more serious since the number of treatment failure reported within a short period were relatively high (Moore et al., 2001). In addition, a high death rate following melarsoprol treatment was also being recorded (MSF report 2002). The role of resistant trypanosomes in treatment failures is however not known and characterisation of T. b. gambiense isolated from this area is of high priority (Brun et al., 2001). In this study, trypanosomes were isolated from patients admitted in the MSF-F hospitals: Ibba hospital, Maridi County (for second-stage disease) and Kotobi hospital, Mundri County (for first-stage disease). We determined the susceptibility of various mice strains to the isolated trypanosomes.

Materials and Methods

Liquid nitrogen:

            Liquid nitrogen was used for cryopreservation. Three nitrogen tanks (Taylor-Wharton) were used: one tank (type 34HC; capacity of 34 litres) and two dry shippers (CX100; capacity of 4 litres volume). The storage tank was kept in MSF-F laboratory at Maridi from where the dry shippers were refilled. The dry shippers were transported by vehicle to respective hospitals for trypanosome isolation.

Patients:

            Active screening of patients for SS was done by MSF-F in Mundri, Maridi and Yambio counties, Western Equatoria, South Sudan. Patients whose diagnosis had been confirmed were transferred to hospital for treatment; first-stage patients were transferred to Kotobi, Mundri County, and second-stage to Ibba, Maridi County. In the hospital, the patients were informed of the objectives and protocols of the study. Only patients (or their guardians) who gave consent (written/thumb print) were included in this study. The ethical Committees of Both Kantos of Basel (EKBB), Switzerland, and the Ministry of Health of Sudan People's Liberation Army (SPLA) approved the study protocol.

Concentration and cryopreservation of trypanosomes:

            Two to three millilitres of venous blood were collected from positive patients into a heparinised vacutainer tube. Aliquots of 500ml were then transferred into NUNC® ampoules. Stabilates were prepared per patient and stored in a dry shipper containing vapour nitrogen at -150oC. The stabilates were later transported to KETRI in Kenya.

Laboratory animals:

Mastomys natalensis and Swiss White mice were bred at KETRI while Severe Combined Immunodeficient (SCID) were purchased from Charles River Company, Germany. The animals were housed under conventional conditions, and fed on commercial pelleted ration and water ad libitum. The SCID mice have no functional T and B lymphocytes (Bosma et al. 1983). All animals (except for SCID mice) were immunosuppressed prior to infection.

Primary propagation of T. b. gambiense isolates:

            The viability of each isolate was primarily determined by infection of one SCID and two M. natalensis. The stabilates were inoculated into an animal intraperitoneally (ip). Parasitaemia was monitored every other day by examination of tail blood. Parasitaemic animals were euthanised using CO2 and trypanosomes harvested by cardiac puncture. Trypanosomes (passage 1) were cryopreserved. The rodents that remained aparasitemic for at least 60 days after inoculation were euthanised and blood collected by cardiac puncture. For each rodent, eight capillaries were prepared and examined for trypanosomes using the mHCT method. When patent infection was not achieved, repeats (up to three times) was carried out in another pair of M. natalensis.

Secondary propagation in mice:

            Secondary propagation was done to determine the susceptibility of various strains of mice to the T. b. gambiense isolates. Initially, the isolates (passage 1) were inoculated into immunosupressed Swiss white mice. The susceptibility of five strains of mice to the T. b. gambiense isolates was assessed using three of the isolates. Thawing of stabilates, inoculation, and parasitaemia determination were carried out as stated earlier.

Patients:

            Fifty- three confirmed SS patients were enrolled in the study. Blood samples were collected from 50 out of 53 patients:- 42 pateints parasitaemic and eight aparasitaemic.

Primary propagation of isolates from patients:

            The 50 samples that were collected were inoculated into Mastomys natalensis and SCID mice. Of these 18 caused patent infections. Most (12/14) isolates were found viable after first inoculation but only two at the second trial and none at the third repeat.

Correlation between parasitaemia in the patients and in vivo viability:

            Forty two (84%) of the patients had trypanosomes in blood. The parasitaemia was however low (only detectable by mHCT method) and varied between the patients. The percentage of isolates that were viable in rodents for each category of parasitaemia was determined (Table 1). There was direct correlation between parasitaemia in the patient of isolation and viability in rodents’ i.e. the recovery rate was higher in the category that was isolated from patients with high parasitaemia than in the aparasitemic group.

Susceptibility of mice to T. b. gambiense:

            All the 18 isolates inoculated into immunosuppressed Swiss white mice caused patent infection, with a pre-patent period of 4 days. Most of the isolates caused low and inconsistent parasitaemia (less than 104/ml) but some isolates grew to high (over 106 trypanosomes/ml of blood) parasitaemia.

            The susceptibility of five species of mice to T. b. gambiense isolates was further determined. The isolates were infective in various mice strains. The prepatent periods were similar but the parasitaemia pattern differed between the mouse strains.

Table 1: Comparison between the level of parasitaemia in SS patients and viability in rodents.

The parasitaemia was determined using HCT method. In lymph node positive patient
**, parasitaemia was not assessed.

Discussion

            This study was carried in Maridi and Mundri counties western Equatoria, Southern Sudan, a highly endemic foci for sleeping sickness. Control of the disease has been largely depends on chemotherapy and mainly aims at reducing the parasite reservoir through case detection (active and passive screening) and subsequent treatment. Chemotherapy has however been constrained by increased (>20%) relapses (Moore et al., 2001) and high mortality following melarsoprol treatment. These factors led to a change of approach to SS treatment - from melarsoprol to DFMO as the first line drug for the second stage disease. Studies to establish the causes of treatment failures as well as drug sensitivity of infecting parasites should be a priority (WHO report 2001).

            In this study, 18 (43%) T. b. gambiense were successfully isolated from HAT patients in Western Equatorial, south Sudan using as the cryopreservative. In previous studies, lower recovery or complete losses of T. b. gambiense following cryopreservation with 10% glycerol have been reported (Burri et al., 2001, Matovu et al., 2001, Enyaru, Personal communication). The relatively higher success rate achieved in this study might be due to the superior cryopreservation medium.

            Viability of the trypanosomes would also have been affected by other factors that prevent/inhibit trypanosome growth including antiparasitic drugs. Patients in this study had had malaria treatment(s) and some (7/42) had previously been treated with trypanocides. Studies indicate that prior drug administration in the host reduces trypanosome viability. In this study, viability of the isolates in rodents appeared to depend on the parasitaemia in the patients i.e., the higher the parasitaemia, the higher the chances of causing patent infection in rodents. Apart from low parasitaemia, earlier reports also noted that the limited infectivity of T. b. gambiense to rodents led to low isolation success (Aerts et al., 1992 and Duke et al., 1989).

            In the current study we used an African rodent, M. natalensis for primary propagation of the isolates. The parasitaemia attained were higher in SCID than in M. natalensis. Inoue et al (1998) reported similar susceptibility of SCID mice to T. b. gambiense isolates. This study further shows the importance of SCID mouse in propagation of T. b. gambiense to large numbers from samples with low parasitaemia is further indicated. The use of this model is however restricted by the cost, necessitating the search for a cheaper rodent model for isolation of T. b. gambiense.

            Mice are easier to handle and breed frequently than M. natalensis. The former, are generally thought to be more susceptible to T. b. rhodesiense than T. b. gambiense. In the current study the susceptibility of various strains of mice to T. b. gambiense was also investigated. The findings of this study could imply that of some strains of mice are susceptibility to T. b. gambiense and would be used for propagation studies. Preliminary studies also indicate that Swiss White mice (TRC colony) may be as good as the M. natalensis in isolation of T. gambiense from SS patients.

            In this study 18 T. b. gambiense were successfully isolated from an area with reported high treatment failures. Characterisation of such T. b. gambiense is of high priority (Brun et al., 2001) since the role of resistant trypanosomes in treatment failures is not known. This may assist in identifying the most appropriate backup trypanocide for use in Southern Sudan, thus improving the chemotherapy of sleeping sickness.

Acknowledgements

            The authors would like to thank the Eastern African Network for Trypanosomosis (EANETT) and Swiss Development Co-operation (SDC) for funding this study. We are also grateful to MSF-F for allowing us to work under their sleeping sickness control Programme in south Sudan, and efficient logistic planning of the field trip. The technical assistance provided by Rashid Farah, Peter Waweru and the staff of primate Division of TRC is appreciated. This paper has been published with the permission of the Centre Director TRC.

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