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- Materials and methods
Objective To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania.
Method We first screened 35 T. b. rhodesiense strains in the mouse model, for sensitivity to melarsoprol (1.8, 3.6 and 7.2 mg/kg), diminazene aceturate (3.5, 7 and 14 mg/kg), suramin (5, 10 and 20 mg/kg) and isometamidium (0.1, 1.0 and 2 mg/kg). A 13 isolates suspected to be resistant were selected for further testing in vitro and in vivo. From the in vitro testing, IC50 values were determined by short-term viability assay, and MIC values were calculated by long-term viability assay. For in vivo testing, doses higher than those in the initial screening test were used.
Results Two T. b rhodesiense stocks expressed resistance in vivo to melarsoprol at 5 mg/kg and at 10 mg/kg. These strains had high IC50 and MIC values consistent with those of the melarsoprol-resistant reference strain. Another isolate relapsed after treatment with 5 mg/kg of melarsoprol although it did not appear resistant in vitro. One isolate was resistant to diminazene at 14 mg/kg and another was resistant at both 14 and 28 mg/kg of diminazene. These two isolates had high IC50 values consistent with the diminazene-resistant reference strain. Two isolates relapsed at a dose of 5 mg/kg of suramin, although no isolate appeared resistant in the in vitro tests. Two isolates were resistant to isometamidium at 1.0 mg/kg and had higher IC50 values. Two isolates were cross-resistant to melarsoprol and diminazene and one isolate was cross-resistant to suramin and isometamidium.
Conclusion The reduced susceptibility of T. b. rhodesiense isolates to these drugs strongly indicates that drug resistance may be emerging in north–western Tanzania.
Objectifs Déterminer la résistance aux médicaments de souches de Trypanosomabruceirhodesiense isolées de patients souffrant de la maladie du sommeil en Tanzanie.
Méthode Nous avons d'abord testé sur un modèle murin, la sensibilité de 35 souches de T. b. rhodesiense au melarsoprol (1.8; 3.6 et 7.2 mg/kg), à l'aceturate de diminazene (3.5; 7 et 14 mg/kg), au suramin (5, 10, et 20 mg/kg) et a l’ isometamidium (0.1; 1.0 et 2 mg/kg). 13 souches suspectées résistantes ont été sélectionnées pour être testées in vitro et in vivo. Les valeurs de IC50in vitro, ont été déterminées par un test de viabilitéà court terme et les CMI ont été calculées selon le test de viabilitéà long terme. Les doses utilisées in vivo, étaient plus élevées que celles initialement testées.
Résultats Deux souches de T. b rhodesiense avaient une résistance in vivoà 5 et à 10 mg/kg melarsoprol. Pour ces 2 souches, les valeurs de IC50 et de CMI du melarsoprol étaient consistantes avec celles d'une souche résistante de référence. Une autre souche a été isolée suite à une rechute après traitement à 5 mg/kg de melarsoprol bien qu'elle n'apparaissait pas résistante in vitro. Une souche était résistante à 14 mg/kg de diminazene et une autre à 14 et 28 mg/kg de diminazene. Les valeurs de IC50 pour ces 2 souches étaient élevées de manière consistante avec celles d'une souche de référence résistante au diminazene. Deux souches ont été isolées suite à une rechute après traitement à 5 mg/kg of suramin, quoiqu’ aucune souche n'apparaissait résistante in vitro. Deux souches étaient résistante à 1.0 mg/kg d'isometamidium et avaient une IC50 plus élevée. Deux souches avaient une résistance croisée au melarsoprol et au diminazene et une souche avait une résistance croisée au suramin et à l'isometamidium.
Conclusion La susceptibilité réduite des souches de T. b. rhodesienseà ces médicaments indique clairement l’émergence de rèsistance aux médicaments dans le nord-ouest de la Tanzanie.
Objetivo Determinar la resistencia a medicamentos en cepas de Trypanosoma brucei rhodesiense aislados de pacientes con enfermedad del sueño en Tanzania.
Método En un principio se evaluaron 35 cepas de T.b. rhodesiense strains en un modelo animal, para determinar la sensibilidad frente al melarsoprol (1.8, 3.6 y 7.2 mg/kg), diaminazina (3.5, 7 y 14 mg/kg), suramin (5, 10, and 20 mg/kg) e isometamidium (0.1, 1.0 and 2 mg/kg). Los 13 aislados aparentemente resistentes, fueron seleccionados para posteriores pruebas tanto in vitro como in vivo. De las pruebas in vitro, se determinaron valores de CI50 mediante ensayos de viabilidad a corto plazo, y los valores de CMI fueron calculados mediante ensayos de viabilidad a largo plazo. Para las pruebas in vivo se utilizaron dosis más altas que las usadas durante el crivaje.
Resultados Dos lineas de T. b rhodesiense presentaron resistencia in vivo al melarsoprol a 5 mg/kg y 10 mg/kg. Estas cepas tenían una CI50 alta y valores de CMI consistentes con aquellos de la cepa de referencia melarsoprol resistante. Otro aislado recayó después de tratamiento con 5 mg/kg de melarsoprol, aunque no mostró resistencia in vitro. Un aislado fué resistente a la diaminazina a 14 mg/kg mientras que otro fue resistente tanto a 14 como a 28 mg/kg of diminazene. Estos dos ailsados tenían valores de CI50 consistentes con la cepa de referencia diminazene-resistente. Dos aislados recayeron con una dosis de 5 mg/kg de suramin, aunque ningún aislado se mostró resistente en las pruebas in vitro. Dos aislados fueron resistentes al isometamidium a 1.0 mg/kg y tenían valores de CI50 más altos. Dos aislados mostraron resistencia cruzada con melarsoprol y diminazene y un aislado tenía resistencia cruzada con suramin and isometamidium.
Conclusión La susceptibilidad reducida de aislados de T. b. rhodesiense a estos medicamentos parecen ser una indicación clara de que las resistencias a medicamentos pueden estar emergiendo en el noreste de Tanzania.
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- Materials and methods
African trypanosomes cause diseases in humans and domestic animals. Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, two subspecies of vector-borne hemoflagellate protozoans, cause Human African Trypanosomiasis (HAT) or sleeping sickness. In animals, trypanosomiasis is caused by various Trypanosoma species, the major ones being Trypanosoma vivax, Trypanosoma congolense and Trypanosoma evansi.
Human African Trypanosomiasis constitutes a serious public health threat in Africa, particularly in east and central Africa, where approximately 60 million people are at risk of contracting the disease. The 45 000 new cases reported to the World Health Organization (WHO) in 1999 do not accurately reflect the real situation – they merely underscore the inadequacy of the current campaign against this disease. It is estimated that 3 00 000–5 00 000 persons are infected with HAT.
Both Gambiense and Rhodesiense forms of HAT or sleeping sickness have been reported from Tanzania. Gambiense sleeping sickness was first recorded around Lake Tanganyika, where an active focus persisted until 1958 (Fairbairn 1948; Ormerod 1961). Rhodesiense sleeping sickness, which appeared in the 1920s and 1930s, was endemic in eight regions of Tanzania: Arusha, Kigoma, Lindi, Mbeya, Kagera, Rukwa, Ruvuma and Tabora. The disease tends to be concentrated in Kigoma (Figure 1). In the past 7 years only five regions have reported sleeping sickness cases; a large number originated in Kigoma (Kibondo and Kasulu districts). Over the past 4 years, the number of cases in Tabora (Urambo district) has been rising. Currently between 4 and 5 million people are thought to be at risk of infection in Tanzania, but only 1% of these are under regular surveillance. In the past 30 years, the number of new cases reported annually has risen above 500 (Annual Sleeping Sickness Reports for Tanzania 1965–1995), although this is likely to be an underestimate.
In Tanzania, as in many other countries, the control of HAT is based primarily on chemotherapy with only a few active drugs available for treatment, a situation that has remained virtually unchanged for more than 40 years. All these drugs have adverse side effects, are expensive or sometimes fail to cure. Recent progress in HAT research suggests that a vaccine against the disease is far from being successful (Atouguia & Costa 1999; Van Nieuwenhove 1999). Early-stage HAT is successfully treated with pentamidine and suramin; but treatment of late-stage HAT, where the trypanosomes have invaded the cerebrospinal fluid (CSF), depends exclusively on the arsenical compound melarsoprol. The one new drug marketed in the past 40 years, Eflornithine (DFMO, Ornidyl®), is only effective against T. b. gambiense and is very expensive.
Chemotherapeutic intervention is facing the problem of emergent resistance, as current drugs have been in continuous use for decades. Consequently, resistant T. brucei species have been reported from various parts of Africa (Matovu et al. 2001). Much less information is available regarding human trypanosomiasis. Relapses after treatment with the first-stage drugs pentamidine and suramin are rare; some early second-stage infections are misdiagnosed as resistance. Drug treatment failures at the second stage are mainly reported for melarsoprol. T. b. rhodesiense is innately refractory to eflornithine while T. b. gambiense is susceptible based on the selective toxicity as a result of different turnover rates for ODC in the two sub-species (Iten et al. 1997). Robertson and Hawking (1960) observed T. b. rhodesiense patients in Uganda who relapsed after two or more courses of melarsoprol; Ruppol and Burke (1977) reported up to 40% melarsoprol relapses in T. b. gambiense patients in Zaire; and Perez Martin et al. (1991) describe melarsoprol-resistant T. b. rhodesiense patients in Mozambique. The overall melarsoprol relapse rate over the past 50 years has been 5%–8% (Pepin et al. 1994), but in recent years this has increased alarmingly. In epidemic T. b. gambiense areas, in Uganda for example, it is 30% (Legros et al. 1999).
Regarding animal trypanosomiasis, there are persistent reports of resistant trypanosomes from all regions of Africa and to all drugs in use (Matovu et al. 2001). Resistance to the major drugs (diminazene, isometamidium, homidium, suramin and quinapyramine) has been reported in T. congolense, T. vivax and T. evansi (Anene et al. 2001); even multiple-drug resistance in T. congolense (Afewerk et al. 2000).
Between 2000 and 2002 we isolated T. b. rhodesiense stocks from sleeping sickness patients to determine whether trypanosome strains with reduced drug sensitivity occurred in Kasulu, Kibondo and Urambo districts in Tanzania, where the number of relapses is increasing. Sensitivity was tested both in vitro and in vivo.
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- Materials and methods
Trypanosoma b. rhodesiense isolates (n = 35) collected from patients in Tanzania between 2000 and 2002 were investigated for sensitivity to melarsoprol, diminazene, suramin and isometamidium. The aim was to find out if these trypanosomes would exhibit reduced drug sensitivity or if other reasons could have caused the relapses in the patients.
Our results probably indicate the presence of drug resistant T. b. rhodesiense in Tanzania. We used in vivo tests involving treating mice with doses higher than the equivalent used to treat trypanosomiasis in the field, the end point indicator of resistance. On this basis, three isolates were resistant to melarsoprol, two to diminazene, two to suramin and three to isometamidium. Although the in vitro culture system provides a convenient way of determining the drug resistance, whereby large number of isolates can be screened under the same conditions, it presents a few obstacles: The first is to isolate trypanosomes constituting a true representative of the population of parasites in humans or animals with the infection. The second obstacle is that trypanosomes which establish in culture may not be representative of the parasites in a sample. There is therefore, inevitably, selection for some trypanosomes which adapt more readily to the culture system, with the faster-growing trypanosomes being more abundant. The in vitro technique will be much improved when bloodstream trypanosomes can be adapted rapidly to culture, and exposed to trypanocides (and possibly their metabolites) for a minimum time to give an easily measurable end-point. The result obtained may then be correlated with results of field treatment (Geerts & Holmes 1998). The amastigote-macrophage assay of Leishmania sp. is currently the only model able to correlate clinical response to the in vitro drug sensitivity of the isolate, as demonstrated in relation to pentavalent antimonials (Croft 2004).
The results of the in vitro tests indicate that, compared with laboratory reference stocks, the test trypanosomes are more, or less, sensitive to a particular concentration of a trypanocide. To interpret these results in the light of clinical findings is not straightforward. It is important to be able to state that a trypanocidal effect exerted in vitro at a concentration of, say, 100 ng/ml melarsoprol is equivalent to a curative dose of, say, 3.6 mg/kg bodyweight. Too few data are currently available for this to be done. In addition, the test does not reflect the role of an unimpaired immune system of the natural host. This is very important because some drugs are cytostatic and their treatment success depends on the immune system (Dee Gee et al. 1983). But even in the absence of immune factor, the in vitro drug sensitivity test can then give a clue of differences in drug sensitivities between different isolates.
For better results, in vitro drug sensitivity test has to be confirmed by in vivo (Enyaru et al. 1998). Therefore, our isolates were probably resistant. Nevertheless, sensitivity from the mouse model does not always reflect that in human patients (Sones et al. 1988). Due to their higher metabolic rate, mice can alter or remove the drug faster than the natural host or vice versa. Hence, in mice the drug may not exert the same trypanocidal effect at the same dose as in humans (Zweygarth & Röttcher 1989). However, the fact that these isolates were obtained from patients who failed to be cured by the drugs tested in the mouse model leaves us with the conclusion that the isolates we found resistant by the mouse model are indeed resistant.
Despite some discrepancies the in vitro test could sometimes corroborate the in vivo test in determining the drug resistance. For example the in vitro and in vivo results support the possibility of resistance of TMRS 12(4) to melarsoprol, since the high dose of 10 mg/kg could not cure it. The maximum dose for humans is 3.6 mg/kg, and it had an IC50 value of 15.8 ng/ml. The relapse of TMRS 11(2) after treatment with 5 mg/kg melarsoprol may also indicate reduced susceptibility considering that the other isolates were completely cured. This is corroborated by its in vitro IC50 and MIC values, which are higher than those of the melarsoprol-resistant control. TMRS 3(11) had low IC50 and MIC values, but unexpectedly relapsed when treated with 5 mg/kg melarsoprol. Thus, the resistance of this isolate requires further investigation. TMRS 12(4), which turned out resistant by all tests used, was isolated from a female patient who was reporting for the third time to Kasulu District Hospital with late-stage sleeping sickness. Isolate TMRS 11(2) was isolated from a late stage patient reported as a first-time relapse case. The trypanosomes from TMRS 12(4) and TMRS 11(2) were confirmed to be melarsoprol-resistant, thus indicating relapse caused by trypanosomes selected against the therapeutic drug level during previous treatment rather than re-infection. In the mouse model experiment the possibility that relapse might be caused by re-invasion of the blood stream by trypanosomes from the CNS is also ruled out because relapse of this nature cannot occur in mice treated earlier than 14 days post-infection (Enyaru et al. 1998). In this experiment mice were treated 36 h post-infection, so trypanosomes had not yet crossed the blood brain barrier.
The relapses after treating infected mice with 14 and 28 mg/kg diminazene also indicate development of resistance to this drug, since the recommended doses for cattle are 3.5–7 mg/kg (Mbwambo et al. 1988; Kaminsky et al. 1989; Enyaru et al. 1998). Diminazene resistance was also indicated in vitro, where the IC50 values were higher than that of the diminazene-resistant control. Therefore, TMRS 11(2) and TMRS 12(4) were resistant to diminazene.
The isolates which relapsed after treatment with 5 mg/kg suramin may not be resistant in humans, as the maximum recommended dose in man is 20 mg/kg. Also, suramin treatment failure has never been observed in the areas where these isolates were obtained (District Medical Officers, personal communication). The relapsed isolates (TMRS 4(1) and TMRS 2(11)) also had low IC50 and MIC values compared to the suramin-resistant control isolate (KETRI 1989). It cannot be said with certainty whether suramin resistance is emerging in the area.
The mice infected with isolates TMRS 2(11), TMRS 1(13) and TMRS 10(6) relapsed after treatment with isometamidium at 1 mg/kg, meaning that it is a partially effective dose, which gave temporary clearance of the parasites. Considering that the recommended dose for cattle is 0.5–1 mg/kg, the data indicate the reduced susceptibility of these isolates to isometamidium. Although Nyeko et al. (1988) did not consider trypanosomes resistant when relapsed at 1 mg/kg isometamidium, the high IC50 values observed in our study do suggest resistance.
Our data curiously indicate cross-resistance between apparently unrelated drugs. For example isolates TMRS 11(2) and TMRS 12(4), which relapsed after 5 and 10 mg/kg melarsoprol, also relapsed after at 14 and 28 mg/kg diminazene. Isolate TMRS 2(11) was resistant to both suramin (5 mg/kg) and isometamidium (1 mg/kg). Although the drugs used to treat trypanosomiasis in humans may be chemically different from those for animals, cross-resistance may develop. Cross-resistance between melamine-based arsenicals and diamidines has been repeatedly observed in trypanosomes with laboratory-induced drug resistance (Rollo & Williamson 1951). Indeed cross-resistance between diminazene (for animal trypanosomiasis) and melarsoprol (for HAT) can be selected with relative ease (Barrett & Fairlamb 1999) because both drugs, and their active metabolites, can enter trypanosomes via the P2 aminopurine transporter, and alteration of this transporter can impair the uptake of both drugs by parasites. It is highly probable that melarsoprol resistance comes by virtue of selection of diminazene resistance of the human trypanosomes through treatment of the animal reservoir (Barrett & Fairlamb 1999).
One shortcoming of this study is that the results of the short-term viability assay were not necessarily reflected in the long-term viability assay, especially for diminazene and isometamidium (Table 2). Iten et al. (1997) made a similar observation whereby differences in susceptibility to melarsoprol in their short-term viability assay (hypoxanthine incorporation) could not be confirmed by the long-term feeder-layer viability assay. A possible explanation of this phenomenon could be the short test duration of the 24-hour assay, which may bias results by differences in drug uptake or metabolism of the drugs by different trypanosome stocks (Matovu et al. 1997).
Reduced susceptibility of T. b. rhodesiense from Tanzania to melarsoprol, diminazene and isometamidium is a clear sign of the emergence of drug resistance in the sleeping sickness endemic areas of Tanzania. The factors responsible for the emergence of resistance cannot be established by this study, although an important one is sub-therapeutic drug concentration. This was not uncommon some years back in Tanzania before the government's decision to provide free treatment for sleeping sickness. At that time most patients could not afford to meet the cost of a complete course of treatment with suramin or melarsoprol due to shortages and the relatively high cost of these drugs.
Under dosing may also occur in treating animal trypanosomiasis, for similar reasons. Block treatment of cattle in endemic areas is another factor, which may impose a high selection pressure; this is exacerbated by its frequency. This may be the reason why so many more resistant trypanosomes are isolated from animals than from humans, who are normally treated under the hospital conditions. Sometimes generic products are used, which are less efficacious (Geerts & Holmes 1998). We conclude that there are conditions present in Tanzania, which can lead to selection of trypanosomes resistant to trypanocides.
Since it is very unlikely that new trypanocidal drugs will be released on the market in the near future, it is essential to try to maintain the efficacy of the currently available drugs. Authorities in Africa need to adopt an integrated disease management strategy to slow down the development of this resistance.