Future Microbiology Review Drug resistance in human African trypanosomiasis

Future Microbiol. (2011) 6(9), 1037–1047

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103710.2217/FMB.11.88 © 2011 Future Medicine Ltd ISSN 1746-0913

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Human African trypanosomiasis (HAT), also known as sleeping sickness in its second stage when the central nervous system is involved, is caused by subspecies of the protozoan parasite Trypanosoma brucei spp. [1]. Many of the basic parameters of the disease have been reviewed recently in Future Microbiology along with recent advances in development of novel agents to treat the disease [2]. There are two forms of HAT caused by two separate subspecies. T. b. gambiense prevalent in Central and West Africa causes a chronic form classically taking around 18 months to progress through the first (hemo-lymphatic) stage to the second (neurological) stage and then another 18 months to death. In Eastern and Southern Africa, T. b. rhodesiense causes an acute disease with shortened first and second stage, leading to death within weeks to

months of infection. Recent evidence, however, indicates that avirulent T. b. gambiense and less acutely virulent forms of T. b. rhodesiense might also exist [3].

Vaccines will not be effective against HAT since the parasites have the ability to sequentially change a surface glycoprotein coat that covers the entire surface. Although attempts to destroy the tsetse fly vectors that transmit the disease have been used effectively in control programs, their continent-wide application is logistically challenging [4]. As a consequence, chemother-apy has been central to efforts to manage HAT and has also been central to efforts to move to a s ituation of disease elimination [5].

However, relatively few drugs are available and they have been used for between 20 and 80 years. The development of resistance to the

Drug resistance in human African trypanosomiasis

Michael P Barrett†1, Isabel M Vincent1, Richard JS Burchmore1, Anne JN Kazibwe2 & Enock Matovu2

1Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland 2Makerere University School of Veterinary Medicine, Kampala, Uganda†Author for correspondence: Tel.: +44 141 330 6904 n Fax: +44 141 330 4600 n [email protected]

Human African trypanosomiasis or ‘sleeping sickness’ is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.

Keywords

n drug resistance n eflornithine transporter n HAPT1 n human African trypanosomiasis n nitroreductase n P2 transporter n sleeping sickness n treatment failure

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current drugs would be calamitous in terms of sustained management of the disease and would reverse the current downward trend in prevalence.

In this review we discuss mechanisms of resis-tance to currently used drugs as determined in the laboratory and also discuss the potential emergence of resistance in the field.

Treatment failure & drug resistance in human African trypanosomiasis

Like all microbes, trypanosomes have the abil-ity to evolve resistance, through genetic muta-tions, to drugs used in their treatment (Figure 1). However, it has proven difficult to link instances of parasite resistance to treatment failure in the field. The difficulties associated with harvest-ing of parasites, especially T. b. gambiense, from infected patients, to allow studies into parasite genotype and phenotype, exacerbate this prob-lem. A recent report that patient cerebrospinal fluid (CSF) from HAT patients yielded parasites capable of robust in vitro and in vivo (rodent)

growth if they were first cultivated over fibro-blast feeder layers [6] might offer improvements in our ability to study field isolates. A number of other important issues need to be considered with regard to treatment failure that do not n ecessarily relate to parasite drug resistance.

Difficulties in drug administration The distribution of HAT drugs today is con-trolled directly by the WHO. In response to requests from endemic country National Programs, or other health workers involved in HAT clinics, drugs are shipped according to a well-regulated schedule with no involvement of the private sector. Problems involving inferior quality or counterfeit drugs (as has been shown to be of significance for malaria [7]) is not an issue for HAT, although it has been a problem in veterinary trypanosomiasis [8]. However, treatments for HAT are notoriously difficult to administer (reviewed in [2,9]), with all current registered drugs requiring protracted parenteral administration (oral nifurtimox being available

Figure 1. The known mechanisms of drug resistance in Trypanosoma brucei. Routes of uptake for each of the currently used trypanocidal drugs are shown. Also, where known, cellular targets are marked, including ODC for eflornithine and the NTR that metabolizes nifurtimox into its active form (N*). Pentamidine binds to kinetoplast DNA (K), although it is not clear that this binding is responsible for its mode of action. Resistance to eflornithine relates to loss of TbAAT6. Resistance to melarsoprol (or its active metabolite melarsen oxide) relates to loss of the TbAT1 (P2) and HAPT1 transporters. Alternatively, upregulation of TbMRPA and can cause resistance when melarsen oxide-trypanothione conjugates are pumped from the cell. Pentamidine resistance also relates to loss of TbAT1 and HAPT1 transporters. Suramin enters by receptor-mediated endocytosis at the flagellar pocket and resistance has been shown to relate to changes in the endocytic pathway. Nifurtimox resistance can come about when the nitroreductase activity involved in its activation is diminished. E: Eflornithine; M: Melarsoprol; N: Nifurtimox; NTR: Nitroreductase; ODC: Ornithine decarboxylase; P: Pentamidine; S: Suramin.

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through the WHO list of essential medicines without being licensed). For stage 1 disease pentamidine is given, usually by intramuscular injection, with once-daily 4 mg kg-1 dosing for 7 days. Suramin is usually given by slow intra-venous injection every 3–7 days, to a total of five dosings over a 4-week period. For stage 2 dis-ease, melarsoprol is administered intravenously in a 3.6% solution in propylene glycol, typically every day for 10 days (although earlier schedules involved inclusion of interrupted courses last-ing a month). Eflornithine as monotherapy, is given over 14 days with four 100 mg kg-1 intra-venous infusions per day. When used in the nifurtimox-eflornithine combination therapy (which is occurring with increasing frequency) eflornithine doses are halved (200 mg/kg twice per day for 7 days) with nifurtimox being given orally three times a day for 10 days. The pro-tocols required to sustain these recommended administration parameters are complex and can inevitably be compromised in some instances, leading to treatment failure (and possibly even promoting selection of resistance).

Pharmacokinetics, pharmacodynamics & difficulties in disease stagingThe general efficacy of the current anti-trypano-somal drugs is variable within populations. This can be due to trypanocidal potency of the drugs themselves or due to variability in pharmacoki-netic behavior in different people. Pentamidine is highly potent, yielding IC

50 values in the order

of 1–10 nM in a typical 3 day in vitro drug sensi-tivity assay. Melarsoprol and suramin also show pronounced activity in these assays killing try-panosomes in the low nanomolar range. By con-trast, nifurtimox has an IC

50 of around 5 µM in

these assays and eflornithine only acts in the tens of µM range [2,9]. The time that parasites must be exposed to drug in order for them to be certain to die is also critical, hence the drug-clearance rate in vivo is of great importance.

Pharmacokinetic properties of the anti-trypanosomal drugs are, therefore, also highly relevant to their activity. Ultimately, in order to be active, parasites must be exposed to drug at lethal concentrations for sufficient time to induce a trypanocidal effect. In stage 2 HAT, when drugs must reach parasites in the CNS and other privileged body compartments, pharmaco-kinetics become even more important. Since dif-ferent drugs have different abilities to distribute to these compartments, correct diagnosis with respect to whether the disease is at stage 1 or at stage 2 is critical, but not straightforward.

Pentamidine displays extensive tissue retention and binds serum proteins, which contributes to a large volume of distribution and long terminal half-life. However, the drug is extensively metab-olized (rates of metabolism can vary between individuals) and it crosses the blood–brain bar-rier at low levels [10]. For suramin, nearly all of the injected drug (>99%) is protein-bound in plasma, which gives it a long terminal half-life (41–78 days) with free drug being available in sufficient quantities to affect parasites due to equilibria in its protein binding. The charged nature of suramin precludes delivery beyond the blood–brain barrier [11]. Melarsoprol is metab-olized rapidly to melarsen oxide, which is the active molecule in vivo. Clearance of the active metabolite is also relatively fast (a half-life of 3.5 h). Accumulation across the blood–brain bar-rier appears to be relatively modest (only 1–2% of maximum plasma levels in CSF [9]). Within the brain, therefore, the drug accumulates to concentrations relatively close to the minimum needed to exert activity. Thus minor shifts in the susceptibility of parasites to the drug can affect efficacy profoundly, and this could have implica-tions in treatment failure where a relatively low drop in sensitivity to drug could render parasites in CSF nonsusceptible to the levels accumulating in this compartment. Eflornithine’s permeation into rodent brain has recently been reported to be low [12]. CSF to plasma ratios between 0.1 and 0.9 have been reported in humans [9], indicating possible variability in brain perme-ation among humans and differences between its ability to enter rodent versus human brains. The mean half-life in plasma following intra-venous i njection of e flornithine is only in the order of 3 h.

In recent years, studies have indicated that the use of white cell count in CSF can act as a surrogate marker for stage 2 infection, and most national programs use this in stag-ing the disease, given the difficulty associated with finding trypanosomes in CSF [1]. In some instances, 5 white cells per µl of CSF is taken as the cut-off point, while in others it is 20. Given the severe toxicity associated with melarsoprol, recommendations were made to use pentami-dine to treat the ‘early–late’ stage of the disease in patients without trypanosomes in CSF and with fewer than 20 white cells per µl. However, Lejon et al. [13] showed that in cases of >10 white cells per µl there was an increased risk of treat-ment failure with pentamidine, and Balasagram et al. [14] showed that patients in which white cell counts were <5 per µl were less likely to fail

Drug resistance in human African trypanosomiasis Review


than patients with CSF containing 5–10 white cells per µl. The relative risk of treatment failure with pentamidine in these cases was considered acceptable when compared with the potential risk of reactive encephalopathy with melarso-prol. However, the switch from melarsorpol to eflornithine or nifurtimox eflornithine combina-tion therapy (NECT) as first-choice treatment for stage 2 means that the rationale for treating ‘early–late’ stage with pentamidine is dimin-ished, since NECT does not carry the toxicity risk associated with melarsoprol [14].

Reports of treatment failure in the fieldTreatment failures with melarsoprol began to cause concern in the late 1990s. Initial reports were treated with some scepticism given the dif-ficulties in distinguishing treatment failures and drug resistance outlined as above. Early efforts also failed to show that parasites from treatment failures were actually drug resistant [15], which contributed to a belief that other phenomena might underlie treatment failures. However, reports from many foci, including those in Northern Uganda, The Democratic Republic of Congo, the Republic of Congo, Southern Sudan and Angola have all indicated elevated levels of melarsoprol treatment failure. A coordinated effort led by the WHO to establish the so-called ‘HAT Sentinel’ program corroborated many of these, and the elevated problem of treatment failures with melarsoprol underpinned recom-mendations to change to eflornithine, and more recently NECT [5]. Once potential molecular mechanisms were proposed (see ‘Melarsoprol resistance’ section) and the TbAT1 gene was implicated as a resistance marker, parasite geno-types were compared in isolates from cured versus relapsed patients in Northern Uganda in 2001 [16]. It was shown that a much higher fre-quency of alleles that were similar in sequence to those found in laboratory-derived drug-resis-tant cells appeared in the relapse patients than in those who were cured. This helped influence a decision to move to eflornithine as first line treatment at the Omugo Treatment Center in Northern Uganda. Interestingly, when parasite genotypes were tested again in samples isolated in 2005, the so-called ‘resistant’ allele could no longer be identified [17], indicating that removal of the selective pressure exerted by melarsoprol led to loss of the resistant alleles. A similar expla-nation could account for a lack of resistant alleles found in trypanosomes isolated from patients in South Sudan in 2003 [18], also several years after the cessation of melarsoprol use (in this

case, however, there was no baseline information on allelotype frequency during the period when treatment failure rates were high). An important conclusion from the Northern Uganda study is that an alternating drug regimen involving sequential use of melarsoprol and then eflorni-thine (or NECT), might restrict the emergence and spread of resistance alleles to either drug. Of note is the fact that zoonotic parasites that can also propagate in animals will be under less selec-tive pressure than anthroponotic parasites, thus resistance selection pressure for gambiense and rhodesiense trypanosomes might also be variable.

Reports from the field about suramin resis-tance are rare. However, since only 14,900 doses of the drug (which is used for stage 1 rhodesiense disease) were distributed by WHO in the last decade [5] selection pressure has been low. The large incidence of reported treatment failures of T. b. gambiense in West Africa in the 1950s could have been due to multiple factors, of which p arasite resistance is just one.

Reports of treatment failures with pentami-dine have also been rare. Mechanistic explana-tions for this are presented in the ‘Pentamidine resistance’ section as it seems relatively difficult to select for parasites with high levels of resis-tance to this drug. However, it should also be noted that increased treatment failure with pentamidine is associated with elevated levels of white cell counts in the CSF (as discussed previously), which indicates that failures likely relate to misdiagnosed stage 2 disease rather than drug resistance.

Eflornithine began to be used in several foci in the early 2000s following the spread of treatment failures with melarsoprol. Trials have shown that the efficacy of eflornithine is limited, with around 10% of those receiving the drug not responding (e.g., 8.1% failures reported in [19]). One trial in Northern Uganda [20] comparing a 7-day and 14-day regimen indicated higher rates of failure (up to 27%) even for the 14-day regi-men, while relapse rates were far lower in other West and Central African countries. Reasons for these exceptional reported relapse rates are difficult to ascertain. Recent evidence in tightly controlled studies [Matovu et al., Unpublished

Observations] indicates that there is a rising inci-dence of treatment failure with eflornithine monotherapy in the Omugo clinic in Northern Uganda, although it has not been possible to determine definitively whether parasite resis-tance is to blame. The accelerated introduction of NECT may help stem the emergence and spread of eflornithine resistance. However, since



nifurtimox is of poor efficacy in monotherapy, eflornithine-resistant parasites have an increased likelihood of surviving NECT treatment too, which might also lead to selection of resistance to nifurtimox.

Molecular mechanisms of resistanceMelarsoprol resistanceOf all the anti-trypanosomal drugs, mecha-nisms of resistance to melarsoprol are the most studied in the laboratory. In 1993 Carter and Fairlamb [21] demonstrated that an adenos-ine transporter, which they termed P2, was probably capable of melarsoprol uptake, and that loss of this transporter was correlated to resistance to the drug in a line, RU15, selected through in vivo selection to sodium melarsen. Melarsoprol is rapidly converted to melarsen oxide in vivo. Since melarsoprol itself is of limited polarity, it can cross membranes via passive diffusion whilst the more polar active metabolite is restricted to carrier-mediated uptake. A first hypothesis proposed that loss of the P2 transporter was in itself responsible for melarsoprol/melarsen oxide resistance. When a gene, TbAT1, encoding the P2 transporter, was cloned [22] and shown to possess a series of point mutations in resistant lines that ren-dered it inactive (or else deleted from other resistant lines) this hypothesis appeared to be corroborated. However, gene knockout experi-ments showed that removal of the TbAT1 gene did lead to functional loss of P2 transporter activity, but these P2 knockout cells were only of modestly reduced sensitivity to melarsen oxide [23]. It appeared, therefore, that loss of P2 was necessary, but insufficient alone, to account for high-level resistance. Using an in vitro lysis assay (in which melarsen oxide rapidly kills trypanosomes) it was shown that pentamidine could retard the lysis of P2 transporter defi-cient cells, and this led to a conclusion that the so called high affinity pentamidine trans-porter (HAPT1) might represent a secondary route for uptake of melamine-based arsenicals. A trypanosome line selected in vitro from the TbAT1 knockout precursor for high-level resis-tance to pentamidine also acquired high-level resistance to the melaminophenylarsenicals and was shown to have functionally lost the HAPT1 transporter [24]. These data support a modified hypothesis whereby loss of both the P2 transporter and HAPT1 is required for high level melaminophenyl arsenical resis-tance. Determining whether still more factors can contribute to resistance will be facilitated

with identification of the HAPT1 carrier’s gene and an assessment of the impact of TbAT1 and HAPT1 double gene knockout.

In addition to P2 transporter deficiencies, it was hypothesized that over-expression of P-glycoprotein efflux pumps could, in principle, cause resistance to melamine-based arsenicals reviewed recently in [25]. The gene encoding one such pump, TbMRPA, when overexpressed in T. brucei, caused a marked decrease in sensitiv-ity to melarsoprol, possibly through its ability to excrete the drug–trypanothione adduct that forms inside the cell. When studying possible synergy between loss of the P2 transporter and expression of the efflux pump, however, it was concluded that the two mechanisms worked in isolation and were strictly additive [26]. The fact that multiple possible routes do exist to selec-tion of resistance, however, must be taken into consideration if diagnostic tests that can report on the likelihood of treatment failure are to be developed.

Pentamidine resistanceThe situation regarding resistance to diamidines including pentamidine was recently reviewed [25,27]. Carter et al. [28] also showed that the P2 transporter was capable of the uptake of pent-amidine, and resistance to this drug was attrib-uted to loss of uptake. However, the RU15 line selected for melarsen resistance was of only marginally increased resistance to pentamidine. Furthermore, a line selected for resistance to pentamidine, PR32.6, had little loss in sensitiv-ity to melaminophenyl arsenicals. The situation regarding diamidine-arsenical cross-resistance thus appeared complex. Part of the complexity was resolved with the discovery that pentamidine is carried by at least two transporters in addition to the P2 transporter [25,27]. HAPT1, named for the fact that its apparent K

m for pentamidine was

in the mid-nanomolar range, and a low-affinity carrier (LAPT1), which had a much lower affin-ity in the tens of µM range, were identified kineti-cally [25]. In order to learn more about the relative contribution to resistance of these other trans-porters a line, termed B48, was selected, starting with the TbAT1-deficient line, for higher level resistance to pentamidine [24]. As discussed above, these cells lost the HAPT1 transporter and were of reduced sensitivity to melamine based arseni-cals. This led to the conclusion that pentamidine and the melaminophenylarsenicals enter via both the P2 and the HAPT1 transporters and that loss of both was necessary for high-level resistance. This hypothesis does satisfy the observations



regarding the B48 cell line. However, the RU15 line (high level melamine-arsenical resistance without appreciable cross-resistance to pentami-dine) and the PR32.6 line (high level pentami-dine resistance without appreciable resistance to melamine-arsenicals) indicates that other fac-tors can also play a role. A variety of different levels of cross-resistance between another series of trypanosome lines selected for resistance to melarsoprol or pentamidine [29] also points to other phenomena, as yet not understood, that contribute to resistance to each drug and the cross-resistance phenotypes.

Further insights into the role of transporters in melaminophenylarsenical & diamidine resistanceAs discussed previously, the TbAT1/HAPT1 double-loss genotype can explain some, but not all, observations made on arsenical-diamidine cross-resistance phenotypes. In addition to the currently used trypanocides (melarsoprol and pentamidine) it was shown that the veterinary trypanocide, diminazene aceturate (berenil), also enters trypanosomes via the P2 transporter. However, a secondary route of uptake was also apparent. It has recently been suggested that this secondary route of uptake is through the HAPT1 transporter [30] as judged in a cell line, selected from the TbAT1 knockout cells, that is of higher level resistance to diminazene and is now also defective in HAPT1-mediated pentamidine uptake. Cross-resistance profiles for pentamidine, diminazene and melaminophenylarsenicals, how-ever, once again point to complexities, since simple cross-resistance profiles consistent with a simple two-transporter explanation [30] are not evident. Another diamidine, DB75, or furamidine, whose methoxy prodrug pafuramidine (DB289), passed through clinical trials before being abandoned due to toxicity issues [27], was also shown to be carried principally via the P2 transporter and loss of the TbAT1 gene also correlated to resistance to that drug, with another minor route, however, also being apparent [31]. In addition, it was shown recently that aza analogs of DB75 (DB820 and DB829) that are active against mouse models of stage 2 disease also enter trypanosomes via the P2 amino purine transporter, but with additional minor routes apparent [32].

A range of lines have now been selected for resistance to melaminophenylarsenical and diamidine drugs. In most cases genetic lesions rendering the P2/TbAT1 transporter inactive have been identified. However, comparison of different lines reveals that the types of mutation

that cause loss of functional P2 activity can be varied. For example, the earliest indications pointed to a series of point mutations in the gene. Other lines from the field were reportedly deficient in the gene, indicating its deletion from the genome. A DB75 resistance-selected line [31] had also deleted the gene, as had a T. brucei gam-biense line selected for arsenical resistance [33]. A separate T. brucei line selected for arseni-cal resistance [34] had kept the gene, however, its transcript could no longer be identified in steady-state RNA purified from these cells. A Trypanosoma equiperdum line selected for dimin-azene resistance had also lost stable RNA without any mutations being present in the coding region of the gene itself [33]. A loss of heterozygosity was apparent in this T. equiperdum line, as the paren-tal strain revealed itself to have two different copies of the gene, differing by only a few base pairs in their flanking regions, while the resistant derivative had only a single allelotype [33]. A key issue relating to the parasite’s ability to mutate in different ways (point mutations, gene deletion or loss of transcription) means that simple genetic tests reporting on the presence and status of the gene might not be applicable in all instances. However, a phenotypic test, in which functional P2 transporter activity can be detected, would be of utility regardless of the underlying genetic changes. Techniques using fluorescence micros-copy to detect uptake of fluorescent diamidines, including DB75 [34] and DAPI [24], have been developed and might find utility in the field, especially as LED-based fluorescence microscopy has been developed in field-usable formats [35].

Much has been learned about the TbAT1 gene that encodes the P2 transporter. Understanding genetic lesions underlying the role of the HAPT1 transporter (and LAPT1) in drug resistance is desirable. It was recently shown by expression in Leishmania parasites that TbNT11.1 and TbNT12.1, members of the equilibrative nucle-oside transporter family, were also capable of carrying pentamidine [36]. Kinetic parameters and substrate recognition profiles [36] were not immediately consistent with these represent-ing HAPT1 or LAPT1, although ongoing work indicates that at least the HAPT1 gene has been identified which should open up ana-lysis into the changes that underlie their roles in drug resistance.

Eflornithine resistanceBacchi et al. [37] studied a series of T. b. rhodesiense isolates that were naturally refractory to eflorni-thine. A range of different observations were made



that might have contributed to the reduced sensi-tivity of these lines. Two of the isolates accumu-lated substantially less drug than sensitive strains. One cloned strain, maintained under eflornithine pressure, showed enhanced putrescine uptake (allowing bypass of constitutively suppressed orni-thine decarboxylase activity). S-adenosyl methio-nine metabolism also appeared to differ between sensitive and resistant lines, the combined pairing of S-adenosylmethionine and its decarboxylated product (which is used as the aminopropyl group donor in polyamine biosynthesis) were greatly ele-vated in sensitive cells after eflornithine treatment. In refractory cells they were still elevated after treatment but to a lesser extent. A more recent study [38] using both eflornithine and RNA inter-ference to inhibit ornithine decarboxylase pointed to elevated decarboxylated S-adenosylmethionine but, in this case, not its precursor. It has also been proposed [39] that T. b. rhodesiense being refrac-tory to eflornithine might relate to its ornithine decarboxylase enzyme having a much shorter half-life than the ornithine decarboxylase of T. b. gambiense, in which case T. b. rhodesiense can gen-erate new enzyme to replenish activity lost after the covalent inactivation of the inhibitor bound enzyme. Consistent results relating to metabolic events associated with eflornithine treatment have been difficult to derive.

Another early study [40] indicated that eflor-nithine’s entry into trypanosomes might be via passive diffusion based on apparent nonsaturabil-ity of uptake (using a 60 min uptake time frame) and a failure of common cationic amino acids to inhibit uptake. However, passive diffusion of the polar zwitterionic eflornithine seems unlikely, as its biophysical characteristics would preclude partitioning into the lipid environment of the membrane, and even in that study temperature sensitivity of uptake indicated that passive diffu-sion was not likely. Two independent studies in procyclic trypanosomes selected for resistance to eflornithine actually pointed to them possessing a transporter for the drug, which was lost in the selection of resistance [41,42]. Recently, two blood-stream form lines were selected for resistance to eflornithine in vitro and both were shown to have decreased their ability to accumulate eflornithine, while changes to ornithine decarboxylase were not apparent and the cellular metabolome was not changed between wild-type and eflornithine-resistant derivatives [43]. The resistance phenotype was maintained in vivo. This appeared to relate to loss of transport of the drug and ana lysis of the amino acid transporter gene repertoire (under-taken because eflornithine is an amino acid and

thus hypothesized to enter via an amino acid car-rier) indicated a single transporter (TbAAT6) was lost, by gene deletion, in both lines. Furthermore, RNAi experiments to downregulate expression of TbAAT6 led to selection of cells resistant to eflornithine and expression of an exogenous copy of TbAAT6 in a cell line selected for eflornithine resistance from which the gene was deleted led to restoration of the eflornithine sensitivity phe-notype. It appears, therefore, that eflornithine is transported into trypanosomes via an amino acid transporter (TbAAT6) and that loss of this trans-porter is sufficient for the selection of high-level resistance to the drug. Two independent studies, described in the ‘new techniques to identify resis-tance genes’ section, using genome scale RNAi also identified a role for TbAAT6 [44,45]. Whether this mechanism is also used in the field has yet to be established, and given the other possible mech-anisms discussed previously, it will be important to distinguish these as parasites from eflornithine treatment failures are isolated for ana lysis.

NifurtimoxFollowing its use off-license in trials, nifurtimox has recently been introduced for treatment of stage 2 HAT as part of the NECT [46]. Nifurtimox alone is a poor trypanocide, its in vitro IC

50 values

against trypanosome growth being around a 1000-fold higher than potent trypanocides including melarsoprol, pentamidine and suramin (although eflornithine is also relatively poor). Nifurtimox is a nitroheterocyclic (nitrofuran) compound. The drug has been used against Trypanosoma cruzi, the causative agent of Chagas disease, since the 1970s [47]. One hypothesis suggested that single electron reduction of the nitro group in nifur-timox would cause oxidative stress due to futile redox cycling whereby a single electron reduction of drug leads to a cycle of reduction–oxidation generating a continuous supply of superoxide that can be further metabolized to hydrogen peroxide. The possibility that nifurtimox generated oxida-tive stress whilst eflornithine caused a reduction in polyamine biosynthesis, and ultimately bio-synthesis of the key redox-protecting metabolite trypanothione, provided a useful rational basis to explain why nifurtimox and eflornithine might act well as a combination chemotherapy. A recent review describes the process in the context of try-panosome drug resistance [48]. However, it tran-spires that nifurtimox and eflornithine are not synergistic in their activity against trypanosomes [43] and, furthermore, there is no indication that either drug increases entry into the CSF for the other in mice [49].



The lack of synergy might be explained by recent advances in understanding of how nifurti-mox actually exerts its activity. It was shown that trypanosomes possess a type 1 nitroreductase enzyme [50], of a type usually found in bacteria. The enzyme is associated with the trypanosome’s mitochondrion and its physiological role has not been described. In T. brucei the gene encoding the enzyme could not be knocked out, indicating it is essential [50], although single knockouts are possible and its expression can be downregulated using RNA interference. Overexpression of the enzyme led to hypersensitivity to nifurtimox and other nitroheterocycles [51] and reduced expres-sion led to reduced sensitivity, indicating a role of the enzyme in the activity of nitroheterocycles. Type 1 nitroreductases are usually involved in two-electron reduction of nitroheterocycles, rather than the single-electron reduction that provokes oxidative stress. Recently it was shown that nifurtimox is indeed subject to two-electron reduction, which leads to further metabolism yielding a highly toxic open-chain nitrile that is now proposed to cause cellular death due to its interaction with a range of cellular targets [50].

Sokalova et al. [51] recently selected trypano-somes resistant to nifurtimox and showed that the parasites were also resistant to fexinidazole, a nitroimiadzole whose trypanocidal activity was determined in the 1980s and is currently in Phase I trials for stage 2 HAT [52]. Cells selected for fexinidazole were also cross-resistant to nifurtimox. Interestingly, absolute degrees of resistance and cross-resistance were not identical in the nifurtimox- or fexinidazole-selected cell lines, which indicated the possibility of different mechanisms of resistance. Furthermore, point mutations in the NTR gene were not identi-fied in resistant lines, although reduced levels of expression could not be ruled out, thus a role for TbNTR in selected resistance to nifurtimox, or fexinidazole, is yet to be proven. The fact that nifurtimox and fexinidazole cross-resistance is easily selected, however, raises an important point: the use of nifurtimox could, in principle, select for genotypes that would yield resistance to fexinidazole even before that latter drug were released in the field. Learning more about nifur-timox (and fexinidazole) resistance is therefore of considerable importance.

New techniques to identify resistance genes Classically, genes involved in drug resistance in trypanosomes have been identified following selection of parasites for resistance to increasing

doses of drug in vitro or in vivo, then seeking individual genes related to resistance based on hypotheses related to likely mechanisms, and finally investigating changes to those genes that distinguish drug-sensitive and drug-resistant cell lines. The last decade has seen an explo-sion in techniques that allow high throughput investigations into cellular changes at the level of the genome, transcriptome, proteome and metabolome that might relate to resistance. To date no reports on the successful identification of genes associated with drug resistance in trypano-somes using these particular functional genom-ics approaches have been published, although all methods are being used and interesting results likely to emerge in the near future.

One area in the functional genomics arena, however, where advances have already been made involves the use of RNAi to identify loss of function mutations that can underlie drug resistance. RNAi is a mechanism distributed patchily throughout the eukaryotes and is thought to have a role in defense against RNA viruses. The process essentially involves recog-nition of dsRNA followed by the generation of siRNA species that bind to target RNAs and lead to their destruction. African trypano-somes possess the cellular machinery enabling RNAi (although T. cruzi does not, and of the Leishmania species only Leishmania brazilien-sis so far appears to be RNAi-competent). By transfecting T. brucei with constructs that allow either production of single RNA molecules that can fold into double-stranded, base-paired stem-loop structures, or else by simultaneously tran-scribing the same inserted DNA molecule on both strands, dsRNA is produced in situ and it is possible to selectively target RNA molecules for knockdown. The method has been widely employed to study gene function in trypano-somes. Furthermore, it is possible to produce a genome-covering collection of fragments cloned into vectors for RNAi [53]. These can then be transfected into trypanosomes and if induced will lead to downregulation of the protein they encode. If the loss of a gene’s function is able to lead to resistance these parasites will be selected for growth in medium containing drug. This method was recently used to great effect in two studies. Baker et al. [45] reported results of selections of trypanosomes transformed with an RNAi library and subsequently exposed to nifurtimox or eflornithine. The parasites selected in eflornithine carried a construct lead-ing to downregulation of TbAAT6 (as described above). In the case of nifurtimox, the NTR gene



was identified. Interestingly, in this latter case the RNAi phenotype’s incomplete knockdown was probably important given that the gene is believed to be essential (i.e., knockdown can reduce activity sufficiently to diminish activa-tion of nifurtimox without leading to cell death that would be associated with total loss of nitro-reductase). In an independent study Schumann-Burkard et al. [44] selected for resistance to eflo-rnithine and melarsoprol. They also identified loss of TbAAT6 gene function in the selection of eflornithine resistance and loss of TbAT1 in selection of melarsoprol resistance.

Recent laboratory data [David Horn, Unpublished

Observations] is indicating that genes encoding proteins involved in endocytosis are frequently lost in RNAi experiments selecting for decreased sensitivity to suramin, which is consistent with the fact that the drug enters via receptor-medi-ated endocytosis bound to serum lipoprotein complexes.

The RNAi approach is therefore validated as having the potential to identify loss of func-tion mutations necessary for drug resistance. Another approach, e.g., systematic overexpres-sion of libraries of trypanosome genes might also allow selection of ‘gain of function’ mutations responsible for resistance, as might be expected, for example, with expression of P-glycoprotein resistance pumps, or else drug targets whose overexpression would increase the amount of drug required to inhibit activity.

Future perspectiveThe incidence of HAT has declined dramatically in the last decade following concerted interna-tional efforts led by the WHO in response to a dramatic surge in the disease at the end of the 20th century. Chemotherapy has been essential

in the fight against HAT. However, resistance to all known drugs can be selected in the labora-tory and the incidence of treatment failures in the field, particularly with melarsoprol, rose sig-nificantly with protracted use of the drug. With no new trypanocides currently in clinical trials beyond Phase I, no new drugs will be on the market for at least 5 years. The most advanced drug, fexinidazole, is a nitroheterocycle; cross-resistance between that drug and nifurtimox (a component of the NECT regimen) is easily selected, as is resistance to eflornithine. Hence it is possible that resistance genes to fexinida-zole could be circulating in the field (after selec-tion with nifurtimox) even prior to its registra-tion. It will be important to continue to study mechanisms of resistance to all drugs in order to develop the diagnostic tools that can identify and trace the emergence and spread of resistance, and to implement policies, for example alternating current drugs if new ones are not forthcoming, to stem the spread of drug resistance.

Financial & competing interests disclosureMP Barrett, E Matovu and AJN Kazibwe thank UBS-Optimus Foundation for funding. MP Barrett, RJS Burchmore and IM Vincent thank the BBSRC and Pfizer for funding. E Matovu and AJN Kazibwe hold a Senior Post-Doctoral Fellowship from the European Foundations Initiative for African Research into Neglected Tropical Diseases (EFINTD). MP Barrett is Chairman of the Kinetoplastids Drug Efficacy Group of the WHO. The authors have no other relevant affi li- The authors have no other relevant affili-ations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Executive summarynConcerted international efforts have brought the prevalence of human African trypanosomiasis to levels beneath 10,000 reported cases

per annum.nPatient treatment with chemotherapy has been the cornerstone of these efforts.nAn increase in treatment failures with melarsoprol has been reported since the 1990s.nLaboratory studies indicate that loss of drug uptake through the TbAT1/P2 and HAPT1 transporters can underlie this resistance.nThe same two transporters are also involved in pentamidine uptake and their loss in resistance to that drug, although field reports of

resistance to pentamidine are scarce.nEflornithine has replaced melarsoprol as first-line treatment for stage 2 disease, and the combination of this drug with nifurtimox is

increasingly implemented.nEflornithine resistance is easily selected in the laboratory through loss of an amino acid transporter. Nifurtimox resistance is also

easily selected.nKnowledge gained from understanding drug resistance in the laboratory should be put to effect in tracking possible emergence and

spread of resistance in the field.nNew drugs that act via mechanisms that will not enable cross resistance to current drugs should be sought.



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Future Microbiology Review Drug resistance in human African trypanosomiasis

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