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Drug resistance and Giardia
ParasitologyToday,vol. 9, no. 5, 1993
187
Drug Resistance and Giardia J.A, Upcroft and P. Upcroft Giardiasis is a worldwide disease that can cause serious morbidity. Metronidazole is the current recommended drug for treatment, and is mostly still effective. However, Giardia duodenalis, the causative agent, is capable of developing resistance to high levels of metronidazole and other drugs, in vitro, via a number of mechanisms. Resistance, in viva, has been reported and many cases of treatment failure have been variously attributed to a number of causes, including resistance. Here, Jacqueline and Peter Upcroft ask" is this the beginning of another chapter of drug resistance? or is the situation likely to remain as a 'few refractory cases'? Should we wait to find out or can we act positively to avert the possibility of yet another valuable drug in our limited pharmacopoeia becoming obsolete?
Enteric pathogenic organisms can be asymptomatic, so that carriers in a community remain undetected. In some communities, the prevalence of organisms such as Giardia duodenalis is very high and in a typical study of Australian aboriginal children, the incidence of Giardia was found to be 32% (Ref. I). It is generally agreed that, although carders of Giardia may be asymptomatic, heavy parasite burdens are detrimental to the well-being of the host. In the extreme, giardiasis can be a debilitating disease resulting in malabsorption and failure-to-thrive syndrome in children 2. Antigiardial
Drugs
The drugs commonly used to treat giardiasis (Box I) are metronidazole (Flagyl, Zion) 3,4 and other members of the 5-nitroimidazoles, especially tinidazole (Fasygyn) which is unavailable in the USA. Furazolidone (Furoxone) 4, a nitrofuran, which is reported to be as effective as metronidazole, is not as widely used, and is unavailable in Australia5. Other drugs, including quinacrine, are effective antigiardials and, more recently, the benzimidazoles, albendazole and mebendazole, have been suggested as suitable alternatives to the 5-nitroimidazoles ~. These drugs are more potent, in vitro, against Giardia than metronidazole, but (from available reports) appear to be similarly effective, in vivoL The macrolide, © 1993,ElsevierSciencePublishersLtd, (UK)
azithromycin, is effective, in vitro, but is not effective in the neonatal mouse model 6. Other drugs have antigiardial activity (Box I) but most have unpleasant side-effects s that may affect compliance. For reasons of efficacy, bioavailability, cost and general tolerance metronidazole is the drug of choice to treat giardiasis. However, it is used worldwide (Box 2) for the treatment of anaerobic protozoan parasites and anaerobic bacteria and is used widely as a prophylaxis pre- and post-operatively in abdominal surgery. Metronidazole has been available for some 30 years, and is now so widely used in veterinary medicine and human clinical medicine that increased metronidazole resistance in a number of organisms that infect humans and a wide range of animals, may well be anticipated. Indeed the situation with Trichomonas vaginalis is serious (see P. Johnson, this issue), and with no effective alternative available to treat trichomoniasis and its associated problems the situation is likely to worsen. Treatment of Enb amoeba histolytica is no less tenuous since metronidazole is the favoured alternative to emetine 7. With a limited choice of effective drugs to treat the anaerobic protozoa, and the enormous expense involved in developing new drugs, prudent administration of valuable chemotherapeutics should be exercised. Among the protozoa, Plasmodiurn falciparum rates as one of the world's top-ten drug-resistant microbes 8, while metronidazole resistance in 71 vaginalis has been widely reported (P. Johnson, this issue). In retrospect, it is clear that resistance in these organisms can often be attributed to the inappropriate use or abuse of cheap, effective and well-tolerated drugs9. With this in mind, it cannot be
too highly stressed that, every time metronidazole is prescribed for prophylaxis in a developing country, there is an excellent chance that T. vaginalis, E. histolytica and G. duodenalis will be exposed. In these situations, the welldocumented variations in drug sensitivities of individual organisms and isolates, in Giardia for example ~°, will select the least sensitive within the natural population, possibly leading to drug-resistant organisms. Standard recommended treatment of giardiasis with metronidazole is administration of 2 g once daily for three days; or 200 mg thrice daily for ten dayss. The drug is rapidly and almost completely absorbed, and peak serum levels of 14 I~g ml ~ following administration of 500 mg of metronidazole can be expected with a half-life of around seven hours (Ref. 3). The concentration of the drug in the gut is unknown, but it is likely to be significantly less than that in the serum. Inhibitory concentrations of antigiardial drugs have been presented in a number of ways. Majewska and colleagues examined a number of laboratory strains and quoted ICs0 values (the concentration of drug which decreased the number of trophozoites to 50% of the control values in three days) which range from as low as 0.01 i~g ml t to I I~g ml I of metronidazole ~°. Boreham, Phillips and Shepherd showed that there was considerable variation in the sensitivity of different stocks of Giardia to metronidazole and other drugs ~. These authors measured the inhibition by the drug radiometrically, and presented ID50 values (the concentration of drug which inhibits the uptake of [3H]thymidine by 50% over a four-hour period during exponential growth) of around I IIM (0.2 I~g ml i) for most stocks. Using the inhibition of adherence
B o x I. Effective A n t i g i a r d i a l A g e n t s M e t r o n i d a z o l e and t i n i d a z o l e - members of the 5-nitroimidazole family of nitroheterocyclic drugs s. F u r a z o l i d o n e - a nitrofuran s. Q u i n a c r i n e - alone or in combination with metronidazoleS, 46. A l b e n d a z o l e and m e b e n d a z o l e - members of the benzimidazole family of anthelmintic drugs, and as effective as metronidazole in clinical trialsL OL-propranolol - in combination with m e t r o n i d a z o l e to treat metronidazoleresistant giardiasis 47. P a r o m o m y c i n - recommended during pregnancy 48. A z i t h r o m y c i n - active in vitro 6,27.
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Parasitology Today, vot. 9, no. 5, 993
Box 2. C u r r e n t Uses for Metronidazole As antiprotozoa/agents: in combination with melarsonyl potassium to treat T~/pan0soma brucei in mice49; to treat Entamoeba histolytica 7, Trichomonas vaginalis s°, Giardia duodenalis s, Blastocystis hominis shs2 and Leishmania s3.
As an antibacterial agent." to treat infections with Helicobacter pylori, the suspected causative agent of duodenal ulcers24,s4; and to treat anaerobes most commonly found in the gastrointestinal tract, including Bacteroides, Eubacterium, Peptococcus, Peptostreptococcus, Clostridium and Fusobacterium species (NB some strains of B. fragilis, B. melaninogenicus and some Clostridium species are metronidazole resistant26). In the treatment of pulmonary acariasis in China, successfully treating most patients~S; periodontitis, a common cause of tooth loss as a result of overgrowth of anaerobic spirochetes and Bacteroides species'6; chronic anaerobic pneumonitisSS; rosacea and acneS6; chronic suppurative otitis mediaST; and Crohn's diseasesS. As a prophylactic: for caesarean sectionSg; and pre-operatively for acute appendicitis 6°.
of trophozoites to monolayers of Caco2 cells, Favennec and colleagues estimated that the concentration of metronidazole required to obtain a 50% reduction in adherence (ID50) was 1.5 I~g ml I (Ref. 12). It can be seen that these values which refer to inhibitory rather than lethal concentrations of drug are I0 I O0-fold less than the serum concentrations of metronidazole following a 500 mg dose. In 1988, Boreham, Phillips and Shepherd reported an eightfold decrease in sensitivity to metronidazole of a laboratory line, BRIS/83/HEPU/IO6-21D m, maintained in a sublethal concentration of metronidazole t3. More recently, we have induced drug resistance in four Giardia strains by a number of methods H. The first method, which involved exposure of parasites to intermittent but increasing concentrations of drug, is analogous to single-dose prophylaxis or therapy. Under these conditions, we were able to induce resistance to metronidazole such that parasites were 400-fold less sensitive to drug, and by repeating earlier work 13 we were able to decrease the sensitivity by more than tenfold. Ultraviolet (UV) mutagenesis enabled us to select lines that were able to grow in an IDs0 that was I00 times that of the parent line 14. While the above discussion presents a range of values, it is logical to assume that maladministration, and a tenfold decrease in sensitivity to metronidazole could lead to the selection of resistant Giardia parasites, in vivo. Doses of metronidazole lower than 500 rag, such as the standard recommended dose of 200 mg thrice daily (and similar low doses given to treat acariasis in China Is and to reduce the need for periodontal surgery in Kuwaitl6), will certainly compromise the effectiveness of this antigiardial drug. The risk posed by the induction of drug resistance in
the other anaerobic protozoa using these regimes is likely to be at least equivalent. Significant Trichomonas resistance already occurs and inactivation of metronidazole by gut flora in the treatment of Entamoeba, as reported in the application of metronidazole as a suppository 17, may greatly reduce the bioavailability when compared with serum levels. Inaccessibility of amoeba in abscesses to the drug will also contribute to a lower therapeutic concentration of drug than is available in the serum. Because it is uncertain whether Giardia is a zoonosis, the same precautions and considerations must be exercised in administering anti-protozoal agents to livestock so as to reduce the chance of selecting drug resistant strains. Drug resistance in Salmonella, for example, has been linked to antimicrobial use in food animals m, Drug-resistant Organisms
Three approaches can be taken to limit selection for drug-resistant Giardia. • The first is to conserve the useful life of presently available drugs by prudent administration, as suggested above. Therapy with combinations of existing agents is a viable approach to preserving the effectiveness of antigiardials. • The second is to produce or design new drugs that may be modifications of existing drugs. We need to understand the mechanisms of drug resistance to prevent or circumvent its occurrence. This aspect of Giardia is covered below. Once the molecular mechanisms are known, modifications of existing drugs may be possible. The production of amikacin, a modified derivative of kanamycin that is unable to bind to bacterial enzymes that destroy kanamycin 8, is an example of
this. Targeting of essential, unique metabolic pathways and proteins presents options for analogue drug design. In Giardia, the arginine-dihydrolase pathway 19 and parasite-specific proteins, such as the cysteine-rich surface proteins 2° and the giardins2L are potential drug targets. • The third approach to limit selection for drug-resistant Giardia, the production of a vaccine, has had a positive boost from Olsen, Morck and Ceri who, at a recent Giardia conference 22, presented the results of immunizing kittens against Giardia. Resistance in organisms other than Giardia against the drugs used to treat giardiasis is not uncommon, and has been reported in Bacteroides 23, Helico bacter pylon 2<2s, Clostndium26 and Tri chomonas (P. Johnson, this issue). One mechanism of resistance to metronidazole is believed to operate via prevention of drug reduction to toxic intermediates by altering the levels or characteristics of the components of electron transport in these organisms (Ref. 27 and P. Johnson, this issue). Pyruvate : ferredoxin oxidoreductase (PFOR), which donates electrons to ferredoxin, is involved in metronidazole resistance, and, in T vaginalis, interception and detoxification of radicals generated via ferredoxin reduction of metronidazole has been reported (P. Johnson, this issue). Among the bacteria, there is evidence that the activation and mechanism of metronidazole resistance occurs via pathways other than that involving PFOR and ferredoxin. Wehnert, Abratt and Woods reported a plasmid, pMTIO0, carrying a Bacteroides fragilis insert that conferred increased resistance to Eschenchia coil, together with increased sensitivity to far-ultraviolet light 28, This plasmid has several small open reading frames, one of which (encoding a 64 amino acid protein) is responsible for the metronidazole resistance. However, this protein has no significant homology with known protein sequences 28. Other systems involved in bacterial metronidazole activation include nitrate and chlorate reduction in E. coil, and a flavodoxin gene, that is linked to a hydrogenase gene, in Clostndium acetobutylicum 29. Furthermore, it has been shown that some strains of bacteria can protect otherwise metronidazolesensitive strains from the drug 3°. Furazolidone is reduced, in vivo, to cytotoxic products, in a similar manner to the 5-nitroimidazoles. The reduction potential of furazolidone is greater than
Parasitology Today, vol. 9, no. 5, 1993
metronidazole and it is reduced via NADPH/NADH oxidase to its nitroanion radical 3~. Resistance to the nitrofurans has been reported in E. coil isolated from patients suffering from urinary tract infections 32 and in Salmonella enteritidis isolated from broiler chickens 33. The mechanism of resistance in S. ententidis was attributed to a decrease in the ability of the bacteria to reduce furazolidone 34. The importance of depletion of the drug-derived toxic radicals by glutathione cycling, for example, has been raised3s, and a mechanism of resistance to nitrofurans, whereby enzyme activities of thiol detoxification pathways are increased, has been proposed 36.
Mechanisms of Drug-resistance in Giardia Failure to treat Giardia successfully with metronidazole and furazolidone has been previously reviewed 27,37. Drug-resistant organisms have been isolated from patients with giardiasis refractive to metronidazole 38 and furazolidone resistant strains have been isolated from patients refractory to furazolidone 39. Our results have shown that resistance in G. duodenalis can readily develop via a number of mechanisms 14. Some of these are similar to those already described and involve the electron-transport systems in Giardia. The negative correlation between PFOR and metronidazole sensitivity of G. intestinalis strains36 has been corroborated by the work of Ellis and colleagues, who showed that resistance of the line BRIS/83/HEPU/106-21D~0 to metronidazole was consistent with reduced production of toxic radicals by decreased PFOR activity ~°. Also consistent with these reports is the purification of ferredoxin from Giardia, which we have shown to reduce metronidazole, in vitro 4t. The involvement of thiol detoxification pathways in furazolidone resistance in Giardia is not clear. Smith and colleagues correlated a decrease in furazolidone sensitivity with an increase in thiol cycling in Giardia, which suggested increased detoxification of furazolidone derived radicals in the less-sensitive strains36. We have been unable to reproduce these data and find that, like E. histolytica 42, cysteine is the only significant low-molecularweight thiol in the parasite grown in TYI-S-33, and that thiol-cycling enzyme activities using exogenous glutathione as substrate were not detected 43.
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Trichomonas also appears to maintain cysteine as the major low-molecularweight thiol (D. Brown and P. Upcroft, unpublished). Thus, without high levels of glutathione, these anaerobic protozoa appear to support novel detoxification mechanisms. Each drug-resistant line that we have examined has undergone chromosomal rearrangement. In the case of the metronidazole-resistant line BRIS/ 83/HEPU/106-21D~0, deletion of a gene from one chromosome has been observed 44. This deletion involves a portion of a partially duplicated chromosome in the strain BRIS/83/ HEPU/106 (Ref. 45). The absence of the gene is also correlated with aberrant growth and the protein that it encodes is a membrane-associated protein. Although this protein has not yet been described in any other organism, it contains highly conserved modules of transport and oxidoreductase proteins, the deletion of which is consistent with the involvement of electron transfer processes in metronidazole resistance. Also consistent with involvement of transport functions is the altered drug-transport and multipledrug-resistant characteristics of the lines that we have examined. All of the resistant lines that were selected in high levels of drug following UV irradiation 14 have partial duplications of at least one chromosome when compared with their parent strains*. In the case of three lines derived from different parent strains and selected in high levels of metronidazole or furazolidone, the same chromosome was involved in the duplication in each case (J.A. Upcroft, N. Chen and P. Upcroft, unpublished).
Conclusion Drug resistance in the pathogenic microorganisms is a serious challenge to the primary health-care system. It is clear from the lessons already learned that we must economize existing effective drugs to extend their useful lives. If we are to learn from previous mistakes in treatment (eg. chloroquine resistance in Plasmodium and metronidazole resistance in Trichomonas), we must anticipate that it is unlikely that devel-
* Induction of drug resistance in these lines involved UV irradiation with 3.5 J m 2 for 10 min, recovery for 48 h, followed by selection in ten times the IDs0 level for furazolidone and 100 times the IDs0 for metronidazole, of the respective parent strains.
opment of new drugs will keep up with the development of resistance and that the cost of producing new drugs will become increasingly prohibitive. Metronidazole is a very effective, cheap drug and is at serious risk of becoming obsolete, if this is not already the case. The obvious precautions that should be exercised by physicians, health workers and veterinarians are not to administer low doses of metronidazole, and to avoid the use of metronidazole as a prophylactic, especially where Giardia, Entamoeba and Trichomonas are likely to be present. Where possible, alternative and combination therapy should be employed. Drug resistance in Giardia is not yet an overt problem, but it is clear that metronidazole resistance occurs, in viva, and can readily be induced, in vitro. We should not be complacent about metronidazole and the other antigiardials and should make every effort to avert the inevitable consequences of maladministration and non-compliance. References
I Reynoldson, J.A., Thompson, R.C.A. and Horton, R.J. (1992) Parasitology Today 8, 412~414 2 Craft, J.C. (1982) Pediatr. Infect. Dis. I, 196 211 3 Scully, B.E. (1988) Meal Clin N. Am. 72, 613 621 4 Chabala, J.C. and Miller, M.W. (1986) in Chemotherapy of Parasitic Diseases (Campbell, W.C. and Rew, RS., eds), pp 25 85, Plenum Press 5 Boreham, P.F.L. (1991) Pharmaceut. J. 234, 271 274 6 Boreham, P.F.L. and Upcroft, J.A. (1991) Trans. R. Sac. Trap. Mad Hyg. 85, 620- 621 7 Knight, R. (1980)J. Antimicrob. Chemothe: 6, 577 593 8 Gibbons, A. (1992) Science 257, 1036 1038 9 Looareesuwan, S., Harinasuta, T. and Chongsuphajaisiddhi, T. (1992) Southeast Asian J. Trap. Mad Public Health 23, 621 -634 10 Majewska, A.C. et al. (1991) Trans. R Sac. Trap. Med. Hyg. 85, 67 69 I Boreham, P.F.L., Phillips, R.E. and Shepherd, R.W. (1984) J. Antimicrob. Chemother 14, 44946 I 2 Favennec, L. et al. (1992) Parasitol. Res. 78, 80 81 3 Boreham, P.F.L., Phillips, R.E. and Shepherd, R.W. (1988) Trans. R. Sac. Trap. Meal Hyg. 82, 104 106 14 Townson, S.M et al. (1992) Trans. R. Sac. Trap. Meal Hyg. 86, 521 522 15 Chen, X.B., Sun, X. and Hu, S.F. (1990) Chung Kuo Chi Sheng Chung Hsueh Yu Chi Sheng Chung Ping Tso Chih 8, 217 219 16 Loesche, W.J. et al. (1991 ) J. Trap. Mad Hyg. 94, 118 122 17 Gerding, D.N. (1987) in Antimicrobial Agents Annual Vol. 2 (Peterson, P.K. and Verhoef, J., eds), pp 131 136, Elsevier 18 Cohen, M.L. and Tauxe, RV, (1986) Science 234, 964 969 19 Schofield, P.J. et al. (1992) Mol. Biochem. Parasitol. 5 I, 29 36
190 20 Nash, T. (1992) Parasitology Today 8, 229 234 21 Peattie, D.A. et a/. (1989)J. Cell Biol. 109, 2323 2335 22 Olson, M.E., Morck, D.W. and Ceri, H. in Giardia: From Molecules to Disease (Thomp son, R.C.A, Reynoldson, J.A. and Lymberry, A.J., eds), CAB International (in press) 23 Britz, M.L. and Wilkinson, R.G. (1979) Antimicrob. Agents Chemother. 16, 19 27 24 Weil, J. et al. (1990) Lancet 336, 1445 25 Haas, C.E., Nix, D.E. and Schentag, J.J. (I 990) Antimicrob. Agents Chemothe~ 34, 1637 164 I 26 Elliot,, T.S. and Stone, J.W. (1990) Aliment~ Pharmacol. Therap. 4, 227 238 27 Upcroft, J.A., Upcroft, P. and Boreham, P.F.L (I 990) Int. J. Parasitol. 20, 489496 28 Wehnert, G.U., Abratt, V.R. and Woods, D.R. (1992) Plasmid 27, 242-245 29 Santangelo, i.D., Jones, D.T. and Woods, D.R. ( 1991 ) J. Bacteriol. 173, 1088 1095 30 Nagy, E. and Foldes, ]. (1991) J. Antimicrob. Chemothe~ 27, 63 70 31 Moreno, S.J., Mason, R.P. and Docampo, R. (1984) J. Biol. Chem. 259, 8252 8259 32 Breeze, A. and Obaseiki-Ebor, E.E. (1983) J. Antimicrob Chemother. 12, 459-467 33 Rampling, A. Upson, R. and Brown, D.F.J. (1990) J. Antimicrob. Chemother. 25, 285 290 34 Brown, D.F.J. et al. (1991) J. Antimicrob. Chemothe~ 27, 23-28 35 Biaglow, J.E. et a/. (1986) Biochem. Pharmacol. 35, 77 90
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36 Smith, N.C., Bryant, C. and Boreham, P.F.L. (1988) Int. J. Parasitol. 18, 991 997 37 Boreham, P.F.L., Upcroft, J.A. and Upcroft, P. (1991) in Biochemical Protozotogy (Coombs, G. and North, M., eds), pp 594 604, Taylor & Francis 38 Kortbeek, L.M. et al. in Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymberry, A.J., eds), CAB International
(in press) 39 Mclntyre, P. et al. (1986) ]. Pediat~ 108, 1005 1010 40 Dis, J.E. et at. Int.j. Parasitol. (in press) 41 Townson, S.M., Upcroft, J.A. and Upcroft, P. (1993) in Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymberry, AJ., eds), CAB International (in press) 42 Fahey, R.C. eta/. (1984) Science 224, 70 72 43 Brown, D.M., Upcroft, J.A. and Upcroft, P. in Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymberry, A.J., eds), CAB International (in press) 44 Upcroft, J.A. et al. (1992) Parasitology 104, 395~t05 45 Upcroft, J.A., Healey, A. and Upcroft, P. lnt~ J. Parasitol. (in press) 46 Taylor, G.D., Wenman, W.M. and Tyrell, D.LJ. (1987) Can. Meal Assoc. J. 136, fl79 1180 47 Popovic, O.S. and Milovic, V. (1990)J. Clin. Gastroenterol. 12, 604--605 48 Kreutner, A.K., Del Bene, V.E. and Amstey,
49 50 51 52 53 54 55 56 57 58 59 60
M.S. (198t) Am. J. Obstet. Gynecol. 140, 895 899 Jennings, F.W. (1991) Trop. Meal Parasitol. 42, 139 142 Lossick, J.G. and Kent, H.L. (1991) Am. ] Obstet. Gynecol. 165, 1217 1222 Lee, M.G. et al. (1990) Ann. Rheum. Dis. 49, 192~ 193 Boreham, P.F.L. and Stenzel, D.J. Adv. Parasitol (in press) Arora, S.K., Sinha, R. and Sehgal, S. (1991) Meal Micrabiol. ImmunoL 180, 21 27 Korovina, N.A. et al. (1991) Antibiot. Khimioter. 36, 46~t8 Shah, A. et al. (1990) Indian ]. Chest Dis. Allied Sci. 32, 117 120 Akamatsu, H. et al. (1990) Arch. DermatoL Res. 282, 449 454 Rotimi, V.O. et al. (I 990) West Af~ J. Med. 9, 89-97 Sutherland, L. et al. (1991) Gut 32, 1071 1075 de Boer, C.N. and Thornton, J.G. (1989) Int. j Gynecot. Obstel_ 28, 103 107 Dahlstrom, B.L., Reiertsen, O. and Rosseland, A.R. (1990) Tidssk~ Non Laegeforen. II0, 1539 1540
]acqueline Upcroft and Peter Upcroft are at The Queensland Institute o f Medical Research, The Bancroft Centre, 3 0 0 Herston Rd, Herston, Queensland 4029, Australia.
Resistance to Clinical Drugs in African Trypanosomes C.J. Bacchi Drug resistance in African trypanosomes continues to confound clinicians and to stymy development of equatorial Africa, taking its toll in lives and economic development. Drugs in current, widespread use have been employed continuously for over 60 years in some instances. The recent studies of Fairlamb and colleagues ~,2 have outlined a defective punne-transport system in drug-resistant trypanosomes, which appears to explain resistance to several established trypanocides and suggests a guide for the development of new drugs. The recently developed agent DL-~-difluoromethylornithine (DFMO) is effective against West African, but not East African, disease3 and its activity may be the result of the unregulated synthesis of S-adenosylmethionine in trypanosomes 4. In this report, Cyrus Bacchi outlines recent developments in the elucidation of mechanisms of resistance to established drugs and naturally occurnng resistance to DFMO. Resistance to clinically effective agents in the treatment of African sleeping
sickness has spread as drugs in use for 40 years or more continue to be used in field clinics3,5. Reliance has centered on the arsenical melarsoprol (Arsobal, Mel B), the diamidines diminazene aceturate (Berenil) and pentamidine, and the sulfonated naphthylamine dye suramin (Fig. I). Suramin is used for early-stage disease when there is no evidence of parasites in the central nervous system (CNS). Pentamidine is used for the treatment of early-stage disease, and for prophylaxis, whereas melarsoprol remains the standby for late-stage, central nervous system disease3,6. Berenil, approved for use in cattle, has replaced pentamidine in some areas for the treatment of earlystage human disease3. The success of early trials and recent approval by the US Food and Drug Administration of the polyamine biosynthesis inhibitor eflomithine (DL-~-difluoromethylomithine; DFMO) has spurred expanded trials of this agent in West Africa, especially against Trypanosoma brucei gambiense in latestage arsenical-refractory disease3,6,7,
D e v e l o p m e n t of Resistance In many trypanosomiasis field clinics, the need to treat patients as quickly as possible, the limited availability of adequate diagnostic tests and the minimal laboratory testing facilities available lead to the rapid deployment of trypanocides. Indices for detecting CNS involvement, and hence the type of treatment, are somewhat arbitrary. Patients tend to leave clinics before completing a course of treatment, returning to receive the same drug after they relapse weeks or months later. Adverse side reactions to some agents or diagnostic procedures (eg. lumbar puncture) do not encourage the completion of therapeutic regimens and hinder follow-up of patients after treatment 3,s. These aspects of clinical reality only partially explain how resistance to trypanocidal drugs develops. Today, resistance to melarsoprol as well as to the diamidines is well recognized, while suramin resistance appears to be less frequently encountered s,8,9, © 1993, Elsevier Science Pubhshers I td, (UK)
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Drug Resistance and Giardia J.A, Upcroft and P. Upcroft Giardiasis is a worldwide disease that can cause serious morbidity. Metronidazole is the current recommended drug for treatment, and is mostly still effective. However, Giardia duodenalis, the causative agent, is capable of developing resistance to high levels of metronidazole and other drugs, in vitro, via a number of mechanisms. Resistance, in viva, has been reported and many cases of treatment failure have been variously attributed to a number of causes, including resistance. Here, Jacqueline and Peter Upcroft ask" is this the beginning of another chapter of drug resistance? or is the situation likely to remain as a 'few refractory cases'? Should we wait to find out or can we act positively to avert the possibility of yet another valuable drug in our limited pharmacopoeia becoming obsolete?
Enteric pathogenic organisms can be asymptomatic, so that carriers in a community remain undetected. In some communities, the prevalence of organisms such as Giardia duodenalis is very high and in a typical study of Australian aboriginal children, the incidence of Giardia was found to be 32% (Ref. I). It is generally agreed that, although carders of Giardia may be asymptomatic, heavy parasite burdens are detrimental to the well-being of the host. In the extreme, giardiasis can be a debilitating disease resulting in malabsorption and failure-to-thrive syndrome in children 2. Antigiardial
Drugs
The drugs commonly used to treat giardiasis (Box I) are metronidazole (Flagyl, Zion) 3,4 and other members of the 5-nitroimidazoles, especially tinidazole (Fasygyn) which is unavailable in the USA. Furazolidone (Furoxone) 4, a nitrofuran, which is reported to be as effective as metronidazole, is not as widely used, and is unavailable in Australia5. Other drugs, including quinacrine, are effective antigiardials and, more recently, the benzimidazoles, albendazole and mebendazole, have been suggested as suitable alternatives to the 5-nitroimidazoles ~. These drugs are more potent, in vitro, against Giardia than metronidazole, but (from available reports) appear to be similarly effective, in vivoL The macrolide, © 1993,ElsevierSciencePublishersLtd, (UK)
azithromycin, is effective, in vitro, but is not effective in the neonatal mouse model 6. Other drugs have antigiardial activity (Box I) but most have unpleasant side-effects s that may affect compliance. For reasons of efficacy, bioavailability, cost and general tolerance metronidazole is the drug of choice to treat giardiasis. However, it is used worldwide (Box 2) for the treatment of anaerobic protozoan parasites and anaerobic bacteria and is used widely as a prophylaxis pre- and post-operatively in abdominal surgery. Metronidazole has been available for some 30 years, and is now so widely used in veterinary medicine and human clinical medicine that increased metronidazole resistance in a number of organisms that infect humans and a wide range of animals, may well be anticipated. Indeed the situation with Trichomonas vaginalis is serious (see P. Johnson, this issue), and with no effective alternative available to treat trichomoniasis and its associated problems the situation is likely to worsen. Treatment of Enb amoeba histolytica is no less tenuous since metronidazole is the favoured alternative to emetine 7. With a limited choice of effective drugs to treat the anaerobic protozoa, and the enormous expense involved in developing new drugs, prudent administration of valuable chemotherapeutics should be exercised. Among the protozoa, Plasmodiurn falciparum rates as one of the world's top-ten drug-resistant microbes 8, while metronidazole resistance in 71 vaginalis has been widely reported (P. Johnson, this issue). In retrospect, it is clear that resistance in these organisms can often be attributed to the inappropriate use or abuse of cheap, effective and well-tolerated drugs9. With this in mind, it cannot be
too highly stressed that, every time metronidazole is prescribed for prophylaxis in a developing country, there is an excellent chance that T. vaginalis, E. histolytica and G. duodenalis will be exposed. In these situations, the welldocumented variations in drug sensitivities of individual organisms and isolates, in Giardia for example ~°, will select the least sensitive within the natural population, possibly leading to drug-resistant organisms. Standard recommended treatment of giardiasis with metronidazole is administration of 2 g once daily for three days; or 200 mg thrice daily for ten dayss. The drug is rapidly and almost completely absorbed, and peak serum levels of 14 I~g ml ~ following administration of 500 mg of metronidazole can be expected with a half-life of around seven hours (Ref. 3). The concentration of the drug in the gut is unknown, but it is likely to be significantly less than that in the serum. Inhibitory concentrations of antigiardial drugs have been presented in a number of ways. Majewska and colleagues examined a number of laboratory strains and quoted ICs0 values (the concentration of drug which decreased the number of trophozoites to 50% of the control values in three days) which range from as low as 0.01 i~g ml t to I I~g ml I of metronidazole ~°. Boreham, Phillips and Shepherd showed that there was considerable variation in the sensitivity of different stocks of Giardia to metronidazole and other drugs ~. These authors measured the inhibition by the drug radiometrically, and presented ID50 values (the concentration of drug which inhibits the uptake of [3H]thymidine by 50% over a four-hour period during exponential growth) of around I IIM (0.2 I~g ml i) for most stocks. Using the inhibition of adherence
B o x I. Effective A n t i g i a r d i a l A g e n t s M e t r o n i d a z o l e and t i n i d a z o l e - members of the 5-nitroimidazole family of nitroheterocyclic drugs s. F u r a z o l i d o n e - a nitrofuran s. Q u i n a c r i n e - alone or in combination with metronidazoleS, 46. A l b e n d a z o l e and m e b e n d a z o l e - members of the benzimidazole family of anthelmintic drugs, and as effective as metronidazole in clinical trialsL OL-propranolol - in combination with m e t r o n i d a z o l e to treat metronidazoleresistant giardiasis 47. P a r o m o m y c i n - recommended during pregnancy 48. A z i t h r o m y c i n - active in vitro 6,27.
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Box 2. C u r r e n t Uses for Metronidazole As antiprotozoa/agents: in combination with melarsonyl potassium to treat T~/pan0soma brucei in mice49; to treat Entamoeba histolytica 7, Trichomonas vaginalis s°, Giardia duodenalis s, Blastocystis hominis shs2 and Leishmania s3.
As an antibacterial agent." to treat infections with Helicobacter pylori, the suspected causative agent of duodenal ulcers24,s4; and to treat anaerobes most commonly found in the gastrointestinal tract, including Bacteroides, Eubacterium, Peptococcus, Peptostreptococcus, Clostridium and Fusobacterium species (NB some strains of B. fragilis, B. melaninogenicus and some Clostridium species are metronidazole resistant26). In the treatment of pulmonary acariasis in China, successfully treating most patients~S; periodontitis, a common cause of tooth loss as a result of overgrowth of anaerobic spirochetes and Bacteroides species'6; chronic anaerobic pneumonitisSS; rosacea and acneS6; chronic suppurative otitis mediaST; and Crohn's diseasesS. As a prophylactic: for caesarean sectionSg; and pre-operatively for acute appendicitis 6°.
of trophozoites to monolayers of Caco2 cells, Favennec and colleagues estimated that the concentration of metronidazole required to obtain a 50% reduction in adherence (ID50) was 1.5 I~g ml I (Ref. 12). It can be seen that these values which refer to inhibitory rather than lethal concentrations of drug are I0 I O0-fold less than the serum concentrations of metronidazole following a 500 mg dose. In 1988, Boreham, Phillips and Shepherd reported an eightfold decrease in sensitivity to metronidazole of a laboratory line, BRIS/83/HEPU/IO6-21D m, maintained in a sublethal concentration of metronidazole t3. More recently, we have induced drug resistance in four Giardia strains by a number of methods H. The first method, which involved exposure of parasites to intermittent but increasing concentrations of drug, is analogous to single-dose prophylaxis or therapy. Under these conditions, we were able to induce resistance to metronidazole such that parasites were 400-fold less sensitive to drug, and by repeating earlier work 13 we were able to decrease the sensitivity by more than tenfold. Ultraviolet (UV) mutagenesis enabled us to select lines that were able to grow in an IDs0 that was I00 times that of the parent line 14. While the above discussion presents a range of values, it is logical to assume that maladministration, and a tenfold decrease in sensitivity to metronidazole could lead to the selection of resistant Giardia parasites, in vivo. Doses of metronidazole lower than 500 rag, such as the standard recommended dose of 200 mg thrice daily (and similar low doses given to treat acariasis in China Is and to reduce the need for periodontal surgery in Kuwaitl6), will certainly compromise the effectiveness of this antigiardial drug. The risk posed by the induction of drug resistance in
the other anaerobic protozoa using these regimes is likely to be at least equivalent. Significant Trichomonas resistance already occurs and inactivation of metronidazole by gut flora in the treatment of Entamoeba, as reported in the application of metronidazole as a suppository 17, may greatly reduce the bioavailability when compared with serum levels. Inaccessibility of amoeba in abscesses to the drug will also contribute to a lower therapeutic concentration of drug than is available in the serum. Because it is uncertain whether Giardia is a zoonosis, the same precautions and considerations must be exercised in administering anti-protozoal agents to livestock so as to reduce the chance of selecting drug resistant strains. Drug resistance in Salmonella, for example, has been linked to antimicrobial use in food animals m, Drug-resistant Organisms
Three approaches can be taken to limit selection for drug-resistant Giardia. • The first is to conserve the useful life of presently available drugs by prudent administration, as suggested above. Therapy with combinations of existing agents is a viable approach to preserving the effectiveness of antigiardials. • The second is to produce or design new drugs that may be modifications of existing drugs. We need to understand the mechanisms of drug resistance to prevent or circumvent its occurrence. This aspect of Giardia is covered below. Once the molecular mechanisms are known, modifications of existing drugs may be possible. The production of amikacin, a modified derivative of kanamycin that is unable to bind to bacterial enzymes that destroy kanamycin 8, is an example of
this. Targeting of essential, unique metabolic pathways and proteins presents options for analogue drug design. In Giardia, the arginine-dihydrolase pathway 19 and parasite-specific proteins, such as the cysteine-rich surface proteins 2° and the giardins2L are potential drug targets. • The third approach to limit selection for drug-resistant Giardia, the production of a vaccine, has had a positive boost from Olsen, Morck and Ceri who, at a recent Giardia conference 22, presented the results of immunizing kittens against Giardia. Resistance in organisms other than Giardia against the drugs used to treat giardiasis is not uncommon, and has been reported in Bacteroides 23, Helico bacter pylon 2<2s, Clostndium26 and Tri chomonas (P. Johnson, this issue). One mechanism of resistance to metronidazole is believed to operate via prevention of drug reduction to toxic intermediates by altering the levels or characteristics of the components of electron transport in these organisms (Ref. 27 and P. Johnson, this issue). Pyruvate : ferredoxin oxidoreductase (PFOR), which donates electrons to ferredoxin, is involved in metronidazole resistance, and, in T vaginalis, interception and detoxification of radicals generated via ferredoxin reduction of metronidazole has been reported (P. Johnson, this issue). Among the bacteria, there is evidence that the activation and mechanism of metronidazole resistance occurs via pathways other than that involving PFOR and ferredoxin. Wehnert, Abratt and Woods reported a plasmid, pMTIO0, carrying a Bacteroides fragilis insert that conferred increased resistance to Eschenchia coil, together with increased sensitivity to far-ultraviolet light 28, This plasmid has several small open reading frames, one of which (encoding a 64 amino acid protein) is responsible for the metronidazole resistance. However, this protein has no significant homology with known protein sequences 28. Other systems involved in bacterial metronidazole activation include nitrate and chlorate reduction in E. coil, and a flavodoxin gene, that is linked to a hydrogenase gene, in Clostndium acetobutylicum 29. Furthermore, it has been shown that some strains of bacteria can protect otherwise metronidazolesensitive strains from the drug 3°. Furazolidone is reduced, in vivo, to cytotoxic products, in a similar manner to the 5-nitroimidazoles. The reduction potential of furazolidone is greater than
Parasitology Today, vol. 9, no. 5, 1993
metronidazole and it is reduced via NADPH/NADH oxidase to its nitroanion radical 3~. Resistance to the nitrofurans has been reported in E. coil isolated from patients suffering from urinary tract infections 32 and in Salmonella enteritidis isolated from broiler chickens 33. The mechanism of resistance in S. ententidis was attributed to a decrease in the ability of the bacteria to reduce furazolidone 34. The importance of depletion of the drug-derived toxic radicals by glutathione cycling, for example, has been raised3s, and a mechanism of resistance to nitrofurans, whereby enzyme activities of thiol detoxification pathways are increased, has been proposed 36.
Mechanisms of Drug-resistance in Giardia Failure to treat Giardia successfully with metronidazole and furazolidone has been previously reviewed 27,37. Drug-resistant organisms have been isolated from patients with giardiasis refractive to metronidazole 38 and furazolidone resistant strains have been isolated from patients refractory to furazolidone 39. Our results have shown that resistance in G. duodenalis can readily develop via a number of mechanisms 14. Some of these are similar to those already described and involve the electron-transport systems in Giardia. The negative correlation between PFOR and metronidazole sensitivity of G. intestinalis strains36 has been corroborated by the work of Ellis and colleagues, who showed that resistance of the line BRIS/83/HEPU/106-21D~0 to metronidazole was consistent with reduced production of toxic radicals by decreased PFOR activity ~°. Also consistent with these reports is the purification of ferredoxin from Giardia, which we have shown to reduce metronidazole, in vitro 4t. The involvement of thiol detoxification pathways in furazolidone resistance in Giardia is not clear. Smith and colleagues correlated a decrease in furazolidone sensitivity with an increase in thiol cycling in Giardia, which suggested increased detoxification of furazolidone derived radicals in the less-sensitive strains36. We have been unable to reproduce these data and find that, like E. histolytica 42, cysteine is the only significant low-molecularweight thiol in the parasite grown in TYI-S-33, and that thiol-cycling enzyme activities using exogenous glutathione as substrate were not detected 43.
89
Trichomonas also appears to maintain cysteine as the major low-molecularweight thiol (D. Brown and P. Upcroft, unpublished). Thus, without high levels of glutathione, these anaerobic protozoa appear to support novel detoxification mechanisms. Each drug-resistant line that we have examined has undergone chromosomal rearrangement. In the case of the metronidazole-resistant line BRIS/ 83/HEPU/106-21D~0, deletion of a gene from one chromosome has been observed 44. This deletion involves a portion of a partially duplicated chromosome in the strain BRIS/83/ HEPU/106 (Ref. 45). The absence of the gene is also correlated with aberrant growth and the protein that it encodes is a membrane-associated protein. Although this protein has not yet been described in any other organism, it contains highly conserved modules of transport and oxidoreductase proteins, the deletion of which is consistent with the involvement of electron transfer processes in metronidazole resistance. Also consistent with involvement of transport functions is the altered drug-transport and multipledrug-resistant characteristics of the lines that we have examined. All of the resistant lines that were selected in high levels of drug following UV irradiation 14 have partial duplications of at least one chromosome when compared with their parent strains*. In the case of three lines derived from different parent strains and selected in high levels of metronidazole or furazolidone, the same chromosome was involved in the duplication in each case (J.A. Upcroft, N. Chen and P. Upcroft, unpublished).
Conclusion Drug resistance in the pathogenic microorganisms is a serious challenge to the primary health-care system. It is clear from the lessons already learned that we must economize existing effective drugs to extend their useful lives. If we are to learn from previous mistakes in treatment (eg. chloroquine resistance in Plasmodium and metronidazole resistance in Trichomonas), we must anticipate that it is unlikely that devel-
* Induction of drug resistance in these lines involved UV irradiation with 3.5 J m 2 for 10 min, recovery for 48 h, followed by selection in ten times the IDs0 level for furazolidone and 100 times the IDs0 for metronidazole, of the respective parent strains.
opment of new drugs will keep up with the development of resistance and that the cost of producing new drugs will become increasingly prohibitive. Metronidazole is a very effective, cheap drug and is at serious risk of becoming obsolete, if this is not already the case. The obvious precautions that should be exercised by physicians, health workers and veterinarians are not to administer low doses of metronidazole, and to avoid the use of metronidazole as a prophylactic, especially where Giardia, Entamoeba and Trichomonas are likely to be present. Where possible, alternative and combination therapy should be employed. Drug resistance in Giardia is not yet an overt problem, but it is clear that metronidazole resistance occurs, in viva, and can readily be induced, in vitro. We should not be complacent about metronidazole and the other antigiardials and should make every effort to avert the inevitable consequences of maladministration and non-compliance. References
I Reynoldson, J.A., Thompson, R.C.A. and Horton, R.J. (1992) Parasitology Today 8, 412~414 2 Craft, J.C. (1982) Pediatr. Infect. Dis. I, 196 211 3 Scully, B.E. (1988) Meal Clin N. Am. 72, 613 621 4 Chabala, J.C. and Miller, M.W. (1986) in Chemotherapy of Parasitic Diseases (Campbell, W.C. and Rew, RS., eds), pp 25 85, Plenum Press 5 Boreham, P.F.L. (1991) Pharmaceut. J. 234, 271 274 6 Boreham, P.F.L. and Upcroft, J.A. (1991) Trans. R. Sac. Trap. Mad Hyg. 85, 620- 621 7 Knight, R. (1980)J. Antimicrob. Chemothe: 6, 577 593 8 Gibbons, A. (1992) Science 257, 1036 1038 9 Looareesuwan, S., Harinasuta, T. and Chongsuphajaisiddhi, T. (1992) Southeast Asian J. Trap. Mad Public Health 23, 621 -634 10 Majewska, A.C. et al. (1991) Trans. R Sac. Trap. Med. Hyg. 85, 67 69 I Boreham, P.F.L., Phillips, R.E. and Shepherd, R.W. (1984) J. Antimicrob. Chemother 14, 44946 I 2 Favennec, L. et al. (1992) Parasitol. Res. 78, 80 81 3 Boreham, P.F.L., Phillips, R.E. and Shepherd, R.W. (1988) Trans. R. Sac. Trap. Meal Hyg. 82, 104 106 14 Townson, S.M et al. (1992) Trans. R. Sac. Trap. Meal Hyg. 86, 521 522 15 Chen, X.B., Sun, X. and Hu, S.F. (1990) Chung Kuo Chi Sheng Chung Hsueh Yu Chi Sheng Chung Ping Tso Chih 8, 217 219 16 Loesche, W.J. et al. (1991 ) J. Trap. Mad Hyg. 94, 118 122 17 Gerding, D.N. (1987) in Antimicrobial Agents Annual Vol. 2 (Peterson, P.K. and Verhoef, J., eds), pp 131 136, Elsevier 18 Cohen, M.L. and Tauxe, RV, (1986) Science 234, 964 969 19 Schofield, P.J. et al. (1992) Mol. Biochem. Parasitol. 5 I, 29 36
190 20 Nash, T. (1992) Parasitology Today 8, 229 234 21 Peattie, D.A. et a/. (1989)J. Cell Biol. 109, 2323 2335 22 Olson, M.E., Morck, D.W. and Ceri, H. in Giardia: From Molecules to Disease (Thomp son, R.C.A, Reynoldson, J.A. and Lymberry, A.J., eds), CAB International (in press) 23 Britz, M.L. and Wilkinson, R.G. (1979) Antimicrob. Agents Chemother. 16, 19 27 24 Weil, J. et al. (1990) Lancet 336, 1445 25 Haas, C.E., Nix, D.E. and Schentag, J.J. (I 990) Antimicrob. Agents Chemothe~ 34, 1637 164 I 26 Elliot,, T.S. and Stone, J.W. (1990) Aliment~ Pharmacol. Therap. 4, 227 238 27 Upcroft, J.A., Upcroft, P. and Boreham, P.F.L (I 990) Int. J. Parasitol. 20, 489496 28 Wehnert, G.U., Abratt, V.R. and Woods, D.R. (1992) Plasmid 27, 242-245 29 Santangelo, i.D., Jones, D.T. and Woods, D.R. ( 1991 ) J. Bacteriol. 173, 1088 1095 30 Nagy, E. and Foldes, ]. (1991) J. Antimicrob. Chemothe~ 27, 63 70 31 Moreno, S.J., Mason, R.P. and Docampo, R. (1984) J. Biol. Chem. 259, 8252 8259 32 Breeze, A. and Obaseiki-Ebor, E.E. (1983) J. Antimicrob Chemother. 12, 459-467 33 Rampling, A. Upson, R. and Brown, D.F.J. (1990) J. Antimicrob. Chemother. 25, 285 290 34 Brown, D.F.J. et al. (1991) J. Antimicrob. Chemothe~ 27, 23-28 35 Biaglow, J.E. et a/. (1986) Biochem. Pharmacol. 35, 77 90
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36 Smith, N.C., Bryant, C. and Boreham, P.F.L. (1988) Int. J. Parasitol. 18, 991 997 37 Boreham, P.F.L., Upcroft, J.A. and Upcroft, P. (1991) in Biochemical Protozotogy (Coombs, G. and North, M., eds), pp 594 604, Taylor & Francis 38 Kortbeek, L.M. et al. in Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymberry, A.J., eds), CAB International
(in press) 39 Mclntyre, P. et al. (1986) ]. Pediat~ 108, 1005 1010 40 Dis, J.E. et at. Int.j. Parasitol. (in press) 41 Townson, S.M., Upcroft, J.A. and Upcroft, P. (1993) in Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymberry, AJ., eds), CAB International (in press) 42 Fahey, R.C. eta/. (1984) Science 224, 70 72 43 Brown, D.M., Upcroft, J.A. and Upcroft, P. in Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymberry, A.J., eds), CAB International (in press) 44 Upcroft, J.A. et al. (1992) Parasitology 104, 395~t05 45 Upcroft, J.A., Healey, A. and Upcroft, P. lnt~ J. Parasitol. (in press) 46 Taylor, G.D., Wenman, W.M. and Tyrell, D.LJ. (1987) Can. Meal Assoc. J. 136, fl79 1180 47 Popovic, O.S. and Milovic, V. (1990)J. Clin. Gastroenterol. 12, 604--605 48 Kreutner, A.K., Del Bene, V.E. and Amstey,
49 50 51 52 53 54 55 56 57 58 59 60
M.S. (198t) Am. J. Obstet. Gynecol. 140, 895 899 Jennings, F.W. (1991) Trop. Meal Parasitol. 42, 139 142 Lossick, J.G. and Kent, H.L. (1991) Am. ] Obstet. Gynecol. 165, 1217 1222 Lee, M.G. et al. (1990) Ann. Rheum. Dis. 49, 192~ 193 Boreham, P.F.L. and Stenzel, D.J. Adv. Parasitol (in press) Arora, S.K., Sinha, R. and Sehgal, S. (1991) Meal Micrabiol. ImmunoL 180, 21 27 Korovina, N.A. et al. (1991) Antibiot. Khimioter. 36, 46~t8 Shah, A. et al. (1990) Indian ]. Chest Dis. Allied Sci. 32, 117 120 Akamatsu, H. et al. (1990) Arch. DermatoL Res. 282, 449 454 Rotimi, V.O. et al. (I 990) West Af~ J. Med. 9, 89-97 Sutherland, L. et al. (1991) Gut 32, 1071 1075 de Boer, C.N. and Thornton, J.G. (1989) Int. j Gynecot. Obstel_ 28, 103 107 Dahlstrom, B.L., Reiertsen, O. and Rosseland, A.R. (1990) Tidssk~ Non Laegeforen. II0, 1539 1540
]acqueline Upcroft and Peter Upcroft are at The Queensland Institute o f Medical Research, The Bancroft Centre, 3 0 0 Herston Rd, Herston, Queensland 4029, Australia.
Resistance to Clinical Drugs in African Trypanosomes C.J. Bacchi Drug resistance in African trypanosomes continues to confound clinicians and to stymy development of equatorial Africa, taking its toll in lives and economic development. Drugs in current, widespread use have been employed continuously for over 60 years in some instances. The recent studies of Fairlamb and colleagues ~,2 have outlined a defective punne-transport system in drug-resistant trypanosomes, which appears to explain resistance to several established trypanocides and suggests a guide for the development of new drugs. The recently developed agent DL-~-difluoromethylornithine (DFMO) is effective against West African, but not East African, disease3 and its activity may be the result of the unregulated synthesis of S-adenosylmethionine in trypanosomes 4. In this report, Cyrus Bacchi outlines recent developments in the elucidation of mechanisms of resistance to established drugs and naturally occurnng resistance to DFMO. Resistance to clinically effective agents in the treatment of African sleeping
sickness has spread as drugs in use for 40 years or more continue to be used in field clinics3,5. Reliance has centered on the arsenical melarsoprol (Arsobal, Mel B), the diamidines diminazene aceturate (Berenil) and pentamidine, and the sulfonated naphthylamine dye suramin (Fig. I). Suramin is used for early-stage disease when there is no evidence of parasites in the central nervous system (CNS). Pentamidine is used for the treatment of early-stage disease, and for prophylaxis, whereas melarsoprol remains the standby for late-stage, central nervous system disease3,6. Berenil, approved for use in cattle, has replaced pentamidine in some areas for the treatment of earlystage human disease3. The success of early trials and recent approval by the US Food and Drug Administration of the polyamine biosynthesis inhibitor eflomithine (DL-~-difluoromethylomithine; DFMO) has spurred expanded trials of this agent in West Africa, especially against Trypanosoma brucei gambiense in latestage arsenical-refractory disease3,6,7,
D e v e l o p m e n t of Resistance In many trypanosomiasis field clinics, the need to treat patients as quickly as possible, the limited availability of adequate diagnostic tests and the minimal laboratory testing facilities available lead to the rapid deployment of trypanocides. Indices for detecting CNS involvement, and hence the type of treatment, are somewhat arbitrary. Patients tend to leave clinics before completing a course of treatment, returning to receive the same drug after they relapse weeks or months later. Adverse side reactions to some agents or diagnostic procedures (eg. lumbar puncture) do not encourage the completion of therapeutic regimens and hinder follow-up of patients after treatment 3,s. These aspects of clinical reality only partially explain how resistance to trypanocidal drugs develops. Today, resistance to melarsoprol as well as to the diamidines is well recognized, while suramin resistance appears to be less frequently encountered s,8,9, © 1993, Elsevier Science Pubhshers I td, (UK)