Chloroquine-resistant Plasmodium falciparum and the MDR phenotype

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the quality of such foundations are the most neglected part of the discussion. Without such caution, models can end up merely reinforcing erroneous assumptions. Improving the accuracy of models: In considering ways to improve the accuracy of models in general, two key phrases to keep in mind are 'adequate description' and 'sufficiently accurate'. The definition of what is adequate and accurate depends on how the results will be used. As explained above, a model of the dynamics of the transmission of a disease depends on the quality of the initial set of values describing key parameters. If a model was used to examine various control options and make recommendations for disease-control policy, then it would be part of the modeler's duty to illustrate how robust the results were. The easiest method of doing this is to conduct sensitivity analyses on key parameters. What happens to the results if a lower/higher value of a parameter is used? Often key parameters have a range of commonly accepted values that can readily be used for an initial set of sensitivity analyses. When a reliable range is unavailable, the modeler can increase/decrease the

value of the parameter(s) by set amounts or percentages. An alternative sensitivity analysis methodology is to 'reverse engineer'; that is, identify a result that represents a significant departure from the original run, and then work backwards to find parameter values that would make that result 'exist'. Once such a 'reverse engineered' parameter has been calculated, researchers can consider whether the calculated value is realistic/possible, and if further- research is warranted to check the value. The value of models: The problems and potential remedies associated with tick-borne disease transmission models do not suggest that such models are of limited value. The potentially largest benefit from modeling is to determine the relative importance of various factors influencing the epidemiology of the disease. By identifying the most critical parameter(s), models can help decide priorities. These priorities can be used to set research agendas and formulate disease-control policy. In the latter case, disease-control policies would logically focus on controlling those factors identified by the model as the most critical in ensuring the successful transmission of the disease.

When setting research priorities based on model estimations, there is always a danger that the delicacies and intricacies of modeling become the main focus of research. However, knowledge of the epidemiology of the disease is not the only factor contributing to the success of a disease-control strategy. Thus, there must be a point at which improving the accuracy of a model will have decreasing returns. Once the biological dynamics of disease transmission have been 'adequately' modeled, attention should be paid to the socio-economic factors that may affect a disease control strategy. Indeed, the disease-transmission model can become a part of a larger model describing the complete biological and socio-economic aspects of a system. References

I Medley, G.F., Perry. B.D. and Young, A.S. (1993) Parasitology 106,25 I- 264 2 lukhebi, A.W., Perry, B.D. and Kruska, R. (I 992) Prey. Vet.Meal 12. 73 85 Note: See Letters, this issue.

Martin Meltzer and R. Andrew Norval are at the Department of Infectious Diseases, College of Vetennary Medicine, University of Florida, PO Box 110880, Gainesville, FL 326t 1-0880, USA.

Chloroquine-resistant Plasmodium falciparum and the MDR Phenotype S.K. Martin The initial evidence linking chloroquineresistant malaria parasites to the multidrug resistant (MDR) phenotype was the observed reversal of resistance, in vitro, by verapamil I. At that time, all of the other MDR characteristics (namely, the presence of multiple copies of mdrgenes which encode a P-glycoproteinlike membrane protein capable of mediating active drug efflux from the resistant parasite) were mere speculation. However, the practical implication of such an association far outweighed the paucity of experimental data, and the idea gained wide acceptance. The techniques used to ascertain drug interactions against malaria parasites, in vitro, are well worked out. Hence, the potentiation of chloroquine toxicity against resistant malaria parasites, in vitro, has generated very little controversy as more compounds are added to the list of so-called reversing

agents. Unfortunately, this has not been the case for all the other criteria needed to make the MDR designation. Conflicting data have been published regarding drug efflux from chloroquineresistant malaria parasites. Krogstad et al. describe a chloroquine-efTlux rate 40-50 times faster from resistant, than from normal sensitive parasites2, and no difference in the chloroquineaccumulation mechanism between sensitive and resistant parasites3. In contrast, Bray et al. 4 found no difference in chloroquine-efflux rates between sensitive and resistant parasites but chloroquine sensitivity correlated with active drug-accumulation rates. Ginsburg and Stein s argue against invoking the MDR phenotype to explain the mechanism of chloroquine resistance in Plasmodium spp. However, they do not offer an alternative explanation for the resistance-reversal phenomenon which

is the only link that has so far stood the test of time. The controversy surrounding the role of mdr-genome amplification, its concomitant effect on P-glycoprotein expression, and chloroquine resistance in P. falciparum has been further highlighted in a recent paper by Barnes et al. 6 In lieu of studying efilux, they wisely used a lesscontroversial technique in parasitology, drug pressure, to select for chloroquine resistance in clones known to be moderately resistant to the drug. Their starting clones had multiple copies of pfmdr and increased amounts of its related Pglycoprotein homologue, pgh I, and both markers were present in quantities commensurate with their levels of chloroquine resistance. In a direct linkage situation, one would have expected increasing copies of pfmdr and more pgh I with increasing levels of chloroquine resistance but, surprisingly, the exact © 1993, ElsevierScience Publ~shePsLtd (UK)

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opposite was found. Although their resuits seem to suggest that chloroquine resistance can occur either in the presence or in the absence of multiple copies of pfmdr and elevated pgh/levels, it is unclear whether their data are indicative of non-linkage of chloroquine resistance with the MDR phenotype or provides an argument for two separate mechanisms. Incidentally, the presence of a biphasic response to verapamil in their highly resistant clones can be interpreted as supportive of the latter position, which the authors obviously favor. The relative ease with which successively higher levels of chloroquine resistance were induced, however, raises questions about the uniqueness of the moderately chloroquine-resistant clones used in this study. Would the response to drug pressure be the same if HB3 (a chloroquine sensitive) and W2 were used in place of FAC8 and W2-MEF? The findings of Barnes et al. provide laboratory equivalents for chloroquine-resistant isolates that were found to possess neither amplified mdr-genomes nor increased amounts of P-glycoprotein. The inverse relationship shown between mefloquine and chloroquine resistances not only mirrors the previously documented decrease in W2 chloroquine ICs0 after induction of mefloquine resistance 7, but also exposes another difference between chloroquine-resistant malaria parasites and MDR cancer cells. By definition, an MDR cell simultaneously gains resistance to multiple chemically unrelated drugs at the time of induction of resistance to one of the drugs8. However, in the case of the malaria parasite, induction of resistance to one

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drug can simultaneously modulate sensitivity to another drug, but resistance to a wide range of MDR related drugs is not gained. This departure from strict MDR requirements has both experimental and clinical relevance as quinine and mefloquine still serve as reliable options for the treatment of chloroquine-resistant malaria. In addition, the ability of Plasmodium spp to change its sensitivity to other antimalarial drugs without direct drug pressure 9 is an MDR-like characteristic, and provides experimental precedence for the occurrence of mefloquine resistant parasites in West Africa. Even though Barnes et at. did not find any other mutations in chromosome 5 to account for the induced highly chloroquine-resistant phenotype, the possibility still exists that this capacity could reside elsewhere in the parasite genome. Wellems et al. I° implicate a locus on chromosome 7 as the plausible site for another gene regulating plasmodial chloroquine resistance, but no membrane protein has so far been linked to it. The work of Barnes et al. shows clearly that chloroquine resistance can be expressed by malaria parasites in the absence of classical MDR characteristics (multiple copies of pfmdr and elevated levels of pgh I). More importantly, their observation that these MDR characteristics could confer negative fitness to malaria parasites under chloroquine drug pressure strengthens rather than weakens the association between a chloroquine-resistant malaria parasite and the MDR phenotype. The preponderance of published data suggests that

A Mitochondrial H e a t Shock Protein from Crithidia fasciculata P.N. Effron, A.F. Tom, D.M. Engman, J.E. Donelson and P,T. Englund Mol. Biochem. Parasitol. 59, 19 1-200 Under normal conditions, heat shock proteins (hsp) help in protein folding and in the assembly and disassembly of macromolecular complexes. They facilitate the translocation of polypeptides into organelles, and help in the refolding of proteins after translocation. How they accomplish these tasks is not known. There are three classes of hsp, characterized by molecular mass (60, 70 and 909 kDa). Some members of the hsp70 family are found in the mitochondrial matrix; the gene for the Trypanosoma © 1993 EqsevierSciencePublishersLtd. (UK)

cruzi mitochondrial

hsp70 protein is called MTP70. Because of the association of the MTP70 gene product with the parasite kinetoplast, Effron and colleagues have looked at the corresponding protein (MCP72) of Crithidia fasciculata. In this paper, they report the cloning and sequencing of the gene and the purification and characterization of its protein product. They also compare the N-terminal sequence of the protein with that specified by the gene, and identify the cleaved mitochondrial

chloroquine-resistant malaria parasites are not typical MDR cells. Nonetheless, the possibility still exists that they could be a variant of the MDR phenotype and the phylogenetic distance between them would allow for this assertion. Moreover, P-glycoprotein belongs to a superfamily of transporters II which include bacterial transport systems, peptide transporters from the major histocompatibility complex (MHC) locus and the cystic fibrosis transmembrane regulator. Recent evidence has even linked an mdr-gene product to a volume-regulated chloride ion channel in epithelial cells 12, and it is conceivable that an MDR-related molecule could also mediate ionic movements across the parasite vacuolar membrane. References I Martin, S.K., Oduola, A.M, and Milhous, W.K. (1987) Science 235,899 90 I 2 Krogstad, D.J. et al. (1987) Science 238,

1283-1285 3 Bray, P.G. et al. (1992) Biochem. Pharmacol. 44, 1317-1324 4 Krogstad,D.J.et al. (1992) Biochem.Pharmacol. 43.57 62 .5 Ginsbung,H. and Stein,W.D. (I 991) Biochem. PharrnacoL 4 I, 1463 1470 6 Barnes, D.A. et al. (1992) EMBO J. II, 3067-3075 70duola. M.J. et al (1988) Exp. Parasitol. 67, 354 360 8 Endicot, J.A. and Ling, V (1989) Annu. Rev. Biochern. 58, 137 17I 9 Rojas-Rivero,L. et al. (1992) Am. J. Trap.Med. Hyg. 47, 372 377 10 WeNems, T.E., Walker-Jonah, A. and Panton, L.J. (1991) Proc. Natl Acad Sci. USA 88, 3382 3386 II Hyde,S.C.et al. (1990) Nature 346, 362-365 12 Valverde, M.A. et al. (1992) Nature 355, 830-833 Samuel K. Martin is at the US Army Medical Research Unit. Unit 64109, Box 401, Kenya.

import peptide sequence; this is the first such sequence identified in a trypanosomatid protozoan. Immunolocalization of MCP72 shows it to be excluded from the kDNA disk-shaped structure; this is similar to the case of the homologous protein in T. cruzi. It is likely that MCP72 functions in the import of nuclear-encoded protein into the mitochondrion. It could also have functions related to kDNA: (dis)assembly of macromolecular complexes related to RNA editing; minicircle or maxicircle replication. To determine the latter will require the development of a C. fasciculata in vitro replication system. •