Invited ReviewResistance to antiparasitic drugs: the role of molecular diagnosis
Introduction
Drug resistance is the most serious barrier in achieving control of many of the parasite species, including protozoa, helminths and arthropods that infect animals and humans. As such, resistance impinges on most branches of animal and health parasitology. The advance of molecular techniques has provided tools for understanding many biological problems and molecular methods have been applied to parasitological problems such as species determination and are now used in several diagnostic applications. This paper looks at the feasibility of applying molecular techniques for the detection and management of resistant parasites. Authors have contributed various sections (Giardia and Trichomonas, Jacqui and Peter Upcroft; Plasmodium, Manoj Duraisingh; Eimeria, H. David Chapman and Martin Shirley; Helminths, Leo Le Jambre and Lucilia, Philip Batterham) as well as contributions throughout the manuscript and this paper is the basis of discussions held at a ‘Round table’ held at the ICC/ASP conference.
Molecular techniques offer some unique advantages in diagnosis: they are highly specific, they can be sensitive even with small quantities of DNA and have the potential to include parasite species identification. Most are based on polymerase chain reaction (PCR). This technology can amplify a desired DNA sequence of any origin hundreds of millions of times in a matter of hours. PCR is especially useful because the reaction conditions can be designed to be highly specific, easily automated and capable of amplifying minute amounts of sample. This approach is ideal for resistances involving point mutations where a single base pair change can be used to design discriminating primers that selectively bind to DNA from resistant or susceptible parasites prior to amplification of the target DNA. Specificity is provided in the design of the primers and greater the number of unique primers greater is the specificity. For example, nested PCR involves at least two amplification steps and potentially improves both sensitivity and specificity. In addition to the use of specific primers for drug resistance other primer combinations can provide a parasite species fingerprint or a positive control for the amplification reactions. The patterns of DNA fragments can be manipulated to reveal whether individuals are heterozygous or homozygous for a particular allele.
Numerous PCR-based methods have been developed to identify the genetic differences. Three examples are provided.
- 1.
Allele-specific PCR (AS-PCR) depends on the design of one of the two primers used in the PCR reaction with the 3′ base complementary to one (e.g. resistant) and not the other (e.g. susceptible) allele. An amplification product is obtained only when polymerisation is initiated on both strands. This method is very sensitive and quite specific but requires optimisation in each laboratory. A further advance has been the changing of bases 5′ to the 3′ base which increases the mismatch at the 3′ end and increases the specificity of the method (Wang et al., 1995).
- 2.
PCR-restriction fragment length polymorphism (PCR-RFLP) involves PCR of the region flanking the polymorphism followed by the cleavage of the PCR product with restriction enzymes that cleave only when the specific allele is present. This method is highly specific because the amplification step (PCR) is separated from the discrimination step (restriction enzyme cleavage), but is not as sensitive as AS-PCR. Controls for complete digestion of the PCR product and allele-specific restriction sites where natural sites are not present can be created by careful engineering of the primers (Duraisingh et al., 1998).
- 3.
Tandem competitive PCR (TC-PCR) is designed to amplify genes such as multidrug resistance genes that are associated with resistance to mefloquine in Plasmodium falciparum. This assay can determine the gene-copy number (Price et al., 1999).
While at present most assay endpoints are the detection of specific size DNA fragments on agarose gels, future applications may use hybridisation and plate readers to automate endpoints. Molecular beacons and microarrays may emerge in future applications.
Although they offer benefits there are several potential pitfalls with molecular diagnosis of resistance (see Sangster, 1990). In brief they are:
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while parasite species identification is often difficult, it is essential in order to make resistance assays valid;
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collections of parasites are often of mixed species which complicates the process;
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parasite collection is often not straightforward;
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tests are best employed as population tests, but in order to test a representative sample from a population, the genotype of several hundred individual parasites might be required;
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tests require detailed knowledge of resistance mechanisms at the molecular level;
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the identified mechanism must be the predominant one in the field;
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molecular tests may not be appropriate for all resistance mechanisms;
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ideally, they need to be offered as a battery of tests so resistance to several available drugs can be measured simultaneously;
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PCR technology is relatively complex.
One aspect of molecular resistance testing that is unmatched by other methods is the potential to detect resistance at very low prevalence. Several mathematical models of resistance in different parasite species indicate that in order to manage resistance by adopting the use of a second chemical before resistance develops to the first, detection of resistance at the level of 1–2% of alleles is essential. A desire for early detection of resistance is thus a universal theme in resistance management. However, the number of parasites that need to be tested may be the most serious limitation for resistance testing. For example, in order to be confident (at the 95% level) that a particular allele was present at less than 1% in a population of diploid organisms, 150 individuals of the same species would have to be genotyped (Sangster and Dobson, 2002).
Another serious obstacle to progress is the difficulty in elucidating genetic mechanisms of resistance. Success has been achieved in P. falciparum partly because of the level of research funding this parasite attracts and because in vitro cultivation of asexual stages is available. In Lucilia, mutagenisation is possible and mutants can be studied in the context of Drosophila genetic information. Some success has also been achieved in helminths partly because resistance models are available. The coccidia fare the worst. Even though they cause important diseases and chemotherapy is central to control, a paucity of genetic maps, limited in vitro culture systems and few resistance bioassays are critical factors slowing progress. Another problem common to resistance research in parasitology is genetic variation within parasite populations. This assumes significance because resistance is often due to small genetic changes that are often difficult to detect against genetic ‘noise’ in the population. Further, laboratory and field isolates can differ in their resistance genotype and the genetic change may be at a site remote from the site of drug action.
How might molecular resistance testing be applied in the field? There are some cases where molecular techniques are sufficiently advanced and the economics of testing such that molecular testing will be applied to individual cases. Human falciparum malaria in areas of low transmission is one example that is discussed in Section 3. Anthelmintic resistance in cattle and horse nematodes are other cases where resistance levels are currently low and diagnosis of resistance would assist in preventing it from becoming widespread. However, for the reasons that we lack the basic knowledge, of cost and the current availability of alternative tests or control methods a ‘cow-side test’ or ‘resistance dipstick’ approach is unlikely to be available for diseases of domestic animals. In these cases, molecular tests are more likely to be used in area-wide monitoring and epidemiological studies to learn the bigger picture of resistance development and management.
Given the difficulties faced by researchers, some advances may be gained by comparing progress on some parasites and identifying opportunities for sharing knowledge or techniques with parasitologists in similar fields. Another common thread is the practical considerations for the application of molecular techniques to the field where parasitologists in different areas face similar problems.
Section snippets
Useful drugs
Giardia duodenalis is the causative organism of giardiosis, a form of diarrhoea in humans and animals and Trichomonas vaginalis causes a genital disease in humans. Nitroimidazoles are the most effective and widely used agents for the control of G. duodenalis and the only drugs available to treat T. vaginalis infections (Upcroft and Upcroft, 2001b). Metronidazole (Flagyl), for example, has a serum half-life of 7–8 h with serum concentrations, using recommended doses, reaching 70–100 μM. Another
The current status of resistance
Malaria threatens over 40% of the world's population and is responsible for at least 300 million infections each year, with over a million deaths. Resistance to antimalarials is one of the most pressing concerns in the control of malaria. In particular, resistance found in P. falciparum, the species which causes the severest form of the disease, is of most concern. The consequences of resistance are an increase in mortality and morbidity with huge knock on economic consequences, borne by the
Anticoccidial drug usage and the status of resistance
Eimeria infection in fowls is a serious and costly disease to the industry. In the USA, 99% of broiler complexes use an anticoccidial drug and 94% of these include them in both starter and grower feeds (Chapman, 2001). Among the anticoccidials, ionophores (principally salinomycin) are most commonly used but synthetic drugs (e.g. nicarbazin, diclazuril) are sometimes used in the starter feed followed by an ionophore in the grower ration. Drugs are used extensively in all countries where chickens
Current status of resistance in nematodes
There are many reviews of the present status of resistance in parasites of small ruminants (see Waller et al., 1996). Table 4 provides a breakdown of resistance in countries within regions. Resistance is virtually ubiquitous although those regions such as Australia and South America that are noted for their extensive livestock industries have the highest levels of resistance. For the same reason, there appears to be a Southern Hemisphere bias that follows extensive grazing, high levels of
Arthropods – Lucilia in sheep
Lucilia cuprina is a major ectoparasite of sheep. Under favourable conditions its larvae initiate myiasis (strikes) on sheep causing severe economic loss to the wool industry and distress to the host animal. A variety of insecticides have been used to control L. cuprina. In this discussion three classes of chemical control agents, the cyclodienes, the organophosphates and the s-triazines will be considered.
Conclusions
It is likely that accurate and timely diagnosis of resistance has potential to improve resistance management in several parasite species. Although it is too late for diagnostic many chemicals, other applications remain. The number of particular instances where molecular diagnosis is desirable far exceeds the number of cases where diagnosis is currently feasible. In addition to the problems imposed by parasite species mix, epidemiology, social infrastructure and economics, there are technical
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