Publications

OBJECTIVE: To assess the efficacy of antimalarial treatment and molecular markers of Plasmodium falciparum resistance in the Chittagong Hill Tracts of Bangladesh.

METHODS: A total of 203 patients infected with P. falciparum were treated with quinine 3 days plus sulphadoxine/pyrimethamine (SP) combination therapy, and followed up during a 4-week period. Blood samples collected before treatment were genotyped for parasite mutations related to chloroquine (pfcrt and pfmdr1 genes) or SP resistance (dhfr and dhps).

RESULTS: Of 186 patients who completed follow-up, 32 patients (17.2%) failed to clear parasitaemia or became positive again within 28 days after treatment. Recurring parasitaemia was related to age (chi(2) = 4.8, P < 0.05) and parasite rates on admission (t = 3.1, P < 0.01). PCR analysis showed that some of these cases were novel infections. The adjusted recrudescence rate was 12.9% (95% CI 8.1-17.7) overall, and 16.6% (95% CI 3.5-29.7), 15.5% (95% CI 8.3-22.7) and 6.9% (95% CI 0.4-13.4) in three age groups (<5 years, 5-14, > or =15). The majority of infections carried mutations associated with chloroquine resistance: 94% at pfcrt and 70% at pfmdr. Sp-resistant genotypes were also frequent: 99% and 73% of parasites carried two or more mutations at dhfr and dhps, respectively. The frequency of alleles at dhfr, dhps and pfmdr was similar in cases that were successfully treated and those that recrudesced.

CONCLUSIONS: The clinical trial showed that quinine 3-days combined to SP is still relatively effective in the Chittagong Hill Tracts. However, if this regimen is continued to be widely used, further development of SP resistance and reduced quinine sensitivity are to be expected. The genotyping results suggest that neither chloroquine nor SP can be considered a reliable treatment for P. falciparum malaria any longer in this area of Bangladesh.

AIMS: To assess resistance to chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) in a Sudanese parasite population.

METHODS: Recurrent security problems in Akuem, Sudan, prevented us from conducting a classical in vivo treatment efficacy study. Instead we genotyped key mutations in the chloroquine resistance transporter (pfcrt), the multidrug resistance gene (pfmdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps). We genotyped the K76T mutation in pfcrt and the N86Y mutation in (pfmdr) by restriction digestion of fluorescent end-labelled polymerase chain reaction (PCR) products, while we genotyped codons 16, 51, 59, 108 and 164 in dhfr and codons 436, 437, 540, 581 and 613 in dhps by primer extension in 100 blood samples.

RESULTS: Sixty-three percent of parasites carried the 76T mutation at pfcrt critical for CQ resistance, while 31% carried the 86Y mutation at pfmdr that is associated with, although not essential, for CQ resistance. We found five dhfr alleles: 60% of infections contained wild-type dhfr alleles, 3% had one mutation, 34% had two mutations, while 3% had three mutations. We found three dhps alleles: 47% were wild type, 44% had one mutation, while 9% had two mutations.

CONCLUSIONS: We expect high levels of treatment failure (RI-RIII) with CQ (20-40%) and predict efficient treatment with SP. However, dhfr alleles with three mutations (51I, 59R, 108N) are present as are dhps alleles with two mutations (437G, 540E). Successful treatment with SP is therefore likely to be short-lived.

Females of many invertebrates contain stored sperm or fertilized eggs or both, causing potential genotyping errors. We investigated errors caused by male DNA contamination by amplifying 5 microsatellites in DNA isolated from various tissue types in the nematode Ascaris lumbricoides. We observed additional alleles in 30/135 uterus-derived samples when compared with muscle controls, resulting in 20/135 (15%) incorrect genotypes and an underestimation of inbreeding. In contrast, we observed additional alleles in only 5/143 ovary-derived samples, resulting in 4/143 (3%) incorrect genotypes and no significant influence on inbreeding estimates. Because uterus constitutes approximately 17% of a female's organ weight, a substantial proportion of samples isolated from female tissue may contain male-derived DNA. Male contamination is easily avoided when using large nematodes such as A. lumbricoides. However, we urge caution for studies using DNA isolated from small invertebrates that store sperm or fertilized eggs or both.

If parasite genotype influences the clinical course of malaria, we expect that isolates from patients with similar pathology would be more closely related than would be expected by chance. To explore this prediction, we typed nine microsatellite markers in sympatric Plasmodium falciparum isolates from cerebral and uncomplicated malaria patients from Vietnam. Temporal structure and linkage disequilibrium were also examined in this data set.

Current methods used to genotype point mutations in Plasmodium falciparum genes involved in resistance to antifolate drugs include restriction digestion of PCR products, allele-specific amplification or sequencing. Here we demonstrate that known point mutations in dihydrofolate reductase and dihydropteroate synthase can be scored quickly and accurately by single-nucleotide primer extension and detection of florescent products on a capillary sequencer. We use this method to genotype parasites in natural infections from the Thai-Myanmar border. This approach could greatly simplify large-scale screening of resistance mutations of the type required for evaluating and updating antimalarial drug treatment policies. The method can be easily adapted to other P. falciparum genes and will greatly simplify scoring of point mutations in this and other parasitic organisms.

Benzimidazole resistance has evolved in a variety of organisms and typically results from mutations in the beta-tubulin locus at specific amino acid sites. Despite widespread treatment of human intestinal nematodes with benzimidazole drugs, there have been no unambiguous reports of resistance. However, since beta-tubulin mutations conferring resistance are generally recessive, frequencies of resistance alleles less than 30% would be difficult to detect on the basis of drug treatment failures. Here we investigate sequence variation in a 1079 bp segment of the beta-tubulin locus in the human whipworm Trichuris trichiura from 72 individual nematodes from seven countries. We did not observe any alleles with amino acid mutations indicative of resistance, and of 40 point mutations there were only four non-synonymous mutations all of which were singletons. Estimated effective population sizes are an order of magnitude lower than those from another nematode species in which benzimidazole resistance has developed (Haemonchus contortus). Both the lower diversity and reduced population sizes suggest that benzimidazole resistance is likely to evolve less rapidly in Trichuris than in trichostrongyle parasites of livestock. We observed moderate levels of population subdivision (Phi(ST)=0.26) comparable with that previously observed in Ascaris lumbricoides, and identical alleles were frequently found in parasites from different continents, suggestive of recent admixture. A particularly interesting feature of the data is the high nucleotide diversities observed in nematodes from the Caribbean. This genetic complexity may be a direct result of extensive admixture and complex history of human populations in this region of the world. These data should encourage (but not make complacent) those involved in large-scale benzimidazole treatment of human intestinal nematodes.

Molecular markers are used widely to discriminate between closely related species of parasites, and in many cases a single locus is used for this purpose. This article aims to show how molecular data derived from a single genetic marker or linkage group - in this case mitochondrial DNA - can lead to ambiguous conclusions and to illustrate how a multilocus approach has enhanced our understanding of the epidemiology of two closely related parasites, the nematodes Ascaris suum, which infects pigs, and Ascaris lumbicoides, which infects humans.

Infection with the endosymbiotic bacteria Wolbachia is widespread in filarial nematodes. Previous studies have suggested concordance between the phylogeny of Wolbachia with that of their nematode hosts. However, there is only one published molecular phylogenetic study of filarial species, based on the 5S rRNA gene spacer. The phylogeny proposed by this study is partially incongruent with previous classifications of filarial nematodes, based on morphological characters. Furthermore, both traditional classifications and molecular phylogenies are, in part, inconsistent with the phylogeny of Wolbachia. Here we report mitochondrial cytochrome oxidase I (COI) gene sequences for 11 species of filaria and for another spirurid nematode which was included as an outgroup. In addition, 16S rRNA, wsp and ftsZ gene sequences were generated for the Wolbachia of several filarial species, in order to complete the available data sets and further resolve the phylogeny of Wolbachia in nematodes. We used these data to evaluate whether nematode and Wolbachia phylogenies are concordant. Some of the possible phylogenetic reconstructions based on COI gene were congruent with the phylogeny of Wolbachia and supported the grouping of the rodent filaria Litomosoides sigmodontis with the lymphatic filariae (i.e. Brugia spp. and Wuchereria spp.) and the sister group relationship of Dirofilaria spp. and Onchocerca spp. However, the placement of the Wolbachia-free filaria Acanthocheilonema viteae is ambiguous and dependent on the phylogenetic methods used.