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The dynamics of gametocyte production in Plasmodium falciparum clones were studied in inhabitants of an area of highly seasonal malaria transmission in eastern Sudan. Reverse-transcriptase polymerase chain reaction was used to detect expression of 2 genes that encode gametocyte-specific proteins, pfs25 and pfg377, in parasites sampled from individuals throughout one year. Some patients who acquired infections during the wet season were found to harbor subpatent gametocytemia through the following dry season in the apparent absence of mosquito transmission. Genotyping of parasites in multiclonal infections showed considerable fluctuation of gametocyte production by individual clones. The gametocytes present at the end of the dry season provide the most probable source of the genetically complex cyclical malaria outbreaks following the rainy season in this region.

We investigated the evolution of drug-resistant Plasmodium falciparum in a village in eastern Sudan. The frequencies of alleles of 4 genes thought to be determinants of drug resistance were monitored from 1990 through 2001. Changes in frequencies of drug-resistance genes between wet and dry seasons were monitored from 1998 through 2000. Parasites were also typed for 3 putatively neutral microsatellite loci. No significant variation in frequencies was observed for the microsatellite loci over the whole study period or between seasons. However, genes involved in resistance to chloroquine showed consistent, significant increases in frequencies over time (rate of annual increase, 0.027/year for pfcrt and 0.018/year for pfmdr1). Genes involved in resistance to the second-line drug used in the area (Fansidar) remained at low frequencies between 1990 and 1993 but increased dramatically between 1998 and 2000, which is consistent with the advent of Fansidar usage during this period. For mutant alleles of the primary drug-resistance targets for chloroquine and pyrimethamine, higher frequencies were seen during the dry season than during the wet season. This cyclical fluctuation in drug-resistance genes most likely reflects seasonal variation in drug pressure and differences in the fitness of resistant and sensitive parasites.

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We monitored post-treatment Plasmodium falciparum among patients treated with chloroquine (CQ) and sulfadoxine-pyrimethamine (SP; Fansidar in a village in eastern Sudan. Parasites were examined on day 0 (pre-treatment), day 7, day 14 and day 21 (post-treatment) during the transmission season. A further sample was taken 2 months later (day 80) at the start of the dry season. Asexual forms and gametocytes were detected by microscopy, and reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect expression of gametocyte-specific proteins pfs 25 and pfg 377. Gametocyte carriage, as revealed by microscopy, increased significantly following CQ and SP treatment, reaching a maximum between days 7 and 14. When measured by RT-PCR, however, there was no significant difference in gametocyte rate between day 0 and days 7 or 14. RT-PCR gametocyte rates dropped dramatically by day 80 post treatment but were still 33% and 8% in the CQ- and SP-treated group at this time. Alleles associated with drug resistance of P. falciparum to chloroquine (the chloroquine resistance transporter, pfcrt, and multidrug resistance, pfmdr1) and to pyrimethamine (dihydrofolate reductase, dhfr) were seen at a high frequency at the beginning of treatment and increased further through time following both drug treatments. Infections with drug-resistant parasites tended to have higher gametocyte prevalence than drug-sensitive infections.

Studies of population genetic structure of parasites can be used to infer which parasite genes are under selection. Here, the population structure of 4 genes associated with drug resistance of Plasmodium falciparum (the chloroquine resistance transporter, pfcrt, dihydrofolate reductase, dhfr, dihydropteroate synthase, dhps, and multi-drug resistance, pfmdr-1) were examined in parasite populations in 3 villages in eastern Sudan and in an urban area of Khartoum, the capital. In order to differentiate the effects of drug selection from neutral influences on population structure, parasites were also genotyped for 3 putatively neutral microsatellite loci (polyalpha, TA81 and pfg377), and for 2 antigenic loci that are either under balancing selection or neutral, merozoite surface protein 1 and 2, (MSP-1 and MSP-2). Cross-sectional surveys were carried out during the peak transmission (wet) season and in the ensuing dry season. No significant variation in frequencies of MSP-1 and MSP-2 alleles was seen among villages in the eastern region and between the villages and Khartoum, nor between the wet and dry season. However, the drug resistance genes, pfmdr-1, pfcrt and dhfr and to a lesser extent the microsatellite loci showed high FST values when comparing villages with Khartoum, indicating strong geographical differentiation at these loci. Moreover, variation in frequencies of the drug resistance genes, pfmdr-1, pfcrt and dhfr, was observed between the wet and dry season. These differences most probably reflect the variation in drug pressure between each region, and in drug usage between the wet and dry season in a given region.

Negative Duffy expression on the surface of human red blood cells was believed to be a barrier for Plasmodium vivax infection in most Africans. However, P. vivax has been demonstrated to infect Duffy-negative individuals in several Central and East African countries. In this study, we investigated the distribution of Duffy blood group phenotypes with regard to P. vivax infection and parasitemia in Sudan. Out of 992 microscopic-positive malaria samples, 190 were identified as P. vivax positive infections. Among them, 186 were P. vivax mono-infections and 4 were mixed P. vivax and Plasmodium falciparum infections. A subset of 77 samples was estimated with parasitemia by quantitative real-time PCR. Duffy codons were sequenced from the 190 P. vivax positive samples. We found that the Duffy Fy(a-b+) phenotype was the most prevalent, accounting for 67.9% of all P. vivax infections, while homozygous Duffy-negative Fy(a-b-) accounted for 17.9% of the P. vivax infections. The prevalence of infection in Fy(a-b+) and Fy(a+b-)were significantly higher than Fy(a-b-) phenotypes (p = 0.01 and p < 0.01, respectively). A significantly low proportion of P. vivax infection was observed in Duffy negative individuals Fy(a-b-). This study highlights the prevalence of P. vivax in Duffy-negatives in Sudan and indicates low parasitemia among the Duffy-negative individuals.

Abstract. There is a need for a specific, sensitive, robust, and large-scale method for diagnosis of drug resistance genes in natural Plasmodium falciparum infections. Established polymerase chain reaction (PCR)-based methods may be compromised by the multiplicity of P. falciparum genotypes in natural infections. Here we adopt a dot-blot method to detect point mutations at nucleotide 323 (residue 108) in the P. falciparum dihydrofolate reductase (dhfr) gene using allele-specific oligonucleotide probes. Serine (Ser) or threonine (Thr) at this position are associated with sensitivity to pyrimethamine while asparagine (Asn) is associated with resistance. The method combines PCR amplification and hybridization of amplified products with radiolabeled allele-specific probes. This technique is specific and sensitive; it detects parasitemia of less than 100 parasites/l of blood, and can identify a minority parasite genotype down to 1% in a mixture. Analysis of P. falciparum isolates from Sudan, of known response to pyrimethamine, has demonstrated the sensitivity and specificity of the method and its ability to detect multiple genotypes in single infections. Furthermore, it has confirmed the association between pyrimethamine responses and dhfr alleles. The method has been successfully extended for analysis of other point mutations in dhfr at residues 51 and 59, which are associated with a high level of pyrimethamine resistance.