V. E. Do Rosario

Malaria Parasites Can Develop Stable Resistance to Artemisinin but Lack Mutations in Candidate Genes atp6 (Encoding the Sarcoplasmic and Endoplasmic Reticulum Ca2+ ATPase), tctp, mdr1, and cg10

ABSTRACT Resistance of Plasmodium falciparum to drugs such as chloroquine and sulfadoxine-pyrimethamine is a major problem in malaria control. Artemisinin (ART) derivatives, particularly in combination with other drugs, are thus increasingly used to treat malaria, reducing the probability that parasites resistant to the components will emerge. Although stable resistance to artemisinin has yet to be reported from laboratory or field studies, its emergence would be disastrous because of the lack of alternative treatments. Here, we report for the first time, to our knowledge, genetically stable and transmissible ART and artesunate (ATN)-resistant malaria parasites. Each of two lines of the rodent malaria parasite Plosmodium chabaudi chabaudi, grown in the presence of increasing concentrations of ART or ATN, showed 15-fold and 6-fold increased resistance to ART and ATN, respectively. Resistance remained stable after cloning, freeze-thawing, after passage in the absence of drug, and transmission through mosquitoes. The nucleotide sequences of the possible genetic modulators of ART resistance (mdr1, cg10, tctp, and atp6) of sensitive and resistant parasites were compared. No mutations in these genes were identified. In addition we investigated whether changes in the copy number of these genes could account for resistance but found that resistant parasites retained the same number of copies as their sensitive progenitors. We believe that this is the first report of a malaria parasite with genetically stable and transmissible resistance to artemisinin or its derivatives.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon

Abstract.  Point mutations in the voltage‐gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine–phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine–serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S‐form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild‐type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.

A fatal case of human babesiosis in Portugal: molecular and phylogenetic analysis

We report the first case of human babesiosis in Portugal. A 66‐year‐old splenectomized man was admitted to a Lisbon hospital after 1 week of fever, abdominal pain, anorexia and nausea. A high parasitaemia (30%) of Babesia parasites was found in Giemsa‐stained blood smears and, despite treatment, the patient died several weeks later of renal failure. Ethylenediaminetetraacetic acid blood samples were processed for polymerase chain reaction (PCR) and reverse line blot hybridization to confirm and characterize the Babesia infection. The amplified PCR product was cloned and subsequently sequenced. Molecular analysis showed that the infection was caused by Babesia divergens and that other blood parasites were not involved. Phylogenetic analysis showed that the 18 S ribosomal RNA gene sequence was similar to three other European isolates of B. divergens. In view of the high risk for splenectomized individuals, strict measures should be taken to avoid tick bites.

Metabolism of primaquine by liver homogenate fractions. Evidence for monoamine oxidase and cytochrome P450 involvement in the oxidative deamination of primaquine to carboxyprimaquine.

The role of monoamine oxidase (MAO) and cytochrome P450 (P450) in the oxidative deamination of primaquine by rat liver fractions was studied. Rat liver fractions including liver homogenate, mitochondria, microsomes and 100,000 g supematant fractions were prepared from a pool of rat livers and characterised using benzylamine as a probe for MAO activity and N,N-dimethylbenzamide as a probe for P450 N-dealkylation activity. Incubation of all fractions with primaquine yielded carboxyprimaquine as the only metabolite detectable by HPLC. The mitochondrial fraction, which contained MAO activity but not P450 activity, presented the highest Vmax/K(M) value for the formation of carboxyprimaquine (8.5 x 10(-6) dm3mg(-1)h(-1). A substantially lower Vmax/K(M) value (1.3 x 10(-6) dm3mg(-1)h(-1)) was obtained in the microsomal fraction, which contained P450 but not MAO activity. The liver homogenate fraction presented a similar value (1.8 x 10(-6) dm3mg(-1)h(-1), though it contained both enzyme systems. Incubations of all the fractions that presented MAO activity, in presence of the MAO inhibitor pargiline, resulted in a marked inhibition of primaquine oxidation. P450 inhibitor SKF 525-A effectively inhibited primaquine metabolism in the microsomal fraction but inhibition in the liver homogenate was less effective. The results are consistent with an important role for MAO in primaquine biotransformation, though clearly metabolism by P450 has a contribution role.

Male size does not affect mating success (of Anopheles gambiae in São Tomé)

Abstract For malaria control, the utility of transgenic vector Anopheles mosquitoes (Diptera: Culicidae) refractory to Plasmodium transmission, will depend on their interbreeding with the wild vector population. In many species, larger males are more successful in obtaining mates. In São Tomé island, we determined that size did not affect mating success of male Anopheles gambiae Giles sensu stricto, the main malaria vector in tropical Africa. Also we showed that larval intraspecific competition is probably insignificant in this population of An. gambiae. Thus, the potential success of transgenic An. gambiae is unlikely to be affected by size selection under field conditions.

Prospective risk of morbidity in relation to multiplicity of infection with Plasmodium falciparum in São Tomé.

The prospective risk of acute morbidity was analysed in relation to multiplicity of Plasmodium falciparum infection in 491 individuals in a peri-urban community in São Tomé. In an initial cross-sectional survey, 40.5% of individuals were recorded by microscopy as infected with P. falciparum, and by PCR 60.5%, with the maximum prevalence in children aged 5-10 years. PCR-RFLP typing of the msp-2 gene of P. falciparum found a mean of 2.4 parasite genotypes per infected person, with little age dependence in this multiplicity and a total of 43 different msp-2 alleles identified. None of these were unique for São Tomé. Study participants were encouraged to report to a project worker whenever they suffered a febrile illness. During the 3 months following the parasitological survey the recorded incidence rates decreased with increasing baseline msp-2 multiplicity, both for P. falciparum-positive episodes and for fever without parasitaemia. While this is consistent with suggestions that multiple P. falciparum infections may protect against super-infecting parasites, confounding by patterns of health service usage is an alternative explanation. The incidence of clinical malaria episodes was only a little higher in children than in adults. This weak age-dependence in clinical immunity might be a consequence of a cohort effect resulting from resurgence of the disease after the breakdown of malaria control programs in the 1980s.

A clonal Plasmodium falciparum population in an isolated outbreak of malaria in the Republic of Cabo Verde

We present the first parasitological, molecular and longitudinal analysis of an isolated outbreak of malaria. This outbreak occurred on Santiago Island (Republic of Cabo Verde), a region where malaria is hypoendemic and controlled, and thus the population is considered non-immune. Blood samples were collected from the inhabitants over 1 month and during cross-sectional surveys in the following year. The presence and nature of the parasites was determined by PCR. Plasmodium falciparum was the only species detected. Genetic analysis revealed that the circulating parasites were genetically homogeneous, and probably clonal. Gametocytes were found throughout this period. Our data suggest that this represented a focal outbreak, resulting in the infection of at least 40% of the villagers with a clonal parasite line. Thus, P. falciparum infections can persist for at least 1 year in a substantial proportion (10%) of the hosts. Implications for malaria control and the interpretation of epidemiological data are discussed.

Plasmodium falciparum: diversity studies of isolates from two Colombian regions with different endemicity.

The population structure of Plasmodium falciparum has been widely studied in diverse epidemiological contexts, but emphasis has been made in regions with high and stable transmission. In order to establish the genetic structure of P. falciparum in areas of Colombia with different degree of endemicity, we studied 100 samples from malaria patients of two different municipalities. The frequency of multiclonal infection in these areas and the correlation with the endemicity were carried out by comparison of the amplified products from polymorphic segments of MSP-1, MSP-2, and GLURP genes. We found low size polymorphism of the studied genes: 1 MSP-1 allele, 3 MSP-2 alleles, and 4 GLURP alleles. We conclude that the P. falciparum population in the regions studied is genetically homogeneous.

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea

Objectives  To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector.