Geographical distribution of Toxoplasma gondii genotypes in Asia: A link with neighboring continents.

Defining the pattern of genetic diversity of Toxoplasma gondii is important to understand its worldwide distribution. During the last decades, a large number of studies have been published on Toxoplasma genotypes circulating in Europe, in North and South America. Two continents are still largely unexplored, Africa and, to a less extent, Asia. In this last continent, an increasing number of publications reported genotypes circulating in diverse provinces of China, but very few data are available for other Asian countries. After a systematic database search, 47 papers related to T. gondii genotypes in Asia were analyzed. Genetic characterization of DNA was performed by microsatellite markers, or more usually by a multiplex PCR using 11 PCR-RFLP markers, allowing data comparison to draw a first global picture of the population structure of this parasite throughout Asia. Overall, 390 isolates or DNA extracts were completely typed by PCR-RFLP and/or microsatellite marker methods, revealing 36 different PCR-RFLP or equivalent microsatellite genotypes: 15 genotypes identified by a ToxoDB number and 21 atypical or unique genotypes. The most common genotype found in Asia is the genotype ToxoDB#9 (Chinese 1). The clonal types I, II and II variant, and III were also commonly found in Asia. The geographical distribution of these genotypes across Asia may reflect either a continuum with Europe for the western part of Asia (presence of Type II), or the circulation of strains through animal migration or human activities between Africa and the Southwestern part of Asia (Africa 1 genotype in Turkey or ToxoDB#20 both I Sri-Lanka and in Ethiopia or Egypt). Although there are some indications of a genetic population structure in Southeast Asian countries different from the rest of Asia, more studies in this tropical part of Asia will be necessary for a region which represent as well as Africa one of the missing links of the T. gondii genetic diversity.

Sarcoptes scabiei mites in humans are distributed into three genetically distinct clades.

Scabies is an ectoparasitic infestation caused by the mite Sarcoptes scabiei. Currently, S. scabiei is taxonomically divided into different varieties on the basis of host origin. Genetics-based research on scabies has been conducted, but the data on genetic diversity of populations of this mite in humans in Europe are lacking. We evaluated the genetic diversity of populations of S. scabiei. A large series of mites obtained from humans in France and the data of mites from various hosts and geographical areas retrieved from GenBank were included to investigate whether mites are divided into distinct populations. The study of cytochrome c oxidase subunit 1 gene polymorphisms were found to be best suited for phylogenetic analysis. S. scabiei mites were distributed into three genetically distinct clades, with most mites clustering in clades B and C. The Fst value and the Nm value calculated for mites included in clades B and C indicated a strong population structure and a very low gene flow between mites of those clades. The results of the present study not only support the rejection of the hypothesis of panmixia for S. scabiei in humans but also suggest that mites belonging to different clades are genetically isolated. Moreover, the results suggest that the subdivision of S. scabies in varieties according to animal or human hosts is not warranted. In conclusion, S. scabiei mites in humans do not constitute a homogeneous population. Further investigations are now required to assess whether different clinical forms of scabies are associated with particular haplotypes or clades.

Wombats acquired scabies from humans and/or dogs from outside Australia.

According to previous studies, Sarcoptes mites of wombats were relatively recently introduced into Australia by colonizers and/or their dogs. However, that affirmation has been called into question due to apparent flaws in the design of the phylogenetic studies. With the aim of providing a definitive answer to this question, a part of the mitochondrial gene coding for 12S rRNA of S. scabiei mites from 23 humans and one dog collected in France was sequenced and a phylogenetic analysis including the sequences previously deposited in Genbank was performed. Phylogenetic analysis did not show host segregation or geographical isolation of the mites. Conversely, the present work suggested that mange in wombats is indeed due to the introduction of S. scabiei into Australia by immigrating individuals and/or their companion animals.

Ape Plasmodium parasites as a source of human outbreaks.

Recent studies have revealed a remarkable molecular diversity of Plasmodium parasites in great apes in Africa, as well as parasite exchange events between these primates and humans. We review the different points of view proposed on the origin of human malaria, and discuss ape Plasmodium parasites as a source of human outbreaks.

[Surveillance of falciparum malaria susceptibility to antimalarial drugs and policy change in the Comoros]. / Surveillance de la chimiosensibilite du paludisme do à Plasmodium falciparum et changement de politique dans l'Union des Comores.

Between May and June 2001, efficacy of chloroquine was assessed in 5 sentinel sites in the 3 Comoro islands. Among the 183 children, age range between 6 and 59 months, followed up for 14 days, clinical failure rates ranged between 31.2 and 73.1% and the total failure rate (clinical and parasitological) between 50 and 88.5%. Failures were mainly early treatment failures. The Ministry of health, during a consensus meeting decided to change the first line drug and to gather baseline information on the efficacy and the tolerance of the combination artemether-lumefantrine. Between June and September 2004, among the 164 children, age range between 6 and 59 months included, the success rate of the combination was 99.4% in the 3 sites with a follow-up of 28 days. No serious drug related adverse event was reported.

[French language training course: malaria workshop organized by Institut Pasteur de Madagascar]. / Formation francophone Sud/Sud: l'atelier paludisme de l'Institut Pasteur de Madagascar.

The Malaria Workshop organized by Institut Pasteur de Madagascar is an original course that applies innovative concepts to training of health professionals involved in malaria control in endemic countries. Course objectives are to enhance the skills needed to fight malaria (transversal competencies, critical approach, and position statement), to reinforce project cycle management proficiency, and to demonstrate how the Internet can be used as a source of documentation to compensate for geographical isolation. The Malaria Workshop is a six-consecutive-week full-day course that has been presented once a year since 2003. Seventy-six researchers, physicians or health ministry officials have already benefited from this training. Teaching methods emphasize andragogy that facilitates a learner/mentor relationship promoting exchange rather than transmission of knowledge and problem-based learning that engages learners to take an active part in gathering information. These methods in combination with the diverse backgrounds and experience of course participants foster a positive dynamic environment for learning that is monitored by weekly progress evaluation. Follow-up surveys have confirmed the positive effect of this training on the professional performance of former participants who become more involved in program development and fund-raising efforts. A professional network is growing and learners are starting to their experience. In this report workshop organizers describe the course's origins and concepts and present the conclusions drawn based on the first five yearly sessions.

[Evidence of an urban, local transmission of malaria in Antananarivo, Madagascar]. / Mise en évidence d'une transmission urbaine autochtone du paludisme à Antananarivo, Madagascar.

Madagascar presents a large heterogeneity in terms of climate and altitude, which explains the uneven spread of malaria throughout the island. The capital, Antananarivo, counts more than one million inhabitants, altitude between 1250 and 1470 m, in an area where the transmission is low but malaria may cause deadly epidemic outbreaks. Numerous malaria cases are reported, without biological confirmation, and reliable data about urban malaria transmission are lacking. The " Institut Pasteur de Madagascar" together with the Malagasy Ministry of Health performed in 2003 a study about malaria transmission in Antananarivo. A prevalence survey of malaria among fever syndromes, with data collected from 43 urban dispensaries, showed that confirmed malaria cases represented only 2% of the total fever cases (15 cases out of 779 fever syndromes). The vast majority was imported from costal areas (13 cases out of 15), where malaria is hyperendemic. However, a local urban transmission was found for two patients and five other subjects identified during a proximity survey. Vectors A. arabiensis and A. funestus were found inside the patient houses, located in close proximity of flooded rice fields. Genetic analysis of P. falciparum strains allowed to distinguish three genotypes, aggregated by house. The analysis of parasite genome polymorphism proves here its validity for epidemic surveys in areas where malaria is unstable, with no premunition in the local urban population.

Mise en evidence d'une transmission urbaine autochtone du paludisme a Antananarivo; Madagascar

La repartition du paludisme a Madagascar est caracterisee par son heterogeneite; du fait des vari ations climatiques et geographiques marquees dans l'ile. La capitale; Antananarivo; regroupe plus d'un million d'habitants entre 1 250 et 1 470 m d'altitude; dans une zone ou la transmission est irreguliere; mais le paludisme peut parfois sevir par vagues epidemiques meurtri res. De nombreux cas de paludisme sont declares dans la capitale sans confimation biologi u e; et des donnees fiables sur l'existence d'une transmission urbaine font defaut. C'est pourquoi l'Institut Pasteur de Madagascar et le Ministere de la Sante de Madagascar ont realise en 2003 une etude sur la transmission palustre dans la Communaute Urbaine d'Antananarivo. Une enquete de prevalence du paludisme parmi les syndromes febriles; realisee dans 43 dispensaires urbains; a montre que les cas de paludisme confi rmes ne representaient que 2des cas de fievre inclus dans l'etude (15 cas sur 779 syndromes febriles). La majorite etait importee des zones cotieres (13 cas sur 15); ou la transmission est pere n n e. Cependant; une transmission urbaine autochtone a ete constatee chez 2 des consultants et 5 autres sujets identifies lors d'une enquete de proximite. Les vecteurs Anopheles arabiensis et Anopheles funestus ont ete retrouves dans les maisons des malades; toutes situees a proximite de rizieres. L'analyse genetique des souches de Plasmodium falciparum a permis de distinguer trois genotypes; agreges par groupe d'habitation. L'analyse du polymorphisme des genomes parasitaires prouve son interet dans le cadre de la surveillance du risque epidemique dans une zone de paludisme instable; ou la population citadine ne presente pas de premunition

Efficacy of artemether-lumefantrine treatment in patients with acute uncomplicated Falciparum malaria in Mayotte, a French collectivity of the Comoros Archipelago.

Mayotte is a French island located in the Comoros archipelago in the Indian Ocean. Due to the high level of resistance to chloroquine and sulfadoxine-pyrimethamine in this area, new therapeutic strategies are required. The aim was to assess and to document the efficacy of artemether-lumefantrine (AL) combination in four oral dosages. The follow-up was carried out during 21 days to monitor the antimalarial drug efficacy in an open trial in April-May, 2002. Results were obtained from 51 patients, aged from three to 46 years (12% less than five years). No case of therapeutic failure was observed. At day 2 after treatment, all the patients were apyretic and none of them had parasitaemia until day 21. This first therapeutic trial of the AL combination in the Indian Ocean sub-region shows that this association is safe, effective and rapid. AL should be an alternative treatment of uncomplicated malaria attacks in Comoros Archipelago, and will be of help to manage imported chloroquine-resistant falciparum malaria strains in Madagascar.

Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros.

We report the outcome of chloroquine treatment and the prevalence of mutations at codon 86 of the pfmdr1 gene, at codon 76 of the pfcrt gene, and at codon 108 of the pfdhfr gene in clinical isolates of Plasmodium falciparum collected from 30 children under 10 years of age living in the Comoros Union. This in vivo study was carried out in February and March 2001 in Moroni. Chloroquine treatment failed in 23 children (76.6%; 95% confidence interval: 57.7 to 90.1%). Subsequent genotyping showed that all P. falciparum isolates (100%) harboured a tyrosine residue at position 86 in pfMDR1. 83.3% (25/30) of these isolates harboured a mutation at position 76 in pfCRT and half (15/30) of these isolates also harboured a mutation at position 108 in pfDHFR. Chloroquine resistance is a real concern in the Comoros Union. The prevalence of pfDHFR mutant parasites is alarming. The alternative drugs proposed as a replacement for chloroquine as first-line treatment in Comoros, and the strategy to monitor the drug susceptibility of Plasmodium sp in this part of the Indian Ocean sub-region are discussed.

[Molecular characterization of mosquitoes of the Anopheles gambiae complex from Mayotte and Great Comoro]. / Caractérisation moléculaire des moustiques du complexe Anopheles gambiae à Mayotte et à Grande Comore.

The mosquitoes of the Anopheles gambiae complex have been characterised at specific and sub-specific levels in two islands of the Comoros archipelago: the island of Mayotte (French departmental collectivity) and the island of Grande Comore (Comoros Union). Results are similar in the two islands and are presented together. The species An. gambiae s.s. was observed alone (determination performed on 149 specimens by PCR product of IGS of rDNA). The molecular form observed alone was S, and corresponds in this geographic area to the chromosomal form Savanna (determination performed on 123 specimens by another PCR product of IGS of rDNA). The haplotype IB was observed alone (determination performed on ten specimens, by sequencing the ITS of rDNA, with special attention at the position 871 of ITS), as previously observed by other authors in East Africa. Finally, in Mayotte and Grande Comore the An. gambiae complex is only composed by An. gambiae s.s. from the molecular form S/type IB.

Sex ratio of Plasmodium falciparum gametocytes in inhabitants of Dielmo, Senegal.

An epidemiological survey was conducted during a 4-month period of intense malaria transmission in Dielmo, a holoendemic Senegalese village. Two thick blood smears per inhabitant were collected weekly. The sex ratio of Plasmodium falciparum gametocytes (gamete precursors) was studied in 50 gametocyte carriers. All age classes were represented (mean 19.7 years; range: 2 months-75 years); 42 (84%) of them did not receive antimalarial treatment. Overall 668 thick smears were examined until 100 gametocytes had been counted or for 40 min. A total of 11204 gametocytes were observed with a mean sex ratio of 0.346 (95% CI 0.317-0.374), i.e. 2.89 females per 1 male. Among the 284 thick smears in which at least 10 gametocytes were observed, the mean percentage of male gametocytes was 27.8%, with a range of 0-82%. Great variability was observed between gametocyte carriers and also between thick smears from the same gametocyte carrier. A multivariate analysis was performed which highlighted the fact that only 2 variables had a significant effect on the sex ratio. Anaemia was associated with an increased percentage of males (Prevalence Rate Ratio [PPR] of male gametocytes was multiplied by 1.65 if haematocrit rate < 32%) and a wave of gametocytes was associated with an increased percentage of female gametocytes (PRR was multiplied by 0.48 during the peak of gametocytaemia and for the 2 weeks following this peak). The variables without significant effect on sex ratio were: age, sex, clinical status and sickle cell trait status of the gametocyte carrier, density of asexual parasites, quinine treatment, and gametocyte density (when taking account of its waves). These results are discussed in regard of possible differential production, mortality or sequestration of one gametocyte sex and selective advantages for the transmission of parasites.

[Hemoparasites of bats in Madagascar]. / Hémoparasites des chauves-souris à Madagascar.

This study aims to evaluate the prevalence and density of haemoparasites in wild malagasy bats. Among the 440 bats, belonging to 14 species sampled in 5 localities in different bio-climatic zones of the island, 93 (21%) showed at least 1 haemoparasite with, by order of frequency, Haemoproteidae (15.7% of 440 bats), microfilariae (7.0%) and Trypanosoma (0.7%). Among these 93 bats, 92 (99%) belonged to the family Vespertilionidae. Four bat species, all endemic to the Madagascar region (Madagascar and Comoros), were found to harbour parasites: Miniopterus manavi with Haemoproteidae (38% of 129 individuals), microfilariae (23%) and Trypanosoma (2%); Myotis goudoti with Haemoproteidae (24% of 68 individuals) and microfilariae (1%); Miniopterus gleni with Haemoproteidae (23% of 13 individuals); and Triaenops furculus with Haemoproteidae (4% of 28 individuals). The sex of bats was not linked to parasite prevalence. Within Miniopterus manavi, those individuals with greater weight also had a higher prevalence of microfilariae; and within the individuals harbouring microfilariae the greatest weights corresponded to the highest density of microfilariae. Ten bat species (with 202 individuals examined) were negative for any haemoparasite. This study is the first to provide evidence of haemoparasites in Malagasy bats; it provides interesting insights, especially concerning the parasite distribution per bat species and families, the pathogenicity of this type of parasitism and the parasite transmission by arthropod vectors.

Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests.

The dissemination of mutant and resistant strains of Plasmodium falciparum makes a considerable contribution to the spread of drug-resistant malaria. Populations around harbours and airports could be particularly exposed to Plasmodium isolates introduced with imported cases of malaria. The use of chloroquine as well as the use of and sulfadoxine/pyrimethamine is currently an effective method for treating uncomplicated cases of malaria in Madagascar. As part of a monitoring programme, in vitro methods were used to assess the sensitivity of P. falciparum isolates in two coastal towns in Madagascar: Mahajanga on the west coast and Toamasina on the east coast. All of the isolates from both sites were sensitive to amodiaquine, quinine, pyrimethamine and cycloguanil. All of the isolates from Mahajanga were sensitive to chloroquine (n = 25; mean IC50 = 22.6 nM, 95% confidence interval: 16.8-28.7 nM), whereas three of the isolates from Toamasina were resistant to chloroquine (n = 18; mean IC50 = 66.3 nM; 95% confidence interval: 42.6-90 nM). The frequency of the Pfcrt Thr-76 and the dhfr Asn-108 mutations was estimated by PCR/RFLP. The 43 P. falciparum isolates examined, including the three in vitro chloroquine-resistant isolates from Toamasina were all wild-type (Lys-76). Phenotyping and genotyping studies suggested that the prevalence of chloroquine- and pyrimethamine-resistant isolates and of mutant strains of P. falciparum is very low. These results showed that in vitro test and genotyping of resistance markers approaches could be successfully used to monitor the emergence of drug-resistant malaria and to try to alleviate the lack of medical teams able to carry out in vivo test. The possible hazard/risk associated with imported cases of malaria is discussed.

South-East Asian ovalocytosis among the population of the Highlands of Madagascar: a vestigé of the island's settlement.

In the Madagascar Highlands, 0.76% of children from 168 random primary schools, and 19 of 150 families from 3 villages, had oval-shaped erythrocytes. Most harboured the deletion in the band 3 gene characteristic of South-East Asian ovalocytosis. This genetic trait supports the Indonesian origin of the Madagascar settlement.

[National Network study to perpetuate the surveillance of Plasmodium falciparum sensitivity to antimalarials in Madagascar]. / Réseau d'Etude de la Résistance (RER) pour pérenniser la surveillance de la sensibilité de Plasmodium falciparum aux antipaludiques à Madagascar.

To redefine strategy and policy to cure or to prevent malaria, there is a need to get relevant and updated data on Plasmodium sp sensitivity level to antimalarial drugs. Thus, in September 1999, the Madagascan Ministry of Health and the Institut Pasteur de Madagascar (IPM) formed a network named RER for malaria resistance surveillance. To alleviate the lack of experienced medical teams within the health centres, and due to technical and logistic matters, as part of the network activities, it was decided to give a start with the in vitro studies which are carried out at IPM. In vitro sensitivity testing is done by use of the isotopic method. Results from the study done in 2001 demonstrate that the Madagascan P. falciparum isolates are susceptible to amodiaquine (n = 215), to cycloguanil (n = 56), to pyrimethamine (n = 98) and to quinine (n = 214). One isolate (1/110 i.e. 0.9%) of mefloquine-resistant phenotype is detected from the Eastern region. P. falciparum susceptibility to chloroquine is satisfactory with 95.4% (206/216) of in vitro sensitive isolates. RER arises from the partnership and collaboration between the Madagascan Ministry of Health and the IPM. The network set-up is presented. The usefulness of the in vivo approach, and the in vitro investigations (chemosusceptibility test and screening of mutations accounting for resistance to chloroquine) to monitor the emergence and the dissemination of drug-resistant parasites in Madagascar as well as in the subregion of the Indian Ocean is discussed.

[Malaria study in the cyclone risk zone: entomological , diagnostic and therapeutic approach in the southeastern region of Madagascar]. / Etude du paludisme en zone de risque cyclonique: approche entomologique, diagnostique et thérapeutique dans la région Sud-Est de Madagascar.

Madagascar is a tropical island affected by many natural disasters. The eastern coastal zone--an area of perennial malaria transmission--is regularly exposed to cyclones. Few malaria studies have been done in this area of Madagascar, and none have examined the potential relationship between malaria and natural disasters. A mobile team spent six weeks in the fields doing three lines of research: an entomological study by catching mosquitoes and determining their species: a therapeutic study of chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) according to a 14 days WHO protocol and also a study of physician's diagnostic ability. Physicians were asked to make a presumptive clinical diagnosis of all febrile patients, and these results were compared to those obtained from blood smear examinations. The entomological study found three major vectors species: Anopheles gambiae, An. funestus and An. mascarensis. The therapeutic study showed that SP was 100% effective (n = 13) and only one case of CQ treatment failure was recorded (1/15). Finally the diagnostic study demonstrated that presumptive diagnosis of malaria based on the only clinical signs leads to an over-estimation of malaria frequency. Over 68% (102/149) of febrile patients were diagnosed by physicians to have malaria while only 52 (34.9%) were proven positive. Of the 47 patients diagnosed clinically as malaria-negative, 12 (25.5%) turned out to be positive. Outbreaks of malaria during or after natural disasters in Madagascar can be successfully treated with either CQ or SP, but compliance may be better with SP since it requires only one dose. Perhaps equally important in the context of natural disasters is to have the capacity to make a definitive diagnosis, and the dipsticks should be made available.

[Hemoparasites in wild birds in Madagascar]. / Hémoparasites des oiseaux sauvages à Madagascar.

This study aims to evaluate the prevalence and density of haemoparasites in native Malagasy birds. Among the 387 birds, belonging to 43 species sampled at six localities in different bio-climatic zones of the island, 139 (35.9%) showed at least 1 hemoparasite with, by order of frequency, Plasmodium and/or Haemoproteus (19.9%), microfilariae (13.7% of 387 birds), Leucocytozoon (11.1%) and Trypanosoma (1.0%). An analysis to further elucidate these observations took into account the interaction of different environmental variables (altitude, season, site of collection) or aspects of the birds (age, weight, sex). There is evidence that some parasites preferentially infect some bird species or families. The largest male birds harboured the highest prevalences and densities of haemoparasite, regardless of species. These findings extend knowledge of bird/blood parasite relationships of Malagasy birds and provide interesting insights, especially concerning the pathogenicity of this type of parasitism and the parasite transmission by insect vectors.