Abstract: Resistance of head lice to pyrethroids induces difficult therapeutic problems. Previous studies demonstrated that this resistance was present in a French urban area, but its prevalence needed to be more precisely evaluated in terms of genotyping lice collected from more infested children over a certain period of time. We monitored the presence of the head lice kdr-like haplotype of the voltage-gated sodium channel alpha-subunit gene in schoolchildren seen three times on a 6-wk period. The prevalence of pediculosis was 2.39% (n = 1551). Genotyped lice (n = 167) were homozygous resistant in all but one pupil. The high frequency of the mutant haplotype (0.93) advocated for the abandonment of pyrethroid insecticides in this area and for the consideration of other treatment options.
Abstract: Little is known about severe imported Plasmodium falciparum malaria in industrialized countries where the disease is not endemic because most studies have been case reports or have included <200 patients. To identify factors independently associated with the severity of P. falciparum, we conducted a retrospective study using surveillance data obtained from 21,888 P. falciparum patients in France during 1996-2003; 832 were classified as having severe malaria. The global case-fatality rate was 0.4% and the rate of severe malaria was ≈3.8%. Factors independently associated with severe imported P. falciparum malaria were older age, European origin, travel to eastern Africa, absence of chemoprophylaxis, initial visit to a general practitioner, time to diagnosis of 4 to 12 days, and diagnosis during the fall-winter season. Pretravel advice should take into account these factors and promote the use of antimalarial chemoprophylaxis for every traveler, with a particular focus on nonimmune travelers and elderly persons.
Abstract: Malaria remains a major health problem in Madagascar. Over past decades, the burden of malarial disease has fluctuated over time, partly in line with the successes and failures of antimalarial policy. In the 1950s and 1960s, a sharp decline in malaria transmission was observed in the central highlands due to indoor spraying with DDT and to the massive use of chloroquine by the population. Following this, the discontinuation of the 'nivaquinization' policy was followed by devastating outbreaks in the central highlands in the 1980s. Currently, the rate of in vitro chloroquine-resistant Plasmodium falciparum isolates does not exceed 5%. This figure appears disconnected from the high level of clinical treatment failure (near 40%). pfcrt mutant isolates are found in less than 1% of isolates on the Island. Conversely, pfmdr1 mutant isolates are found in more than 60% of isolates and may be responsible for the bulk of resistance to chloroquine in Madagascar. Other antimalarials remain generally effective in Madagascar. Recent clinical and in vitro data support the complete efficacy of the combination artesunate-amodiaquine in Madagascar. As such, this artemisinin combination therapy should play a central role in the control and possible elimination of P. falciparum malaria in Madagascar
Abstract: The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the Plasmodium falciparum dhfr (pfdhfr) haplotypes and flanking microsatellites in 322 P. falciparum isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type pfdhfr allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral pfdhfr triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation pfdhfr allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.
Abstract: We sought to test the association of polymorphisms in Plasmodium falciparum nhe-1 (Pfnhe-1, gene PF13_0019) with in vitro susceptibility to quinine, which was previously reported in a limited number of reference strains or culture-adapted isolates. Determination of in vitro susceptibility to quinine, genotyping of Pfnhe-1 ms4760 microsatellite and polymorphism in codon 76 of Pfcrt were performed for 83 isolates obtained from symptomatic malaria-infected travelers returning from various African countries to France or from subjects living in Madagascar. Nineteen different ms4760 microsatellite profiles of Pfnhe-1 were found including 14 not previously described. Multivariate analysis showed no significant association between the in vitro susceptibility to quinine with particular ms4760 profiles. Contrary to previous reports, we only observed that the number of NHNDNHNNDDD repeats was positively associated with the increased IC50 of QN (P = 0.01). We concluded that the studied polymorphisms in Pfnhe-1 did not appear as valid molecular markers of in vitro susceptibility to quinine in P. falciparum isolates from Africa. Because we did not include any isolate of Asian origin in our series, these results did not exclude the possibility of regional associations, for example in South-East Asia.
Abstract: Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important, particularly regarding the evolution and spread of Pfmdr-1 alleles in P. falciparum populations under changing drug pressure which may have important consequences in terms of antimalarial use management.
Abstract: Large studies on severe imported malaria in non-endemic industrialized countries are lacking. We sought to describe the clinical spectrum of severe imported malaria in French adults and to identify risk factors for mortality at admission to the intensive care unit.
Abstract: We have developed a High-Resolution DNA Melting method to detect mutations related to Plasmodium falciparum resistance. This method is based on real-time PCR followed by High Resolution Melting ramping from 67 degrees C to 80 degrees C with fluorescence data acquisition set at 0.1 degrees C increments. The accuracy of the technique was assessed using 177 P. falciparum clinical isolates and two reference strains. Results perfectly matched those obtained by DNA sequencing for some important genetic markers of P. falciparum resistance. This technique could be of great value for epidemiological studies, especially in developing countries.
Abstract: The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.
Abstract: In Gabon, following the adoption of amodiaquine/artesunate combination (AQ/AS) as first-line treatment of malaria and of sulphadoxine/pyrimethamine (SP) for preventive intermittent treatment of pregnant women, a clinical trial of SP versus AQ was conducted in a sub-urban area. This is the first study carried out in Gabon following the WHO guidelines.
Abstract: Post-malaria neurological syndrome (PMNS) defined by a post-infective encephalopathy occurring within 2 months after an episode of Plasmodium falciparum infection is still a debated entity. We describe 2 cases of PMNS in 2 patients of African origin, born and living in France. Both patients had severe P. falciparum infection, followed by PMNS. They recovered with no sequelae. These are the first-reported cases of PMNS in patients of African ethnicity and living in France.
Abstract: We noticed overrepresentation of atovaquone-proguanil therapeutic failures among Plasmodium falciparum-infected travelers weighing >100 kg. We report here 1 of these cases, which was not due to resistant parasites or impaired drug bioavailability. The follow-up of such patients should be strengthened.
Abstract: The adhesion of infected red blood cells (IRBCs) to the cell lining of microvasculature is thought to play a central role in the pathogenesis of severe malaria. Individual IRBC can bind to more than one host receptor and parasites with multiple binding phenotypes may cause severe disease more frequently. However, as most clinical isolates are multiclonal, previous studies were hampered by the difficulty to distinguish whether a multiadherent phenotype was due to one or more parasite population(s). We have developed a tool, based on cytoadhesion assay and GeneScan genotyping technology, which enabled us to assess on fresh isolates the capacity of adherence of individual P. falciparum genotypes to human receptors expressed on CHO transfected cells. The cytoadhesion to ICAM-1 and CD36 of IRBCs from uncomplicated and severe malaria attacks was evaluated using this methodology. In this preliminary series conducted in non immune travelers, IRBCs from severe malaria appeared to adhere more frequently and/or strongly to ICAM-1 and CD36 in comparison with uncomplicated cases. In addition, a majority genotype able to strongly adhere to CD36 was found more frequently in isolates from severe malaria cases. Further investigations are needed to confirm the clinical relevance of these data.
Abstract: To investigate whether the severity of Plasmodium falciparum attack in endemic areas was associated with the multiplicity of infection (MOI) and/or with a particular genotype(s).
Abstract: Plasmodium falciparum malaria is a serious health hazard for travelers to malaria-endemic areas and is often diagnosed on return to the country of residence. We conducted a retrospective study of imported falciparum malaria among travelers returning to France from malaria-endemic areas from 1996 through 2003. Epidemiologic, clinical, and parasitologic data were collected by a network of 120 laboratories. Factors associated with fatal malaria were identified by logistic regression analysis. During the study period, 21,888 falciparum malaria cases were reported. There were 96 deaths, for a case-fatality rate of 4.4 per 1,000 cases of falciparum malaria. In multivariate analysis, risk factors independently associated with death from imported malaria were older age, European origin, travel to East Africa, and absence of chemoprophylaxis. Fatal imported malaria remains rare and preventable. Pretravel advice and malaria management should take into account these risk factors, particularly for senior travelers.
Abstract: Inadequate treatment practices with antimalarials are considered major contributors to Plasmodium falciparum resistance to chloroquine, pyrimethamine and sulfadoxine. The longitudinal survey conducted in Dielmo, a rural Senegalese community, offers a unique frame to explore the impact of strictly controlled and quantified antimalarial use for diagnosed malaria on drug resistance.
Abstract: The head louse, Pediculus humanus capitis (De Geer), is an hematophagous ectoparasite that affects mainly children. Resistance to insecticides belonging to pyrethroids and other pediculicides, such as malathion, is responsible for frequently reported treatment failures. Recent studies showed that a M815I-T929I-L932F kdr-like mutation in the voltage-gated sodium channel alpha-subunit gene was associated with permethrin resistance in head lice from several countries worldwide. We searched for the presence ofpyrethroid resistance gene in head lice populations obtained in schoolchildren in an urban area of France. All the 15 primary schools of Bobigny, a city located 3 km north of Paris, were selected to participate. Of 3,493 children enrolled, 3,345 (95.8%) children were screened for head lice by using fine-toothed antilouse combs. Live head lice were detected in 112 (3.3%) of children screened. A subsample of 90 lice was processed for DNA study. The amplification of a 332-bp portion of the kdr-like gene spanning the codon 929 was performed, and polymerase chain reaction products were submitted to the restriction enzyme SspI. Twenty of these lice (22.2%) were homozygous susceptible, 33 (36.7%) were homozygous resistant, and 37 (41.1%) were heterozygotes. Globally, the frequency of the T929I mutation was 0.57. The prevalence of pediculosis in schoolchildren of Bobigny seemed relatively low in comparison with findings of other European studies. The presence of the T929I mutation associated with permethrin resistance probably reflected the frequent local use of this insecticide. Further studies are now required to evaluate the prevalence of the kdr-like mutant allele in head lice in French schools.
Abstract: Plasmodium falciparum drug resistance represents a major health problem in malaria endemic countries. The mechanisms of resistance are not fully elucidated. Recently, an association between putative transporter gene polymorphisms and in vitro response to chloroquine (CQ) and quinine has been reported in culture-adapted, cloned isolates from various geographical origins. However, this was not confirmed in another study performed on isolates from a defined region in Thailand.
Abstract: We examined the atovaquone in vitro susceptibility and the cytochrome b (cytb) gene polymorphism of African Plasmodium falciparum isolates during the first years of atovaquone/proguanil use.
Abstract: Because of its dramatic public health impact, Plasmodium falciparum resistance to chloroquine (CQ) has been documented early on. Chloroquine-resistance (CQR) emerged in the late 1950's independently in South East Asia and South America and progressively spread over all malaria areas. CQR was reported in East Africa in the 1970's, and has since invaded the African continent. Many questions remain about the actual selection and spreading process of CQR parasites, and about the evolution of the ancestral mutant gene(s) during spreading.
Abstract: Procalcitonin (PCT) has been found elevated in complicated forms of Plasmodium falciparum malaria. Its usefulness has almost never been assessed in uncomplicated falciparum malaria.
Abstract: Controversy exists about which antimalarial chemoprophylaxis regimen should be used among travellers to Africa: the WHO and other experts recommend the use of mefloquine throughout sub-Saharan Africa, whereas French experts still support the combination of chloroquine and proguanil in most of West Africa (the so-called zone 2 countries). In this case-control study based at a travel clinic, we examined the compliance with antimalarial chemoprophylaxis and its efficacy among travellers to tropical areas. Cases were patients with Plasmodium falciparum malaria (n = 131). Controls were patients who had a negative malaria film (n = 158). Of all controls, only 36 (22.8%) were adequately protected (i.e. compliant with an adapted regimen of chemoprophylaxis). In zone 2 countries, the efficacy of the combined chloroquine and proguanil was 58% (95% CI 22-78%) for all users, but increased to 100% (95% CI 89-100%) for compliant users. In zone 3 countries, the efficacy of mefloquine was 90% (95% CI 51-98%) and 100% (95% CI 58-100%) for all users and compliant users, respectively.
Abstract: Among populations living in areas endemic for malaria, repeated parasite exposure leads to a gradual increase in protective immunity to the disease. In contrast, this immunity is assumed to disappear after several years of non-exposure. This study was designed to investigate long-term immunity in subjects removed from the risk of exposure. Plasmodium falciparum malaria attacks occurring after short trips to sub-Saharan Africa were compared between 99 European patients and 252 African immigrants who had been resident in Europe for at least four years. Relative to the European patients, those originating from Africa had lower mean +/- SD parasite densities (0.8 +/- 1.5/100 red blood cells versus 1.4 +/- 2.8/100 red blood cells; P = 0.007), less frequent severe disease (4.4% versus 15.2%; P = 0.0005), accelerated parasite clearance and defervescence, and higher levels of antibodies to P. falciparum. These results suggest the persistence of acquired immunity to P. falciparum malaria after several years of non-exposure in African immigrants.
Abstract: We have developed a new fragment-analysis method to enumerate the clones and to quantify their proportions within Plasmodium falciparum isolates. We prospectively enrolled 20 adult patients with uncomplicated malaria who were returning to France from various sub-Saharan countries, from January 2000 through July 2001. The analysis of clonal populations was performed on blood samples obtained at 10 times: 1 before treatment with oral quinine and 9 during the first 96 h of the treatment. The resistance genotypes pfcrt and dhfr were determined for chloroquine and antifolinics. Multiple P. falciparum genotypes were detected in 19 (95%) of 20 patients: 2, 3, 4, and 5 genotypes were found in 4, 9, 4 and 2 patients, respectively. Disappearance and reappearance of some clones within a few hours was observed. Individual clones represented 0.4%-99.4% of total parasitemia. Surprisingly, in 10 of 15 subjects tested, resistance genotypes varied according to the time of blood collection. These findings may have important implications with regard to the interpretations of resistance studies.
Abstract: Msp-1 and Msp-2 genes, each present as a unique copy in the genome of Plasmodium, contain polymorphic repeats in bloc 2. We studied allelic polymorphism of Msp-1 and Msp-2 by amplifying bloc 2 with a fluorescent primer, and analysing the fragment generated. We validated this method by mixing two cloned strains: chloroquine-susceptible HB3-Honduras and chloroquine-resistant FCM29-Cameroon. This method was then used to quantify the clones in natural isolates of 19 infected persons during quinine treatment. The fragment analysis method detects efficiently clone numbers and the proportions of each in isolates.
Abstract: Little is known about severe imported malaria in nonendemic industrialized countries. The purpose of this retrospective study was to describe the clinical spectrum of severe imported malaria in adults and to determine factors that were present at admission and were associated with in-intensive care unit mortality. This retrospective study evaluated the 188 patients who were admitted to our intensive care unit in 1988-1999 with severe and/or complicated imported malaria. Among them, 93 had strictly defined severe malaria, and 95 had less severe malaria. The mean age was 38 years, 51% of patients were nonimmune whites, 94% acquired Plasmodium falciparum in sub-Saharan Africa, and 96% had taken inadequate antimalarial chemoprophylaxis. Mortality was 11% (10 patients) in the severe malaria group, whereas no patients died in the less severe malaria group (p = 0.002). In the bivariable analysis, the main factors associated with death in the severe malaria group were the Simplified Acute Physiology Score, shock, acidosis, coma, pulmonary edema (p < 0.001 for each), and coagulation disorders (p = 0.002). Bacterial coinfection is not infrequent and may contribute to death. Severe imported malaria remains a major threat to travelers. In our population, the most relevant World Health Organization major defining criteria were coma, shock, pulmonary edema, and acidosis.
Abstract: Drug-resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South-east Asia. It causes severe fever or anaemia that leads to more than a million deaths each year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions. The rationale for chemoprophylaxis is weakening as multiple-drug resistance develops against well-tolerated drugs. Plasmodium falciparum drug-resistant malaria originates from chromosome mutations. Analysis by molecular, genetic and biochemical approaches has shown that (i). impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii). one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii). the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv). treatment with sulphadoxine-pyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD). The mechanisms of resistance for amino-alcohols (quinine, mefloquine and halofantrine) are still unclear. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries. Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrug-resistance encountered in the forests of South-east Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilize at the same level as chloroquine-sensitive malaria. Nevertheless, resistance levels may differ according to place and time. In vivo and in vitro tests do not provide an adequate accurate map of resistance. Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance.
Abstract: The number of travellers in malaria striken areas increases each year (2). The risk of infection is high in Sub-Saharan Africa, but appropriate chemoprophylaxis can reduce the morbidity and mortality rate of malaria. Half of the samples of malaria cases received by the National reference centre of malaria chemosensibility (CNRCP) for chemosensibility analysis came from two hospitals in the north of Paris: Bichat Claude Bernard in Paris and Delafontaine in Saint-Denis. In 2000, quite all the malaria cases (n = 387) observed at the Bichat and Delafontaine Hospitals came from Africa (99%). Plasmodium falciparum remains the most represented (87.6%) species, with an average parasitic density of 0.3%. Patients with P falciparum came for medical advice on the tenth day after return (median, extremes 0-174 days). More than half of the patients (58%) did not take any medication for chemoprophylaxis and even if they took some, it was irregular or inappropriate. The most used drug chemoprophylaxis is the association of chloroquine and proguanil or Savarine. In 15% of the cases, the travellers took chloroquine as a prophylaxis and 4% other medicine not recommended by the French authorities. An average of 43.7% of these travellers took inappropriate chemoprophylaxis. In total, 27 chemoprophylaxis failures are reported. Some patients (22%) have already taken self treatment which was readjusted during admission at hospital. The first treatment of malaria in 2000 was monotherapy with quinine (P. falciparum) and chloroquine (P. ovale, malariae, vivax). The treatment associations in case of suspicious resistance were quinine + doxycycline and atovaquone + proguanil. Treatment failure was infrequent and resulted above all from a bad observance. More information should be given to travellers as well as doctors about recommendations and treatments.
Abstract: To assess the relationship between the presence of DHFR and DHPS mutations in Plasmodium falciparum, parasite in vitro resistance, and in vivo efficacy of sulfadoxine-pyrimethamine (SP) treatment.
Abstract: In south-eastern Iran, the sulfadoxine-pyrimethamine (SP) combination has been used to treat malaria caused by chloroquine-resistant Plasmodium falciparum. To explore the molecular basis of antimalarial resistance in this region, the dhfr, dhps and pfcrt genes of 50 clinical isolates of P. falciparum, collected from cases of uncomplicated malaria in 2000-2001, were checked for the point mutations that appear associated with SP or chloroquine (CQ) resistance. The results of the study, which was based on a PCR followed by DNA sequencing, indicated that all 50 isolates presented the DHFR S108N mutation associated with pyrimethamine resistance. Seven isolates (14%) had a triple DHFR mutation (S108N, N51I, C59R) and 32 (64%) had a double mutation in this domain. Thirty-nine isolates (78%) had the wild-type DHFR 51 codon but only 15 (30%) had the wild-type DHFR 59 codon. Eleven isolates (22%) only had the DHFR S108N mutation. All isolates had the wild-type DHPS 436 and 540 codons and all but two of the isolates had the wild-type DHPS 437 codon. All isolates but one had the PFCRT K76T mutation associated with CQ treatment failure. The results of this preliminary investigation indicate that SP may remain the treatment of choice for cases of uncomplicated malaria in south-eastern Iran.
Abstract: Four airport malaria cases have been observed in the vicinity of the Roissy-Charles-de-Gaulle International Airport, Paris, France. These cases were geographically very close to each other and clustered in a short period of time during the summer of 1999. The phenotype and genotype of the Plasmodium falciparum isolates obtained from these patients were determined in order to know whether a single mosquito could have infected more than one subject. The genomic characterisation of isolates was performed using the polymorphic markers merozoite surface protein 1 (Msp 1) and merozoite surface protein 2 (Msp 2) genes, the kappa and omega repeats domains of cg2 and the dihydrofolate reductase (DHFR) genotypes. Results showed identical genotypes for isolates 1, 2 and 4 whereas the genotype of isolate 3 differed at one locus. The molecular analysis was consistent with the hypothesis that all patients could have been bitten by the same mosquito and that patient 3, may have received a different clone and an additional species. In vitro susceptibility data did not confirm or rule out this hypothesis because isolates had the same profile of susceptibility to the tested drugs.
Abstract: A PCR-based technique using molecular beacons was developed to detect the chloroquine resistance-associated pfcrt K76T point mutation in Plasmodium falciparum. One hundred thirty African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This rapid and inexpensive genomic assay could expand the possibilities for monitoring chloroquine resistance.
Abstract: In Liberia, little information is available on the efficacy of antimalarials against Plasmodium falciparum malaria. We measured parasitological resistance to chloroquine and sulfadoxine-pyrimethamine (SP) in Harper, south-west Liberia in a 28-d study in vivo. A total of 50 patients completed follow-up in the chloroquine group, and 66 in the SP group. The chloroquine failure rate was 74.0% (95% confidence interval [95% CI] 59.7-85.4%) after 14 d of follow-up and 84.0% (95% CI 70.9-92.8%) after 28 d (no polymerase chain reaction [PCR] analysis was performed to detect reinfections in this group). In the SP group, the failure rate was 48.5% (95% CI 36.2-61.0%) after 14 d and 69.7% (95% CI 57.1-80.4%) after 28 d, readjusted to 51.5% (95% CI 38.9-64.0%) after taking into account reinfections detected by PCR. Genomic analysis of parasite isolates was also performed to look for point mutations associated with resistance. Genotyping of parasite isolates revealed that all carried chloroquine-resistant K-76T mutations at gene pfcrt, whereas the triple mutation (S108N, N511, C59R) at dhfr and the A437G mutation at dhps, both associated with resistance to SP, were present in 84% and 79% of pretreatment isolates respectively. These results seriously question the continued use of chloroquine and SP in Harper and highlight the urgency of making alternative antimalarial therapies available. Our study confirms that resistance to chloroquine may be high in Liberia and yields hitherto missing information on SP.
Abstract: In 2000, the chemosusceptibility of imported malaria was stable in France. All countries of infection considered, the bi-resistance to chloroquine and cycloguanil has not changed from 1996 to 2000. The monotherapy using quinine or mefloquine remains the first-line treatment to falciparum malaria. Resistance to these two antimalarials is rare in Africa and has not evolved over the past 15 years.
Abstract: Drug resistant malaria is mostly due to Plasmodium falciparum, a species highly prevalent in tropical Africa, Amazon and Southeast Asia. P. falciparum is responsible for severe involvement of fever or anaemia prompting more than a million deaths per year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality in some human populations from endemic regions. Rationale for chemoprophylaxis is becoming week as multiple drug resistance against well tolerated drugs develops. Plasmodium falciparum drug resistant malaria originate from chromosomal mutations. Analysis using molecular, genetic and biochemical approaches has shown that Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among parasites isolates populations while resistance to antifolinics is highly prevalent in most malarial endemic countries. An established and strong drug pressure and a low antiparasitic immunity probably explains the multidrug-resistance encountered in forests of Southeast Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilise at an equal level as chloroquine-sensitive malaria. Nevertheless, resistance levels may differs according to places and time. In vivo and in vitro tests are insufficient to give an accurate map of resistance. Biochemical tools at a low cost are urgently needed for a prospective monitoring of resistance.
Abstract: Recent transfection based studies demonstrated that cg2, a candidate gene for chloroquine resistance in Plasmodium faiciparum, was not the resistance determinant. A further analysis of the initial 36 kb locus comprising the cg2 gene led to the discovery of another gene, pfcrt, which was absolutely associated with chloroquine resistance in forty parasite lines. The aim of this study was to evaluate, in 146 unselected clinical isolates obtained mostly from non-immune travellers returning from various endemic countries to France in years 1995-1999, the association between in vitro chloroquine resistance and the sequence of a part of the pfcrt gene. For comparison, the determination of the cg2 kappa and the pfmdr1 codon 86 genotypes were also performed on the same isolates. As determined by an isotopic semi-microtest, 70 isolates were susceptible to chloroquine (50% inhibitory concentration < 80 nM) and 76 were resistant. The amplification of a portion of the pfcrt gene spanning codons 72 to 76, followed by sequencing showed three distinct genotypes: one type associated with susceptible isolates, one type associated mostly with resistant isolates and one type found in a resistant isolate originating from South America. Three different zones could be defined according to the status of codon 76. For 50% inhibitory concentration values < or = 40 nM (n = 47), all isolates but one had K76 (wild type). For 50% inhibitory concentration values located between 40 and 60 nM, isolates had either K76 (n = 5) or K76T (mutant type) (n = 6). For 50% inhibitory concentration values > 60 nM (n = 88), all isolates had K76T. A lack of a strong association between the pfmdr1 N86Y mutation and in vitro chloroquine resistance was observed. Cg2 genotypes were less strongly linked than pfcrt genotypes with in vitro chloroquine susceptibility in isolates located below 40 nM and above 60 nM. Further studies are needed to determine the reliability of the pfcrt gene as a genetic marker for chloroquine resistance.
Abstract: The parasitic loads of mouse livers experimentally infected with Leishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The Leishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.
Abstract: The purpose of this prospective study was to update epidemiological data on cutaneous larva migrans (CLM) and to assess the therapeutic efficacy of ivermectin. We performed the study between June 1994 and December 1998 at our travel clinic. Ivermectin (a single dose of 200 microg/kg) was offered to all the patients with CLM, and its efficacy and tolerability were assessed by a questionnaire. Sixty-four patients were enrolled. All were European and had stayed in tropical areas. After the patients had returned from their destinations, 55% had lesions occur within a mean of 16 days (range, 1-120 days; >1 month in 7 patients). The initial diagnosis was wrong in 55% of patients. The mean number of lesions was 3 (range, 1-15), and the main sites were the feet (48%) and buttocks (23%). The cure rate after a single dose of ivermectin was 77%. In 14 patients, 1 or 2 supplementary doses were necessary, and the overall cure rate was 97%. The median time required for pruritus and lesions to disappear was 3 and 7 days, respectively. No systemic adverse effects were reported. Physicians' knowledge of CLM, which can have a long incubation period, is poor. Single-dose ivermectin therapy appears to be effective and well tolerated, even if several treatments are sometimes necessary.
Abstract: A PCR-based technique using new fluorescent probes, called molecular beacons, was developed to detect the antifolate resistance-associated S108N point mutation in Plasmodium falciparum. One hundred African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This new molecular technique appears to be a promising tool for epidemiological studies.
Abstract: The incidence of cycloguanil resistance in 501 Plasmodium falciparum isolates from individuals entering France from Africa was estimated by a method based on PCR-restriction-fragment-length polymorphisms. None of the subjects had taken antifol prophylaxis. Annual incidence of the resistance, detected as a point mutation at codon 108 in the parasite's dihydrofolate-reductase gene, increased from 19.8% in 1995 to 43.6% in 1997 (P < 0.001). The proportion of isolates found to be susceptible (i.e. wild-type) among travellers returning from the African countries known as Group 2 in France (i.e. Burkina Faso, Côte d'Ivoire, Gambia, Ghana, Guinea, Liberia, Madagascar, Mali, Mauritania, Niger, Senegal, Sierra Leone, Tchad and Togo) was reasonably high (62.9%) and much higher than in the other subjects returning from other identifiable countries in Africa (35.3%). The antimalarial prophylaxis recommended in France to those travelling to Group-2 countries, chloroquine-proguanil, therefore still seems reasonable, although cycloguanil resistance may seriously undermine the efficacy of this drug combination in the future.
Abstract: The correlation between the structure of two short sequences from the Plasmodium falciparum cg2 gene and parasite chloroquine susceptibility was evaluated in unselected clinical isolates obtained from travellers returning mainly from Africa to France in 1995 and 1996. As determined by an isotopic semi-microtest, 74 isolates were susceptible to chloroquine (50% inhibitory concentration < 80 nM), 13 were intermediate (80 nM < 50% inhibitory concentration < 110 nM) and 53 were resistant (50% inhibitory concentration > 110 nM). Two polymerase chain reaction assays were developed, one for the kappa and one for the omega repeat domains of cg2 gene. The kappa and the omega repeat domains of 99 isolates were sequenced. A variation in the unit number of kappa and omega repeats was observed. Variations in repetitive sequences, which were not previously described, were found: three for the kappa repeat region: kappa9; kappa10 and kappa11 and three for the omega repeat region: omega8; omega9 and omega22. A polymorphism was observed inside the repeat units of kappa and omega regions. There were six possible kappa repeat units and seven possible omega repeat units. The presence of a particular pattern, containing kappa14 and omega16 repeat units, was associated with a lack of chloroquine susceptibility in 44 out of 46 cases. However, not all resistant isolates had this 'resistant' genotype. Among 43 resistant isolates, 36 (84%) had the kappa14 repeats sequence and 36 had the omega16 repeats sequence. These results lend further support to linkage between cg2 polymorphisms and chloroquine resistance without excluding the existence of other resistance component(s).
Abstract: Amphotericin B (AmB) has been used as a second-line treatment of visceral leishmaniasis, particularly in human immunodeficiency virus-positive patients. AmB median effective doses (ED50s) were determined on an isolate obtained before any treatment and on a second isolate obtained 4 years later from the same AmB-treated patient. ED50s were similar (0.059 and 0.067 mg/kg of body weight, respectively), demonstrating the first evidence of AmB ED50 stability of Leishmania infantum after a long-term drug exposure. An isoenzymatic study was performed in order to verify that the second isolate originated from the same parasite as the first isolate. The present case report showed that treatment failure was not due to parasite resistance in spite of a prolonged exposure to the drug.
Abstract: The in vitro susceptibility of chloroquine and the genomic profile of dihydrofolate reductase (DHFR) codon 108 was determined against african isolates of P. falciparum (Pf) from imported malaria cases without previous drug intake by an isotopic microtest or PCR + RFLP. Pf resistance to chloroquine or to the DHFR inhibitor was present in 49% and 46% of isolates, respectively. Pf drug resistance was more frequent in permanent than in seasonal malarial transmission areas and chloroquine plus DHFR resistance reached 28% in years 1995-97. Updating the guidelines for the prevention of malaria in travellers to Africa is necessary.
Abstract: Drug targeting enhances drug efficacy. This principle was tested in the treatment of an experimental visceral leishmaniasis. Using transmission electron microscopy (TEM) we localized pentamidine-loaded polymethocrylate nanoparticles in the liver of mice infected with Leishmania major and compared the ultrastructural changes in the parasites of these mice when they were treated with bound versus free pentamidine. Between days 13 and 17 after infection, loaded nanoparticles treated group were injected i.v. with 3 doses of 0.17 mg/kg bound pentamidine loaded on 2 x 10(11) nanospheres; control groups received 2 x 10(11) unloaded nanospheres. Drug reference control groups received five doses of 200 mg/kg pentavalent antimony (Glucantime) or three doses of free pentamidine (0.17 mg/kg or 2.28 mg/kg). Mice treated with bound pentamidine displayed a 77% reduction in their parasite burden versus the untreated controls. Nanoparticles were located by TEM inside parasitized Küpffer cells, in the phagolysosomes without entering the Leishmania. The low dose of 0.17 mg/kg bound pentamidine damaged the Leishmania to the same extent as 2.28 mg/kg of free pentamidine (the usual dose in human chemotherapy). In the parasites inside the Küpffer cells, TEM showed a swollen mitochondrian with loss of cristae, destruction or fragmentation of the kinetoplast, loss of ribosomes and destruction of parasite structures except for the subpellicular microtubules. This study therefore shows that a dose of bound pentamidine 13 times smaller than the usual dose of free pentamidine has a similar effect on the parasite.
Abstract: The use of drug delivery systems may reduce the toxicity and improve the activity of antileishmanial compounds. In view of such a strategy, we loaded the antileishmanial agent pentamidine on polymethacrylate nanoparticles. The activity of pentamidine-loaded nanoparticles was compared with that of free pentamidine in a BALB/c mice model of visceral leishmaniasis induced by Leishmania infantum. On day 0, mice were infected intravenously with 10(7) promastigotes and then treated via the tail vein on days 14, 16 and 18 with bound pentamidine, free drug or isotonic saline (control group). On day 21, liver parasite burdens were evaluated using the Stauber method. Livers and spleens were removed and weighed. Effective doses (ED) were determined using the Michaelis-Menten representation relating the percentage of parasite suppression to the dose. The ED50 of bound pentamidine was six times lower than that of free pentamidine (0.17 mg kg-1 vs 1.06 mg kg-1). The ED90 value calculated for bound pentamidine was 1 mg kg-1. It was not possible to obtain the ED90 for free pentamidine because the dose-response curve reached a plateau near 60% of parasite suppression. A significant decrease in liver and spleen weights, probably reflecting the leishmanicidal activity, was observed for treated mice with bound pentamidine. These results showed that bound pentamidine was more potent than the free drug against L. infantum in our BALB/c mice model.
Abstract: A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.
Abstract: Visceral leishmaniasis is caused by hemoflagellate protozoa which are obligatory parasites of the mononuclear phagocyte system. Leishmaniasis causes high morbidity and mortality worldwide. The treatment of choice remains pentavalent antimonials, but high toxicity and failures have been reported. An alternative to conventional treatment is delivery anti-leishmania agents using colloidal carrier systems. Carriers improve drug activity against intracellular disease involving the mononuclear phagocyte system. The principle of drug delivery by carrier systems has been applied successfully for anticancer drugs. Recently complete remission of polyresistant visceral leishmaniasis was obtained by injection of liposomal amphotericin B. At present, no colloidal drug carrier for antimony derivatives is available, but pentamidine can be linked experimentally to methacrylate polymer nano-particles. Drug-loaded nanoparticles have been shown to be effective against amastigote leishmania both in vitro and in vivo. Another colloidal system of major interest for drug delivery, the liposome has already been loaded with amphotericin B and used for human therapy. The concept of particulate drug carriers opens the way for new chemotherapeutic approaches in the field of parasitology.
Abstract: The efficiency of antileishmanial agents may be enhanced by improving their bioavailability with a colloidal drug carrier. We have investigated the action of free pentamidine, compared with pentamidine bound to polymethacrylate nanoparticles, in a rodent model. BALB/c mice were infected, via the tail vein, with 4 x 10(7) L. major (MON 74) promastigotes. Twelve days after infection, seven groups of mice were treated respectively with methylglucamine antimoniate (Glucantime) 5.56 mg/kg i.p. x 5 d., pentamidine bound nanoparticles (100 microM), unloaded polymethacrylate nanoparticles, unloaded nanoparticles associated with free pentamidine (100 microM) 0.1 ml i.v. x 3 d and free pentamidine isethionate (2.28 mg/kg and 0.17 mg/kg i.v. x 3 d.). Twenty-one days post infection, the mice were sacrificed and the Leishmania load in the liver calculated from the number of amastigotes/500 liver cells and total liver weight in treated and untreated mice. Results demonstrated a 77% amastigote reduction in the group treated with targeted pentamidine relative to the control group. The ratio free pentamidine/bound-pentamidine was approx. 12.
Abstract: Antileishmanial chemotherapy is hampered by the location of parasites within lysosomal vacuoles of the macrophages which restricts the bioavailability of many potential antileishmanial compounds. In this study, the effectiveness of pentamidine targeted to the infected cells by a linkage to a colloidal drug carrier, methacrylate polymer nanoparticles was explored. In the same way, polyisoalkylcyanoacrylate nanospheres which have, in vitro, trypanolytic properties were also tested. The study was performed in an in vitro model using Leishmania major amastigote stages within the U 937 human monohistiocytic cell line. The antileishmanial activities of unloaded or pentamidine-loaded nanoparticles were compared to those of the free drugs. The 50% effective concentration of targeted pentamidine was 0.10 microgram/ml, while it was up to 2.7 micrograms/ml with the free drug after a 24-hour incubation time. The pentamidine-bound nanoparticles proved to be 25 times more active than the free drug. Unloaded polyisoalkylcyanoacrylate nanoparticles destroyed intracellular amastigote stages (50% EC = 15 micrograms/ml) but at a level close to the cytotoxic concentration.
