Plasmodium vivax est-il encore le paradigme d'un paludisme simple ?

P. vivax is supposed to be involved in benign tertian fever, responsible for a non-complicated disease that could be easily treated by standard antimalarial drug regimen. This could be considered as a long-standing paradigm of a non-virulent malaria parasite. When a patient exhibits severe malaria with the vivax parasite, the issue is often to find falciparum. However, with the implementation of molecular diagnosis, it has becoming more evident that vivax parasites could be involved in severe disease with probably a different pathogenesis. Mixed infections are frequent in various parts of Southeast Asian endemic areas and it was speculated that drugs used to treat falciparum could be involved in the development of vivax drug resistance. How should primaquine be used today for the treatment and prophylaxis of vivax malaria? Considering the re-emergence of vivax malaria in several areas, improving the treatment for this disease is certainly an important issue to avoid late episodes and transmission potential.     

Protection against malaria by immunization with non-attenuated sporozoites under single-dose piperaquine-tetraphosphate chemoprophylaxis

Experimental whole-parasite immunization through concurrent administration of infectious Plasmodium sporozoites with drugs that prevent pathogenic blood-stage infection represents the current benchmark in malaria vaccine development. Key questions concerning translation remain, including the requirement for single-dose drug regimens that can reliably prevent breakthrough infections. We assessed the feasibility and efficacy of immunization with single-dose piperaquine chemoprophylaxis and concurrent sporozoite administration (PPQ-CPS) in the murine P. berghei ANKA/C57BL/6 infection model. We demonstrate that PPQ-CPS is protective with an efficacy comparable to previous findings using whole-parasite immunization under chloroquine chemoprophylaxis. PPQ-CPS immunization resulted in an expansion of intrahepatic and intrasplenic effector memory CD8⁺ T cells. In summary, PPQ-CPS appears to be a safe and efficacious immunization regimen in the rodent malaria model and may thus become an important improvement regarding the translation of whole-parasite immunization toward a human malaria vaccine.     

Plasmodium falciparum Mutant Haplotype Infection during Pregnancy Associated with Reduced Birthweight,Tanzania

Intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine (IPTp-SP) is a key strategy in the control of pregnancy-associated malaria. However, this strategy is compromised by widespread drug resistance from single-nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. During September 2008–October 2010, we monitored a cohort of 924 pregnant women in an area of Tanzania with declining malaria transmission. P. falciparum parasites were genotyped, and the effect of infecting haplotypes on birthweight was assessed. Of the genotyped parasites, 9.3%, 46.3%, and 44.4% had quadruple or less, quintuple, and sextuple mutated haplotypes, respectively. Mutant haplotypes were unrelated to SP doses. Compared with infections with the less-mutated haplotypes, infections with the sextuple haplotype mutation were associated with lower (359 g) birthweights. Continued use of the suboptimal IPTp-SP regimen should be reevaluated, and alternative strategies (e.g., intermittent screening and treatment or intermittent treatment with safe and effective alternative drugs) should be evaluated.     

Sensitivity to quinine and mefloquine of Plasmodium falciparum in Thailand

Between 1982 and 1984 a regimen consisting of quinine and tetracycline was routinely used in Thailand to treat outpatients with microscopically confirmed falciparum malaria. Due to compliance problems associated with the 7-day multiple-dose regimen, there was a recrudescence rate of approximately 30%. Studies carried out in 1982 and 1984 in four areas of Thailand indicated that there was a significant decrease in the sensitivity of Plasmodium falciparum to quinine. A significant, though less marked, reduction in the sensitivity of P. falciparum to the structurally related drug mefloquine was also observed, although this compound was not operationally deployed in Thailand before 1985. These findings emphasize the need to replace the long multiple-dose quinine regimen by an effective, acceptable, single-dose treatment.     

Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.     

DNA sequence polymorphisms of the pfmdr1 gene and association of mutations with the pfcrt gene in Indian Plasmodium falciparum isolates

Mutations in the Plasmodium falciparum multidrug resistance (pfmdr1) gene are known to provide compensatory fitness benefits to the chloroquine (CQ)-resistant malaria parasites and are often associated with specific mutations in the P. falciparum CQ resistant transporter (pfcrt) gene. Prevalence of the specific mutations in these two genes across different malaria endemic regions was mostly studies. However, reports on mutations in the pfmdr1 gene and their genetic associations with mutations in the pfcrt gene in Indian P. falciparum field isolates are scarce. We have sequenced a 560 bp region of pfmdr1 coding sequence in 64 P. falciparum isolates collected from different malaria endemic populations in India. Twenty out of these 64 isolates were laboratory cultured with known in vitro CQ sensitiveness (10 sensitive and 10 resistant). Three low frequency mutations (two non-synonymous and one synonymous) in the pfmdr1 gene were segregating in Indian isolates in addition to the predominant Y₈₆ and Y₁₈₄ ones, with high haplotype and nucleotide diversity in the field isolates in comparison to the cultured ones. No statistically significant genetic association between the mutations in the pfmdr1 and pfcrt gene could be detected; almost all observed associations were intragenic in nature. The results on the genetic diversity of the pfmdr1 gene were discussed in term of evolutionary perspectives in Indian P. falciparum, with possible future potential of gaining further insights on this gene in view of evolving malaria parasites resistant to artemisinin partner drugs.     

Emergence of sulfadoxine–pyrimethamine resistance in Indian isolates of Plasmodium falciparum in the last two decades

Genotyping the sulfadoxine–pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country.     

Sulfalene with pyrimethamine and chloroquine with pyrimethamine in single-dose treatment of Plasmodium falciparum infections. A trial in a rural population in northern Nigeria

A comparative trial was carried out in northern Nigeria of the ability of the drug combinations chloroquine—pyrimethamine and sulfalene—pyrimethamine to clear the peripheral blood stream of asexual forms of P. falciparum within 7 days. The reappearance of asexual P. falciparum forms within the 70-day follow-up period and the occurrence of vomiting during the 2-3 hours following administration of the drugs were also recorded. The purpose of the trial was to choose the more suitable of the two drug combinations for repeated mass administration in the intervention phase of a collaborative field research project in the epidemiology and control of malaria in the African savannah. No differences were observed between the two drug combinations from a parasitological point of view. However, the sulfalene—pyrimethamine combination was found easier to administer and occasioned fewer records of vomiting. It was therefore recommended for use in the project.     

Analysis of genetic diversity in the chloroquine-resistant gene Pfcrt in field Plasmodium falciparum isolates from five regions of the southern Cameroon

Understanding the population genetics of genes which shape resistance to antimalarial drugs can help in devising novel control strategies. The high spread of the resistant strains of the malaria parasite Plasmodium falciparum pose a greater challenge than before to the control programs across the world. Specific mutations in the P. falciparum chloroquine resistant transporter gene “Pfcrt” have been associated with resistance to not only chloroquine, but also to amodiaquine, one of the artemisinin partners used in Cameroon for the treatment of uncomplicated malaria. We here present data on genetic variation at the Single Nucleotide Polymorphisms (SNPs) level in the Pfcrt gene in five distinct geographical settings of the Southern-Cameroon (the most malaria endemic part), i.e. Ebolowa, Yaounde, Bertoua, Douala and Kye-ossi (a city bordering Cameroon and two others African countries). Two novel mutations, hitherto unreported (in Cameroon) were found in the Pfcrt gene and variable genetic diversity was observed across the populations. High linkage disequilibrium was found between few SNPs including one of the novel mutations suggesting a synergistic work for conferring/maintaining a higher level of resistance. The inference of evolutionary pattern of this gene in Cameroon based on the genetic diversity data depicts a signature of Darwinian positive natural selection on these loci. Observation of novel mutations might traduce new variants in chloroquine/or amodiaquine resistance (proposal awaiting an experimental verification) and signal of positive selection can be the result of drug pressure exerted by misuse of chloroquine (though officially banned from the country) and/or amodiaquine. Our findings thus, provide a baseline understanding of the evolution of a malaria drug resistant gene in Cameroon and suggest a successful establishment of chloroquine-resistant strains which requires urgent attention of the malaria control program in Cameroon.     

Absence of association between Plasmodium falciparum malaria and human immunodeficiency virus infection in children in Kinshasa, Zaire

The possible associations between Plasmodium falciparum malaria and HIV (human immunodeficiency virus) seropositivity were investigated in 1986 at the Mama Yemo Hospital in Kinshasa, Zaire. No significant difference was found in the HIV seropositivity rate of 164 children presenting with P. falciparum malaria (1.2%) and 169 healthy controls (0.6%). Secondly, no association was found between P. falciparum slide positivity (51.6%) and HIV seropositivity (3.8%) among 1046 children presenting to the hospital with medical complaints. Infants less than 6 months old had the lowest slide-positivity rate, but among infected children the younger ones more frequently had high parasitaemias. HIV seropositivity rates were highest for children less than 6 months old. In older children, seropositivity was strongly associated with a history of blood transfusion. Thus, in Kinshasa children, P. falciparum malaria is a major public health problem; perinatal transmission and blood transfusions constitute important mechanisms of HIV infection; and P. falciparum does not appear to act as an opportunistic agent in children infected with HIV.     

Heterozygous mutants of TIRAP (S180L) polymorphism protect adult patients with Plasmodium falciparum infection against severe disease and mortality

Toll-interleukin-1 receptor domain containing adapter protein (TIRAP) plays a crucial role in TLR2 and TLR4 signaling pathways. Glycosylphospatidylinositol (GPI), considered a toxin molecule of Plasmodium falciparum, interacts with TLR2 and 4 to induce an immune inflammatory response. A single nucleotide polymorphism at coding region of TIRAP (S180L) has been reported to influence TLRs signaling. In the present study, we investigated the association of TIRAP (S180L) polymorphism with susceptibility/resistance to severe P. falciparum malaria in a cohort of adult patients from India. TIRAP S180L polymorphism was typed in 347 cases of severe malaria (SM), 232 uncomplicated malaria and 150 healthy controls. Plasma levels of TNF-α was quantified by ELISA. Heterozygous mutation (S/L) conferred significant protection against MOD (multi organ dysfunction), NCSM (non-cerebral severe malaria) as well as mortality. Interestingly, homozygous mutants (L/L) had 16 fold higher susceptibility to death. TIRAP mutants (S/L and L/L) were associated with significantly higher plasma TNF-α levels compared to wild type (S/S). The results of the present study demonstrate that TIRAP S180L heterozygous mutation may protect patients against severe malaria and mortality.     

Effects of Mefloquine Use on Plasmodium vivax Multidrug Resistance

Numerous studies have indicated a strong association between amplification of the multidrug resistance-1 gene and in vivo and in vitro mefloquine resistance of Plasmodium falciparum. Although falciparum infection usually is not treated with mefloquine, incorrect diagnosis, high frequency of undetected mixed infections, or relapses of P. vivax infection triggered by P. falciparum infections expose non–P. falciparum parasites to mefloquine. To assess the consequences of such unintentional treatments on P. vivax, we studied variations in number of Pvmdr-1 (PlasmoDB accession no. PVX_080100, NCBI reference sequence {"type":"entrez-nucleotide","attrs":{"text":"NC_009915.1","term_id":"166093247","term_text":"NC_009915.1"}}NC_009915.1) copies worldwide in 607 samples collected in areas with different histories of mefloquine use from residents and from travelers returning to France. Number of Pvmdr-1 copies correlated with drug use history. Treatment against P. falciparum exerts substantial collateral pressure against sympatric P. vivax, jeopardizing future use of mefloquine against P. vivax. A drug policy is needed that takes into consideration all co-endemic species of malaria parasites.     

Quinine-Resistant Malaria in Traveler Returning from Senegal, 2007

We describe clinical and parasitologic features of in vivo and in vitro Plasmodium falciparum resistance to quinine in a nonimmune traveler who returned to France from Senegal in 2007 with severe imported malaria. Clinical quinine failure was associated with a 50% inhibitory concentration of 829 nmol/L. Increased vigilance is required during treatment follow-up.     

Indirect ELISA test for malaria in blood donors

To determine the potential risk of transfusion malaria at the Hospital Militar Central in Bogota, Colombia, sera from 3114 blood donors were tested for malaria antibodies by the indirect ELISA technique. Positive results were found in 8·6 per thousand of the serum samples using P. falciparum antigen containing more than 60% mature forms as substrate. Three cases of transfusion-induced malaria were confirmed during the study. The first patient developed a P. vivax infection one week after the administration of one unit of infected blood. The other two patients received a red blood cell concentrate and a platelet preparation, respectively, derived from a single donor and developed a P. falciparum infection eight days after transfusion. The application of the ELISA technique would be of use in attempts to control transfusion-induced malaria.     

Prospective double-blind trial of two different doses of mefloquine plus pyrimethamine-sulfadoxine compared with pyrimethamine-sulfadoxine alone in the treatment of falciparum malaria

This double-blind study is based on the treatment of 75 adult male patients suffering from Plasmodium falciparum malaria in Medellín, Colombia, a city in which there is no malaria transmission. The patients, who came from regions with high resistance to antimalarials, were divided into three groups receiving single-dose treatment as follows: a combination of 280 mg mefloquine, 800 mg sulfadoxine and 40 mg pyrimethamine; a combination of 420 mg mefloquine, 1200 mg sulfadoxine and 60 mg pyrimethamine; and a combination of 1500 mg sulfadoxine and 75 mg pyrimethamine. After treatment, follow-up examination was performed daily for I week and then weekly for another 3 weeks. The cure rate in the mefloquine groups (within the study period of 28 days) was 100%, and in the third group 75%. Normal blood levels of the administered drugs were found in 6 patients of the third group who were not cured; they were subsequently cured with a single dose of 1000 mg of mefloquine. Drug tolerance was good and no toxic effects were demonstrated in blood and urine examinations. While the doses in the drug combinations (containing mefloquine) gave very good results, we would recommend a slightly higher dose combination of mefloquine with sulfadoxine—pyrimethamine for the treatment of falciparum malaria in areas with a high prevalence of chloroquine resistance.     

Population-based validation of dihydrofolate reductase gene mutations for the prediction of sulfadoxine-pyrimethamine resistance in Uganda

Mutations in the dihydrofolate reductase gene (dhfr) of Plasmodium falciparum have been proposed as molecular markers for the surveillance of sulfadoxine-pyrimethamine (SP)-resistant malaria, but such proposals have not been validated. At 7 Ugandan sites in 1999, we determined the population-based prevalence of infections with mutations and the mutant allele frequency of dhfr codons 108, 51, and 59 using a random sample of infected individuals aged 1-45 years. Sulfadoxine-pyrimethamine treatment failure was independently estimated by In vivo in 327 children aged 6-59 months with clinical malaria. The prevalence of infections with the single point mutations and the dhfr codons 108 and 51 mutant allele frequency were not correlated to SP treatment failure. However, the dhfr codon 59 mutant allele frequency was positively correlated to SP treatment failure (r= 0.72, P= 0.06). The ratio of the infections with the mutant to wild genotype (M/W) and that of the mutant to wild allele (MA/WA) had the same values. Both dhfr codon 59 M/W and MA/WA ratio were significantly and positively correlated to SP treatment failure (r= 0.73, P= 0.05). Moreover, the prevalence of infections with only 2 mutations (Asn-108 plus Ile-51) was significantly and inversely correlated to the prevalence of infections with 3 mutations (Asn-108 plus Ile-51 plus Arg-59) (r = 0.92, P = 0.004), suggesting the stepwise accumulation of the dhfr mutations is Asn-108 Ile-51 Arg-59 and further supporting the idea of using the dhfr codon 59 M/W ratio as a molecular index for the prediction of SP treatment failure. At the population level, the dhfr codon 59 M/W ratio is a simple and stable index for the estimation of SP treatment failure.     

10. Effect of insecticide-treated bed nets on the dynamics of multiple Plasmodium falciparum infections

The rates of acquisition and loss of individual genotypes belonging to the FC27 family of the Plasmodium falciparum merozoite surface protein 2 (msp2) gene were studied in 120 children aged 5 months to 2·5 years, in a randomized controlled trial of insecticide-treated bed nets (ITNs) in Kiberege village, Tanzania. Analysis of longitudinal changes in positivity for individual alleles in samples collected at intervals of one month indicated that the average duration of infections, allowing for undetected parasite genotypes, was 73 d in those aged <18 months and 160 d in children aged ≥18 months, consistent with a shift from acute to chronic infection with age. Overall, 51% of genotypes infecting the host were estimated to be detected by polymerase chain reaction-restriction fragment length polymorphism analysis in any one sample of 0·5 μL of packed peripheral blood cells. In children less than 18 months old this sensitivity was 61% (SE = 6%) compared with 41% (SE = 6%) in older children. Conversely, the rate of appearance of new parasite genotypes was higher in children <18 months of age than in older children, but this partly reflected the difference in sensitivity. The overall incidence of new infections was estimated to be reduced by 17% in ITN users. There was no statistically significant difference between users and non-users in observed infection multiplicity, sensitivity, recovery rate, or estimated infection rates for individual alleles. This suggests that, in areas of high P. falciparum endemicity, ITNs have little effect on the establishment of chronic malaria infection.     

Comparative assessment on the prevalence of mutations in the Plasmodium falciparum drug-resistant genes in two different ecotypes of Odisha state,India

Considering malaria as a local and focal disease, epidemiological understanding of different ecotypes of malaria can help in devising novel control measures. One of the major hurdles in malaria control lies on the evolution and dispersal of the drug-resistant malaria parasite, Plasmodium falciparum. We herewith present data on genetic variation at the Single Nucleotide Polymorphism (SNP) level in four different genes of P. falciparum (Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps) that confer resistance to different antimalarials in two different eco-epidemiological settings, i.e. Hilly-Forest (HF) and Riverine-Plain (RP), in a high malaria endemic district of Odisha state, India. Greater frequency of antimalarial resistance conferring SNPs and haplotypes was observed in all four genes in P. falciparum, and Pfdhps was the most variable gene among the four. No significant genetic differentiation could be observed in isolates from HF and RP ecotypes. Twelve novel, hitherto unreported nucleotide mutations could be observed in the Pfmdr1 and Pfdhps genes. While the Pfdhps gene presented highest haplotype diversity, the Pfcrt gene displayed the highest nucleotide diversity. When the data on all the four genes were complied, the isolates from HF ecotype were found to harbour higher average nucleotide diversity than those coming from RP ecotype. High and positive Tajima's D values were obtained for the Pfcrt and Pfdhfr genes in isolates from both the HF and RP ecotypes, with statistically significant deviation from neutrality in the RP ecotype. Different patterns of Linkage Disequilibrium (LD) among SNPs located in different drug-resistant genes were found in the isolates collected from HF and RP ecotypes. Whereas in the HF ecotype, SNPs in the Pfmdr1 and Pfdhfr were significantly associated, in the RP ecotype, SNPs located in Pfcrt were associated with Pfmdr1, Pfdhfr and Pfdhps. These findings provide a baseline understanding on how different micro eco-epidemiological settings influence evolution and spread of different drug resistance alleles. Our findings further suggest that drug resistance to chloroquine and sulfadoxine–pyrimethamine is approaching fixation level, which requires urgent attention of malaria control programme in India.     

Molecular inference of sources and spreading patterns of Plasmodium falciparum malaria parasites in internally displaced persons settlements in Myanmar–China border area

In Myanmar, civil unrest and establishment of internally displaced persons (IDP) settlement along the Myanmar–China border have impacted malaria transmission. The growing IDP populations raise deep concerns about health impact on local communities. Microsatellite markers were used to examine the source and spreading patterns of Plasmodium falciparum between IDP settlement and surrounding villages in Myanmar along the China border. Genotypic structure of P. falciparum was compared over the past three years from the same area and the demographic history was inferred to determine the source of recent infections. In addition, we examined if border migration is a factor of P. falciparum infections in China by determining gene flow patterns across borders. Compared to local community, the IDP samples showed a reduced and consistently lower genetic diversity over the past three years. A strong signature of genetic bottleneck was detected in the IDP samples. P. falciparum infections from the border regions in China were genetically similar to Myanmar and parasite gene flow was not constrained by geographical distance. Reduced genetic diversity of P. falciparum suggested intense malaria control within the IDP settlement. Human movement was a key factor to the spread of malaria both locally in Myanmar and across the international border.     

Comparison of Parascreen Pan/Pf, Paracheck Pf and light microscopy for detection of malaria among febrile patients, Northwest Ethiopia

Two malaria rapid diagnostic tests (RDT), Parascreen Pan/Pf^® and Paracheck Pf^®, were tested in rural health centres in Ethiopia against independent expert microscopy (the gold standard). Participants (n =1997) presented with presumptive malaria to ten health centers in Amhara Regional State during the 2007 peak malaria season (October to December). By microscopy, 475 (23.8%) suspected malaria cases were positive, of which 57.7% were P. falciparum; 24.6% P. vivax and 17.7% mixed infections. Parascreen and Paracheck were positive for 442 (22.1%) and 277 (13.9%) febrile patients, respectively. For Parascreen, P. falciparum sensitivity was 79.6%, specificity 97.4%, positive predictive value (PPV) 86.9%, and negative predictive value (NPV) 95.6%. For Parascreen, P. vivax sensitivity was 74.4%, specificity 98.6%, PPV 76.3% and NPV 98.4%. For Paracheck, P. falciparum sensitivity was 73.7%, specificity 99.2%, PPV 95.3%, NPV 94.5%. Sensitivity was significantly higher for both tests (P < 0.05) when parasite density was >100/μl of blood; in these cases Parascreen was 90.7% and 91.5% sensitive for P. falciparum and P. vivax, respectively, while Paracheck was 87.9% sensitive for P. falciparum. Parascreen thus performed adequately for both P. falciparum and P. vivax compared to expert microscopy and is more useful than Paracheck where microscopy is unavailable.