Falciparum Field Isolates Research Articles

K13 Kelch Propeller Domain and mdr1 Sequence Polymorphism in Field Isolates From Northeast Region, India

Emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia has made an intense warning to global malaria control programme since Southeast Asia is playing a key role in the evolution of drug resistant malaria parasites. Several polymorphisms in K13 gene have been designated with developing resistance either in vitro or in vivo . In this study, polymorphisms within K13 kelch propeller domain and Pf mdr1 gene were investigated in a total of 134 P. falciparum field isolates circulating in 3 states of Northeast region of India, namely, Assam, Arunachal Pradesh, and Tripura because information regarding this is limited from this region. Six polymorphisms were detected, out of which 2 were new. A481 V mutation was found in 4.5% of the total field isolates. A675 V and D702 N new mutations were present in 3.7% and 1.5% isolates, respectively. Synonymous polymorphism at codon 703 was highly observed (6.7%) than other polymorphisms. N86 Y allele in Pf mdr1 gene was also noticed in isolates that contained mutation in K13 propeller domain, but N86 wild-type allele was found comparatively higher in frequency within overall isolates. Moreover, both nucleotide and haplotype diversities in K13 gene were high in Arunachal Pradesh isolates than other 2 states. This is the first report from Northeast region of India with evidence of A675 V candidate mutation along with 2 other new mutations which pays attention for further molecular surveillance in this region to document detailed scenario of K13 polymorphism pattern corresponding to artemisinin resistance.

Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites

Background Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample.MethodsIn this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture.ResultsThe cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission.Conclusions Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised.

Prevalence of Multiple Drug-resistant Plasmodium falciparum Malaria Cases in Northeast India

Two numbers of Plasmodium falciparum field isolates from Gossingpara, Runikhata area in Chirang district of Assam had shown multiple mutations in Pfcrt-dhfr-dhps gene (up to seven mutations: One mutation in Pfcrt gene, three mutations in Pfdhfr gene and three mutations in Pfdhps gene). Similarly, two cases in Bat camp, Miao area under Changlang district of Arunachal Pradesh had shown a total of eight mutations, of which one mutation in Pfcrt gene, three mutations in Pfdhfr gene, three mutations in Pfdhps gene and one mutation in PfATPase6gene. One case in 3 Miles, Miao area of Changlang district has shown mutations in Pfcrt (one mutation), Pfdhfr (four mutations) and Pfdhps (three mutations) gene. These results indicated that there is an existence of multiple mutant P. falciparum malaria cases in northeastern region of India.

Genetic Differentiation and Flow of Plasmodium falciparum dhfr Gene Sequences Associated with Pyrimethamine Drug Resistance

The present study was conducted in Chirang, North Cachar Hills and Karbi Anglong district in Assam with an aim to find out the genetic differentiations and flow of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene associated with Pyrimethamine resistance. Therefore, a total of 116 participants were enrolled for the study. Presence of malaria parasites were confirmed by microscopic slide examination method. P. falciparum mono-infection was observed in 69 cases. A 648 base pair portion of Pfdhfr gene containing major single nucleotide polymorphisms were amplified, sequenced, and then analyzed in BioEdit v7.0.9 software, Mega 5 software and DnaSP version v.5.10.01. Three polymorphic sites were observed in the dhfr gene sequences of P. falciparum field isolates in Assam. The value of neutrality test did not show any statistically significant result. The highest level of haplotype diversity (0.38889) was observed in the P. falciparum isolates in Chirang district of Assam. Little to moderate genetic differentiation was observed between the P. falciparum field isolates in Chirang and NC hills. It specifies that the gene flow levels were high enough to counteract the effects of genetic drift.

Mutational prevalence of chloroquine resistance transporter gene among Plasmodium falciparum field isolates in Assam and Arunachal Pradesh, India

Objective: The present study aims to find out the mutational prevalence of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene in Assam and Arunachal Pradesh, India. Methods: To fulfil the objective of the study, a total of 54 P. falciparum malaria positive samples were attempted for genotyping of Pfcrt gene using polymerase chain reaction (PCR) and direct DNA sequencing method. Results: The K76T mutation was observed in 77.78% cases followed by M74I (61.11%), N75E (61.11%) and C72S (16.67%). Triple mutant allele M74I+N75E+K76T was found in 61.11% P. falciparum field isolates. Double mutant allele C72S+K76T was seen among 16.67% samples. Mutation studies have shown existence of three different haplotypes of which two were associated with chloroquine resistance. The haplotype CVIET was found preponderance and circulated in all four districts. The other haplotype SVMNT was observed only in Karbi Anglong district of Assam. Maximum haplotype diversity was also recorded from Karbi Anglong district of Assam. Conclusion: Our study has confirmed high prevalence of mutant Pfcrt genotypes associated with chloroquine resistance in Assam and Arunachal Pradesh, India.

Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens.

Polymorphisms within Plasmodium falciparum vaccine candidate antigens have the potential to compromise vaccine efficacy. Understanding the allele frequencies of polymorphisms in critical binding regions of antigens can help in the designing of strain-transcendent vaccines. Here, we adopt a pooled deep-sequencing approach, originally designed to study P. falciparum drug resistance mutations, to study the diversity of two leading transmission-blocking vaccine candidates, Pfs25 and Pfs48/45. We sequenced 329 P. falciparum field isolates from six different geographic regions. Pfs25 showed little diversity, with only one known polymorphism identified in the region associated with binding of transmission-blocking antibodies among our isolates. However, we identified four new mutations among eight non-synonymous mutations within the presumed antibody-binding region of Pfs48/45. Pooled deep sequencing provides a scalable and cost-effective approach for the targeted study of allele frequencies of P. falciparum candidate vaccine antigens.

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds.

BackgroundMedicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernoniaamygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodiumberghei and to validate the findings on field isolates of Plasmodium falciparum.MethodsAqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V.amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P.falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926.ResultsVer-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An.stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 μg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines.ConclusionsVernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0812-2) contains supplementary material, which is available to authorized users.

Population genetic study of Plasmodium falciparum parasites pertaining to dhps gene sequence in malaria endemic areas of Assam

Plasmodium falciparum malaria parasite had developed resistance to almost all the currently used antimalarial drugs. The purpose of the study was to come across the genetic distances in P. falciparum dhps gene sequences circulating in Assam. A partial fragment of P. falciparum dhps gene containing major single nucleotide polymorphisms associated with sulphadoxine resistance were amplified and sequenced. Thereafter specific bioinformatics tools like BioEdit v7.0.9, ClustalW in Mega 5, DnaSP version v.5.10.01 etc were used for the analysis. A total of 100 P. falciparum positive cases in different malaria endemic areas of Assam were included for the study. Based upon the mutation analysis, a total of seven different P. falciparum dhps genotypes were observed with five variable sites. Maximum five haplotypes were found in the P. falciparum isolates from Jorhat district of Assam. Four polymorphic sites were observed in the P. falciparum dhps gene sequences in Karbi Anglong, NC Hills, Chirang and Jorhat whereas the isolates from other study areas had three polymorphic sites. A statistically significant positive value of Tajima’s D were observed among the P. falciparum field isolates in Assam indicating that there is an excess of intermediate frequency alleles and can result from population bottlenecks, structure and/or balancing selection. Extensive gene flow took place among the P. falciparum population of Jorhat with Sivasagar, Chirang with Sivasagar and Chirang with Karbi Anglong. However, large genetic differentiation was observed among the P. falciparum isolates of NC Hills with Lakhimpur, Tinsukia, Dibrugarh and Golaghat and also the parasite population of Karbi Anglong with Lakhimpur and Tinsukia signifying little gene flow among the population. This finding has shown that mutant Pfdhps gene associated with sulphadoxine resistance is circulating in Assam. It is believed that, the parasite population may have undergone high level of breeding.

Use of Plasmodium falciparum culture-adapted field isolates for in vitro exflagellation-blocking assay.

BackgroundA major requirement for malaria elimination is the development of transmission-blocking interventions. In vitro transmission-blocking bioassays currently mostly rely on the use of very few Plasmodium falciparum reference laboratory strains isolated decades ago. To fill a piece of the gap between laboratory experimental models and natural systems, the purpose of this work was to determine if culture-adapted field isolates of P. falciparum are suitable for in vitro transmission-blocking bioassays targeting functional maturity of male gametocytes: exflagellation.MethodsPlasmodium falciparum isolates were adapted to in vitro culture before being used for in vitro gametocyte production. Maturation was assessed by microscopic observation of gametocyte morphology over time of culture and the functional viability of male gametocytes was assessed by microscopic counting of exflagellating gametocytes. Suitability for in vitro exflagellation-blocking bioassays was determined using dihydroartemisinin and methylene blue.ResultsIn vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia. Functional maturity of male gametocytes was assessed by exflagellation observations and all four isolates could be used in exflagellation-blocking bioassays with adequate response to methylene blue and dihydroartemisinin.ConclusionThis work shows that in vitro culture-adapted P. falciparum field isolates of different genetic background, from South America and Southeast Asia, can successfully be used for bioassays targeting the male gametocyte to gamete transition, exflagellation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0752-x) contains supplementary material, which is available to authorized users.

High prevalence of sulphadoxine–pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo

Intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N+dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N+dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here reported for the first time in the Republic of Congo. The increasing prevalence of multiple mutant alleles observed in this study is alarming and may present a challenge for the future interventions including IPTp-SP in the country.

Limited genetic diversity among Plasmodium falciparium isolates using nested PCR in Jazan Area, Saudi Arabia

ABSTRACT Objective To establish molecular characterization of Plasmodium falciparum field isolates in Jazan Area of Saudi Arabia measured with highly polymorphic genetic marker, i.e. the merozoite surface protein 2 (MSP 2). Methods Blood samples were collected from 128 clinically suspected patients attending both Jazan and Sabia hospitals, Kingdom of Saudi Arabia. Both hospitals reflected urban and rural settings respectively. Analysis of central polymorphic region of MSP 2 (3D7 and FC27 allelic families) was performed using nested PCR for malaria patients. Results For MSP 2 allelic families of Plasmodium falciparum, 16 cases (53.3%) carried FC27 type and 14 cases (46.7%) carried 3D7 type, whereas no malaria cases harbored both allelic types. The present study showed that in urban area, 80% of FC27 fragments were 500 bp while in rural area it was 45.5% (P = 0.08). The FC27 400 bp allele was more prevalent in patients from rural than those from the urban area (P = 0.08). The most prevalent infecting 3D7 allele was the 3D7 300 bp in both areas. In the present study, there were no multiple infections. Conclusions The limited genetic diversity which was observed in Jazan (considered as an endemic area) may be attributed to the small sample size or sustained malaria control program.

Genetic diversity in the msp-1 and msp-2 alleles among Plasmodium falciparum field isolates from Jazan, Saudi Arabia.

The genetic diversity of Plasmodium falciparum infections in human is associated with the pathogenesis of malaria. It is commonly determined through amplification of the polymorphic regions of the merozoite surface proteins -1 (msp-1) and -2 (msp-2) genes. This study aimed to (1) determine the prevalence of the msp-1 and msp-2 allelic familiesand (2) identify the multiplicity of infection (MOI) in P. falciparum field isolates from the Jazan region in the Kingdom of Saudi Arabia (KSA). Blood samples from patients with microscopically confirmed malaria infections (N = 48), collected in 2010, were analysed for msp-1 and msp-2 polymorphisms.K1, MAD20 and RO33 allelic types of the msp-1 gene and 3D7 and FC27 alleles of the msp-2 gene were analysed via nested polymerase chain reaction (PCR) according to band size. The MOI was then calculated. In msp-1, 16 different alleles were identified by examining size differences in the agarose gels. These alleles-representing 5, 5 & 6 alleles-belong to K1 (120bp-420bp), RO22 (180bp-420bp) and MAD 20 (150 bp-410bp), respectively. For msp-2, a larger range of amplicon sizes was detected. A total of 13 different alleles were identified: the FC27 family had 6 alleles (380- bp1280bp), while the 3D7 family had 7 alleles (110 bp-1200bp.MOI was 1.81 for MSP-1 & 2.17 for MSP-2, with overall mean MOI of 1.99). Considerable genetic diversity was evident in the P. falciparum field isolates from the Jazan region of KSA. This diversity represents an essential step in developing effective measures to prevent malaria in KSA, as well as in assessing vaccines derived from these genes.

Assessing the Gametocyte Production in Field Isolates of Plasmodium falciparum

The production of gametocytes in field isolates of Plasmodium falciparum is not clearly understood even though the gametocytes are crucial for disease transmission. Samples collected during the malaria transmission period from two different regions of India were cultured in vitro for gametocyte production and analyzed by PCR and RT-PCR assay for Pfs25 gene. A total of 20 P. falciparum field isolates were collected which showed varying intensity of in vitro gametocyte production. The isolates which produced mature gametocytes in vitro also had an increase in their Pfs25 expression, indicating that the gametocyte produced is directly proportional to the Pfs25 gene expression. The expression ranged from 0.32 to 4.56 fold in field isolates when compared to the reference strain. The in vitro gametocyte production in fresh field isolates directly correlated with the expression of Pfs25 gene in these isolates as shown by ANOVA test.

Genetic polymorphisms associated with sulphadoxine-pyrimethamine drug resistance among Plasmodium falciparum field isolates in malaria endemic areas of Assam.

Background:The emergence of antimalarial drug resistance malaria parasite is widespread in North eastern region of India. During January 2012-December 2013, we conducted active surveillance for detection of antifolate resistance-associated genetic polymorphisms in Plasmodium falciparum malaria parasite from different malaria endemic areas of Assam.Materials and Methods:A total of 281 field samples were collected from suspected malaria patients of which 106 malaria P. falciparum positive cases were detected in microscopic slide examination. A nested PCR was done for amplification of a 648 bp portion of the dhfr gene and 710 bp portion of the dhps gene.Results:Mutation analysis revealed existence of three different haplotypes of the P. falciparum dhfr gene of which ANRNI was highly prevalent (90%). Triple mutant haplotypes AIRNI (N51I + C59R + S108N) of the dhfr gene associated with pyrimethamine resistance were prevalent in Chirang district of Assam. Whereas, dhps mutation study revealed that triple mutant haplotype AGEAA (S436A + A437G + K540E) associated with Sulphadoxine resistance was found among 26% of P. falciparum field isolates. However, P. falciparum dhfr-dhps two locus mutation analysis showed that there were a total of nine dhfr-dhps genotypes.Conclusion:It was noticed that 93.62% (88/94) isolates had mutations in the sequences of both enzymes, which is an indication of prevalence of high grade of Sulphadoxine — pyrimethamine resistance in P. falciparum malaria parasites in Assam.

Endothelial modulation of ICAM-1, CD36 and inflammatory cytokines IL-6, IL-8 and MCP-1 by P. falciparum-infected erythrocytes.

Endothelial modulation of ICAM-1, CD36 and inflammatory cytokines IL-6, IL-8 and MCP-1 by P. falciparum-infected erythrocytes.

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.

Reduced in vitro doxycycline susceptibility in plasmodium falciparum field isolates from Kenya is associated with PfTetQ KYNNNN sequence polymorphism.

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC50 of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50 of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.

Mirincamycin, an old candidate for malaria combination treatment and prophylaxis in the 21st century: in vitro interaction profiles with potential partner drugs in continuous culture and field isolates.

BackgroundSpreading resistance of Plasmodium falciparum to existing drugs calls for the search for novel anti-malarial drugs and combinations for the treatment of falciparum malaria.MethodsIn vitro and ex vivo investigations were conducted with fresh P. falciparum field isolates and culture-adapted P. falciparum clones to evaluate the anti-malarial potential of mirincamycin, a lincosamide, alone and in combination with tafenoquine (TQ), dihydroartemisinin (DHA), and chloroquine (CQ). All samples were tested in a histidine-rich protein 2 (HRP2) drug susceptibility assay.ResultsInteraction analysis showed additive to synergistic interaction profiles with these potential partner drugs, with an overall geometric mean fractional inhibitory concentration at 50% inhibition (FIC50) of 0.78, 0.80 and 0.80 for mirincamycin with TQ, DHA, and CQ, respectively. Antagonism was not found in any of the tested field isolates or clones. The strongest tendency toward synergy (i.e. the lowest FIC) was seen with a combination ratio of 1:0.27 to 1:7.2 (mean 1:2.7) for the combination with tafenoquine. The optimal combination ratios for DHA and CQ were 1:444.4 to 1:36,000 (mean 1:10,755.5) and 1:2.7 to 1:216 (mean 1:64.5), respectively. No evidence of an activity correlation (i.e. potential cross-resistance) with DHA, mefloquine, quinine or chloroquine was seen whereas a significant correlation with the activity of clindamycin and azithromycin was detected.ConclusionsMirincamycin combinations may be promising candidates for further clinical investigations in the therapy and prophylaxis of multidrug-resistant falciparum malaria or in combination with 4 or 8-aminoquinolines for the treatment and relapse prevention of vivax malaria.

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 560bp 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.

Ex vivo anti-malarial drugs sensitivity profile of Plasmodium falciparum field isolates from Burkina Faso five years after the national policy change.

BackgroundThe recent reports on the decreasing susceptibility of Plasmodium falciparum to artemisinin derivatives along the Thailand and Myanmar border are worrying. Indeed it may spread to India and then Africa, repeating the same pattern observed for chloroquine resistance. Therefore, it is essential to start monitoring P. falciparum sensitivity to artemisinin derivatives and its partner drugs in Africa. Efficacy of AL and ASAQ were tested by carrying out an in vivo drug efficacy test, with an ex vivo study against six anti-malarial drugs nested into it. Results of the latter are reported here.MethodsPlasmodium falciparum ex-vivo susceptibility to chloroquine (CQ), quinine (Q), lumefantrine (Lum), monodesethylamodiaquine (MDA), piperaquine (PPQ) and dihydroartemisinin (DHA) was investigated in children (6 months – 15 years) with a parasitaemia of at least ≥4,000/μl. The modified isotopic microtest technique was used. The results of cellular proliferation were analysed using ICEstimator software to determine the 50% inhibitory concentration (IC50) values.ResultsDHA was the most potent among the 6 drugs tested, with IC50 values ranging from 0.8 nM to 0.9 nM (Geometric mean IC50 = 0.8 nM; 95% CI [0.8 - 0.9]). High IC50 values ranged between 0.8 nM to 166.1 nM were reported for lumefantrine (Geometric mean IC50 = 25.1 nM; 95% CI [22.4 - 28.2]). MDA and Q IC50s were significantly higher in CQ-resistant than in CQ-sensitive isolates (P = 0.0001). However, the opposite occurred for Lum and DHA (P < 0.001). No difference was observed for PPQ.ConclusionArtemisinin derivatives are still very efficacious in Burkina Faso and DHA-PPQ seems a valuable alternative ACT. The high lumefantrine IC50 found in this study is worrying as it may indicate a decreasing efficacy of one of the first-line treatments. This should be further investigated and monitored over time with large in vivo and ex vivo studies that will include also plasma drug measurements.