Falciparum-infected Blood Samples Research Articles

Molecular markers of dihydroartemisinin-piperaquine resistance in northwestern Thailand

BackgroundDihydroartemisinin-piperaquine (DHA-PPQ) combination therapy is the current first-line treatment for Plasmodium falciparum malaria in Thailand. Since its introduction in 2015, resistance to this drug combination has emerged in the eastern part of the Greater Mekong Subregion including the eastern part of Thailand near Cambodia. This study aimed to assess whether the resistance genotypes have arisen the western part of country.MethodsFifty-seven P. falciparum-infected blood samples were collected in Tak province of northwestern Thailand between 2013 and 2019. Resistance to DHA was examined through the single nucleotide polymorphisms (SNPs) of kelch13. PPQ resistance was examined through the copy number plasmepsin-2 and the SNPs of Pfcrt.ResultsAmong the samples whose kelch13 were successfully sequenced, approximately half (31/55; 56%) had mutation associated with artemisinin resistance, including G533S (23/55; 42%), C580Y (6/55; 11%), and G538V (2/55; 4%). During the study period, G533S mutation appeared and increased from 20% (4/20) in 2014 to 100% (9/9) in 2019. No plasmepsin-2 gene amplification was observed, but one sample (1/54) had the Pfcrt F145I mutation previously implicated in PPQ resistance.ConclusionsKelch13 mutation was common in Tak Province in 2013–2019. A new mutation G533S emerged in 2014 and rose to dominance in 2019. PPQ resistance marker Pfcrt F145I was also detected in 2019. Continued surveillance of treatment efficacy and drug resistance markers is warranted.

Signatures of selection and drivers for novel mutation on transmission-blocking vaccine candidate Pfs25 gene in western Kenya.

BackgroundLeading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities.MethodsDry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1–4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples.ResultsNine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low nucleotide diversity (0.04×10−2) and low mutation frequencies (1.3% to 7.7%). Chulaimbo had the highest frequency (15.4%) of mutated sites followed by Kisii (6.7%) and Homa Bay (5.1%). Neutrality tests of Pfs25 variations showed significant negative values of Tajima’s D (-2.15, p<0.01) and Fu’s F (-10.91, p<0.001) statistics tests. Three loci pairs (123, 372), (364, 428) and (390, 394) were detected to be under linkage disequilibrium and none had history of recombination. These results suggested that purifying selection and inbreeding might be the drivers of the observed variation in Pfs25.ConclusionGiven the low level of nucleotide diversity, it is unlikely that a Pfs25 antigen-based vaccine would be affected by antigenic variations. However, continued monitoring of Pfs25 immunogenic domain 3 for possible variants that might impact vaccine antibody binding is warranted.

Molecular surveillance of the Pfmdr1 N86Y allele among Congolese pregnant women with asymptomatic malaria

BackgroundMalaria in pregnancy is associated with considerable morbidity and mortality. Regular surveillance of artemisinin-based combination therapy tolerance, or molecular makers of resistance, is vital for effective malaria treatment, control and eradication programmes. Plasmodium falciparum multiple drug resistance-1 gene (Pfmdr1) N86Y mutation is associated with reduced susceptibility to lumefantrine. This study assessed the prevalence of Pfmdr1 N86Y in Brazzaville, Republic of Congo.MethodsA total 1001 of P. falciparum-infected blood samples obtained from asymptomatic malaria pregnant women having a normal child delivery at the Madibou Integrated Health Centre were analysed. Pfmdr1 N86Y genotyping was conducted using PCR-restriction fragment length polymorphism.ResultsThe wild type Pfmdr1 N86 allele was predominant (> 68%) in this study, whereas a few isolates carrying the either the mutant allele (Pfmdr1 86Y) alone or both alleles (mixed genotype). The dominance of the wildtype allele (pfmdr1 N86) indicates the plausible decline P. falciparum susceptibility to lumefantrine.ConclusionThis study gives an update on the prevalence of Pfmdr1 N86Y alleles in Brazzaville, Republic of Congo. It also raises concern on the imminent emergence of resistance against artemether–lumefantrine in this setting. This study underscores the importance to regular artemether–lumefantrine efficacy monitoring to inform the malaria control programme of the Republic of Congo.

Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates

The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries.

Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand

Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum–infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991–2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR–restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype.

Cytochrome b mutation and atovaquone susceptibility in Plasmodium falciparum isolates from the Thai-Cambodian border during 1990–2010

WHO reported that the efficacy of artemisinin combination-based therapies (ACTs) has decreased around the Thai-Cambodian border. To maintain the efficacy and prolong the life span of ACTs, malarone (an atovaquone and proguanil combination) has been administered in Trat and Chanthaburi Provinces of Thailand since 2009. The mutations of codons 133, 258, 268, 272, 275, 280, 283, and 284 in the cytochrome b gene have been reported to be related to malarone resistance. This study investigates the susceptibility in vitro and mutations in the cytochrome b gene before and after the administration of malarone. Plasmodium falciparum infected blood samples obtained from malaria patients attending malaria clinics in these two provinces were included in this study. Fifteen parasite isolates were subjected to in vitro susceptibility tests against atovaquone. Their mean inhibitory concentrations ranged from 5 10 9 -5 10 10 M. The DNA sequences from 37 PCR samples revealed no mutations. Therefore the variation of drug susceptibility among these parasites may be unrelated to point mutations in the cytochrome b gene.

Genetic analysis of the merozoite surface protein-1 block 2 allelic types in Plasmodium falciparum clinical isolates from Lao PDR

BackgroundMSP-1 is one of the potential malarial vaccine candidate antigens. However, extensive genetic polymorphism of this antigen in the field isolates of Plasmodium falciparum represents a major hindrance for the development of an effective vaccine. Therefore, this study aimed to establish the prevalence and genetic polymorphisms of K1, MAD20 and RO33 allelic types of msp-1 block 2 among P. falciparum clinical isolates from Lao PDR.MethodsPlasmodium falciparum isolates were collected from 230 P. falciparum-infected blood samples from three regions of Lao PDR. K1, MAD20 and RO33 were detected by nested PCR; SSCP was used for polymorphism screening. The nested PCR products of each K1, MAD20 and RO33 allelic types that had different banding patterns by SSCP, were sequenced.ResultsThe overall prevalence of K1, MAD20 and RO33 allelic types in P. falciparum isolates from Lao PDR were 66.95%, 46.52% and 31.30%, respectively, of samples under study. Single infections with K1, MAD20 and RO33 allelic types were 27.83%, 11.74% and 5.22%, respectively; the remainders were multiple clonal infections. Neither parasite density nor age was related to MOI. Sequence analysis revealed that there were 11 different types of K1, eight different types of MAD20, and 7 different types of RO33. Most of them were regional specific, except type 1 of each allelic type was common found in 3 regions under study.ConclusionsGenetic polymorphism with diverse allele types was identified in msp-1 block 2 among P. falciparum clinical isolates in Lao PDR. A rather high level of multiple clonal infections was also observed but the multiplicity of infection was rather low as not exceed 2.0. This basic data are useful for treatment and malaria control program in Lao PDR.

Genetic polymorphism of merozoite surface protein-1 (MSP-1) block 2 allelic types in &lt;em&gt;Plasmodium falciparum&lt;/em&gt; field isolates from mountain and coastal area in West Sumatera, Indonesia

Background: The fi eld isolates of P. falciparum may display variant forms and different frequencies. This study was designed to know the diversity of allelic type of MSP-1 block 2 among P. falciparum isolates collected in a mountain and a coastal area in West Sumatera, Indonesia, and compare mountain and coastal area. Methods: A total of 56 P. falciparum infected blood samples, collected from 27 patients attending local health facilities in South Solok district in a mountain region and 29 patients attending a local health facilities in South Coastal district region, West Sumatera, Indonesia were used in this study. The regions flanking the highly polymorphic characters, block 2 for MSP-1, were genotyped by allele-specific nested-PCR to analyse the population diversity of parasite. Sequence analysis of the polymorphic regions of MSP-1 was also conducted to identify allelic diversity in the parasite population. Results: Diverse allelic polymorphism of MSP-1 was identified in P. falciparum isolates from a mountain area and a coastal area in West Sumatera, Indonesia, and most of the infections were determined to be mixed infections. Sequence analysis of MSP-1 block 2 revelaled that 16 different alleles for MSP-1 (3 for K1 type, 2 for MAD20 type and 2 for RO33 type) were identified. Conclusion: Extensive genetic polymorphism with diverse allele type was identified in MSP-1 in P. falciparum field isolates from a mountain and a coastal area. A high level of mixed infections was also obcserved, as was a high degree of multiplicity of infection. (Med J Indones 2011; 20:11-4) Keywords: allelic types, coastal area, mountain area, MSP-1 block 2, Plasmodium falciparum

Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene

BackgroundEffective treatment remains a mainstay of malaria control, but it is unfortunately strongly compromised by drug resistance, particularly in Plasmodium falciparum, the most important human malaria parasite. Although P. falciparum chemoresistance is well recognized all over the world, limited data are available on the distribution and prevalence of pfcrt and pfmdr1 haplotypes that mediate resistance to commonly used drugs and that show distinct geographic differences.MethodsPlasmodium falciparum-infected blood samples collected in 2007 at four municipalities of Luanda, Angola, were genotyped using PCR and direct DNA sequencing. Single nucleotide polymorphisms in the P. falciparum pfcrt and pfmdr1 genes were assessed and haplotype prevalences were determined.Results and DiscussionThe most prevalent pfcrt haplotype was StctVMNT (representing amino acids at codons 72-76). This result was unexpected, since the StctVMNT haplotype has previously been seen mainly in parasites from South America and India. The CVIET, CVMNT and CVINT drug-resistance haplotypes were also found, and one previously undescribed haplotype (CVMDT) was detected. Regarding pfmdr1, the most prevalent haplotype was YEYSNVD (representing amino acids at codons 86, 130, 184, 1034, 1042, 1109 and 1246). Wild haplotypes for pfcrt and pfmdr1 were uncommon; 3% of field isolates harbored wild type pfcrt (CVMNK), whereas 21% had wild type pfmdr1 (NEYSNVD). The observed predominance of the StctVMNT haplotype in Angola could be a result of frequent travel between Brazil and Angola citizens in the context of selective pressure of heavy CQ use.ConclusionsThe high prevalence of the pfcrt SVMNT haplotype and the pfmdr1 86Y mutation confirm high-level chloroquine resistance and might suggest reduced efficacy of amodiaquine in Angola. Further studies must be encouraged to examine the in vitro sensitivity of pfcrt SVMNT parasites to artesunate and amodiaquine for better conclusive data.

Genetic polymorphism of merozoite surface protein-1 and merozoite surface protein-2 in Plasmodium falciparum field isolates from Myanmar

BackgroundMerozoite surface protein-1 (MSP-1) and MSP-2 of Plasmodium falciparum are potential vaccine candidate antigens for malaria vaccine development. However, extensive genetic polymorphism of the antigens in field isolates of P. falciparum represents a major obstacle for the development of an effective vaccine. In this study, genetic polymorphism of MSP-1 and MSP-2 among P. falciparum field isolates from Myanmar was analysed.MethodsA total of 63 P. falciparum infected blood samples, which were collected from patients attending a regional hospital in Mandalay Division, Myanmar, were used in this study. The regions flanking the highly polymorphic characters, block 2 for MSP-1 and block 3 for MSP-2, were genotyped by allele-specific nested-PCR to analyse the population diversity of the parasite. Sequence analysis of the polymorphic regions of MSP-1 and MSP-2 was also conducted to identify allelic diversity in the parasite population.ResultsDiverse allelic polymorphism of MSP-1 and MSP-2 was identified in P. falciparum isolates from Myanmar and most of the infections were determined to be mixed infections. Sequence analysis of MSP-1 block 2 revealed that 14 different alleles for MSP-1 (5 for K1 type and 9 for MAD20 type) were identified. For MSP-2 block 3, a total of 22 alleles (7 for FC27 type and 15 for 3D7 type) were identified.ConclusionExtensive genetic polymorphism with diverse allele types was identified in MSP-1 and MSP-2 in P. falciparum field isolates from Myanmar. A high level of mixed infections was also observed, as was a high degree of multiplicity of infection.

Positive selection on the Plasmodium falciparum sporozoite threonine–asparagine-rich protein: Analysis of isolates mainly from low endemic areas

The sporozoite threonine–asparagine-rich protein (STARP) of Plasmodium falciparum is an attractive target for a pre-erythrocytic stage malaria vaccine because both naturally acquired and experimentally induced anti-STARP antibodies can block sporozoite invasion of hepatocytes. To explore the extent of sequence variation, we surveyed nucleotide polymorphism across the entire gene, encompassing 2 exons and an intron, of 124 P. falciparum-infected blood samples from Thailand and 10 from 4 other endemic areas. In total 24 haplotypes were identified despite low-level nucleotide diversity at this locus. The mean number of nonsynonymous substitutions per nonsynonymous site (dN) significantly exceeded that of synonymous substitutions per synonymous site (dS), suggesting that the STARP gene has evolved under positive selection, probably from host immune pressure. The preponderance of conservative amino acid exchanges and a strongly biased T-nucleotide toward the third position of codons in repeat arrays have reflected simultaneous constraints on this molecule, probably from its respective unknown function and nucleotide composition. Sequence conservation in the STARP locus among clinical isolates from different disease endemic areas would not compromise vaccine incorporation.

Combined Molecular and Clinical Assessment of Plasmodium falciparum Antimalarial Drug Resistance in the Lao People’s Democratic Republic (Laos)

Molecular markers provide a rapid and relatively inexpensive approach for assessing antimalarial drug susceptibility. We collected 884 Plasmodium falciparum-infected blood samples from 17 Lao provinces. Each sample was genotyped for 11 codons in the chloroquine resistance transporter (pfcrt), dihydrofolate reductase (pfdhfr), and dihydropteroate synthase (pfdhps) genes. The samples included 227 collected from patients recruited to clinical trials. The pfcrt K76T mutation was an excellent predictor of treatment failure for both chloroquine and chloroquine plus sulfadoxine-pyrimethamine, and mutations in both pfdhfr and pfdhps were predictive of sulfadoxine-pyrimethamine treatment failure. In multivariate analysis, the presence of the pfdhfr triple mutation (51 + 59 + 108) was strongly and independently correlated with sulfadoxine-pyrimethamine failure (odds ratio = 9.1, 95% confidence interval = 1.4-60.2, P = 0.017). Considerable geographic heterogeneity in allele frequencies occurred at all three loci with lower frequencies of mutant alleles in southern than in northern Laos. These findings suggest that chloroquine and sulfadoxine-pyrimethamine are no longer viable therapy in this country.

Analysis of sulphadoxine/pyrimethamine resistance-conferring mutations of Plasmodium falciparum from Mozambique reveals the absence of the dihydrofolate reductase 164L mutant

BackgroundPlasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate.MethodsA total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa.ResultsThe frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/108N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/108N + 437G/540E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected.ConclusionThe observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.

Comparison of microsatellite and antigen-coding loci for differentiating recrudescing Plasmodium falciparum infections from reinfections in Kenya

We have compared the ability of five Plasmodium falciparum microsatellites and three antigen-coding loci to differentiate recrudescence from reinfection. We used 133 pairs of P. falciparum-infected blood samples collected during in vivo drug efficacy trials from three sites in Kenya with different malaria endemicities. There were no significant differences between the marker subsets in their ability to discriminate recrudescences from new infections across the three sites. Overall, microsatellite loci revealed significantly higher expected heterozygosity and multiplicity of infection levels than antigen-coding loci. The mean expected heterozygosity across all loci in the three populations was significantly higher with microsatellites (0.70, 0.78 and 0.79) than antigen-coding loci (0.53, 0.60 and 0.62) for Mwea, Tiwi and Bondo areas, respectively. These observations can be explained by three non-exclusive hypotheses: (i) microsatellites are more polymorphic than antigenic loci; (ii) partially immune hosts remove certain parasites from infections on the basis of their antigenic alleles; and/or (iii) recombination occurs in vitro or in vivo with microsatellites.

Genetics of drug-resistant Plasmodium falciparum malaria in the Venezuelan state of Bolivar.

The state of Bolivar in Venezuela experiences episodic outbreaks of multidrug-resistant Plasmodium falciparum malaria. We obtained P. falciparum-infected blood samples in Bolivar in 1998-2000, and performed molecular assays for mutations conferring resistance to the antifolate combination of sulfadoxine-pyrimethamine (SP) and to chloroquine. All infections carried the dihydrofolate reductase (dhfr) S108A and N51I mutations, and 45% of the infections had the dhfr C50R mutation, which has been implicated in mid-level resistance to SP. Two dihydropteroate synthase (dhps) mutations also involved in SP resistance, A581G and K540E, were detected in 90% and 67% of the samples, respectively. The dhfr 1164L mutation, which confers high-level resistance, was not identified. The P. falciparum chloroquine resistance transporter (pfcrt) K76T mutation, which is critical for chloroquine resistance, was found in 167 of 168 infections. Six dhfr/dhps allelotypes and four pfcrt-resistant alleles were observed. Their interrelationships suggest a semi-clonal propagation of P. falciparum malaria in Bolivar, and an invasion of multi-resistant pathogens from Brazil. Despite national restrictions on the use of SP and chloroquine, genotypic resistance to these therapies remains widespread in Bolivar.

A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies.

A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus- or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/microl of blood using DNA prepared from 25-microl blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.