Importance of kelch 13 C580Y mutation in the studies of artemisinin resistance in Plasmodium falciparum in Greater Mekong Subregion

The mortality caused by Plasmodium falciparum was reduced by Artemisinin (ART) and ART combination therapy (ACT). However, Artemisinin resistance (ART-R) emerge during 2008 in Cambodia and spread to Greater Mekong Subregion (GMS). ART-R was confirmed not to spread to India, a gateway to whole Africa. The whole genome sequencing approach of P. falciparum assumed the k13 gene encoded Kelch protein was discovered to be associated with ART-R. Of the single nucleotide polymorphisms (SNPs) of k13 gene, C580Y mutant was commonly dominant in Cambodia, Myanmar, Thailand, Laos and Vietnam and assumed to be one of strong molecular markers for ART-R in P. falciparum isolates in GMS. Literatures published between 2017 and 2018 were reviewed in this work. F446I is observed to be doubtful molecular marker as ART-R marker. Transgenic experiment showed that parasite with F446I mutation displayed prolonged clearance in respond to ART while C580Y was applied as positive control mutant. Furthermore, study of C580Y allele in four countries Cambodia, Thailand, Laos resulted in single origin whereas the parasite with this allele showed multi-origin in three Provinces of Vietnam. As artemisinin was short acting drug, the role of long acting partner drug was studied by using transgenic C580Y mutant and C580 to leave recrudescent P. falciparum. Recently, there was treatment failure with ACT in some countries in GMS. In this review, the importance of C580Y mutation in the study of ART-R was discussed.     

Correlation of in vitro Sensitivity of Chloroquine and other Antimalarials with the Partner Drug Resistance to Plasmodium falciparum Malaria in Selected Sites of India

Background: Antimalarial drug resistance is a potential threat for control and elimination of malaria. To ascertain the status of antimalarial drug resistance at the study sites, correlation between in vitro drug sensitivity pattern and drug resistance molecular markers in Plasmodium falciparum malaria was undertaken. Materials and Methods: Polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt) K76T and pfmdr1 N86Y were studied in relation to the in vitro susceptibility of P. falciparum in culture (n = 10) and field isolates (n = 40) to chloroquine (CQ), amodiaquine (AQ), quinine (QN), mefloquine (MQ) and artemisinin (ART). The prevalence of drug resistance molecular markers, pfdhfr (codon S108N, C59R, N51I, I164 L and A16V), pfdhps (codon S436F and A437G), pfATPase6 (codon D639G and E431K) and mutation in the propeller domain of pfK13 gene were also analysed. Chi-square test and parametric Pearson correlation test were performed using SPSS version 17. Results: In vitro assay showed 18% resistance to CQ, 8% to AQ and 4% to QN. However, no resistance was observed towards MQ and ART. The mutations in pfcrt and pfmdr1 were statistically not significantly associated with susceptibility responses for antimalarials; however, increased IC₅₀ values of drugs were reflected as mutant and/or mixed isolates for both gene polymorphisms. CQ was found as independent predictor for other antimalarials, i.e., AQ, QN and ART, with r² score 0.241, 0.241 and 0.091, respectively. Mutation in the pfATPase6 gene at codon E431K was observed in only one sample from Tripura which also had increased IC₅₀ value of 6.28 nM. However, moderate numbers of mutations at codon S108N, C59R and I164 L for pfdhfr gene and S436F and A437G for pfdhps gene were also observed. None of the samples showed mutation in propeller domain of pfK13 gene. Conclusion: The correlation between IC₅₀ and molecular markers for antimalarial drug resistance is reported for the first time through this study. A positive correlation between in vitro drug resistance with molecular markers for antimalarial drug resistance could make in vitro assay a reliable tool to predict drug efficacy which is needed for detection of emerging resistance in the country.     

Hundreds of microsatellites for genotyping Plasmodium yoelii parasites

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.     

Updates on k13 mutant alleles for artemisinin resistance in Plasmodium falciparum

Background

In the fight against malaria caused by Plasmodium falciparum, the successes achieved by artemisinin were endangered by resistance of the parasites to the drug. Whole genome sequencing approach on artemisinin resistant parasite line discovered k13 gene associated with drug resistance. In vitro and in vivo studies indicated mutations in the k13 gene were linked to the artemisinin resistance.

Detection by real-time PCR of the<Emphasis Type="Italic"> Pfcrt</Emphasis> T76 mutation,a molecular marker of chloroquine-resistant<Emphasis Type="Italic"> Plasmodium falciparum</Emphasis> strains

An efficient and fast diagnostic assay was developed to detect the point mutation involved in the resistance of Plasmodium falciparum to chloroquine. The test, based upon a real-time PCR principle and performed with the LightCycler system, was carried out on venous blood isolates from 221 cases of falciparum malaria. The assay provided a quick, sensitive and reliable detection of the T76 Pfcrt mutation and the chloroquine resistance. The results of this study suggest that this LightCycler-based technique could be used to optimise the treatment of imported uncomplicated falciparum malaria, and also for the molecular surveillance of drug resistance among imported cases of this disease.     

Acquisition of Antibodies against Plasmodium falciparum Merozoites and Malaria Immunity in Young Children and the Influence of Age,Force of Infection,and Magnitude of Response

Individuals in areas of Plasmodium falciparum endemicity develop immunity to malaria after repeated exposure. Knowledge of the acquisition and nature of protective immune responses to P. falciparum is presently limited, particularly for young children. We examined antibodies (IgM, IgG, and IgG subclasses) to merozoite antigens and their relationship to the prospective risk of malaria in children 1 to 4 years of age in a region of malaria endemicity in Papua New Guinea. IgG, IgG1, and IgG3 responses generally increased with age, were higher in children with active infection, and reflected geographic heterogeneity in malaria transmission. Antigenic properties, rather than host factors, appeared to be the main determinant of the type of IgG subclass produced. High antibody levels were not associated with protection from malaria; in contrast, they were typically associated with an increased risk of malaria. Adjustment for malaria exposure, using a novel molecular measure of the force of infection by P. falciparum, accounted for much of the increased risk, suggesting that the antibodies were markers of higher exposure to P. falciparum. Comparisons between antibodies in this cohort of young children and in a longitudinal cohort of older children suggested that the lack of protective association was explained by lower antibody levels among young children and that there is a threshold level of antibodies required for protection from malaria. Our results suggest that in populations with low immunity, such as young children, antibodies to merozoite antigens may act as biomarkers of malaria exposure and that, with increasing exposure and responses of higher magnitude, antibodies may act as biomarkers of protective immunity.     

Evaluation of the OptiMAL Test for Rapid Diagnosis of Plasmodium vivax and Plasmodium falciparum Malaria

The development of rapid and specific diagnostic tests to identify individuals infected with malaria is of paramount importance in efforts to control the severe public health impact of this disease. This study evaluated the ability of a newly developed rapid malaria diagnostic test, OptiMAL (Flow Inc., Portland, Oreg.), to detect Plasmodium vivax and Plasmodium falciparum malaria during an outbreak in Honduras. OptiMAL is a rapid (10-min) malaria detection test which utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme parasite lactate dehydrogenase (pLDH). Differentiation of malaria parasites is based on antigenic differences between the pLDH isoforms. Since pLDH is produced only by live Plasmodium parasites, this test has the ability to differentiate live from dead organisms. Results from the OptiMAL test were compared to those obtained by reading 100 fields of traditional Giemsa-stained thick-smear blood films. Whole-blood samples were obtained from 202 patients suspected of having malaria. A total of 96 samples (48%) were positive by blood films, while 91 (45%) were positive by the OptiMAL test. The blood films indicated that 82% (79 of 96) of the patients were positive for P. vivax and 18% (17 of 96) were infected with P. falciparum. The OptiMAL test showed that 81% (74 of 91) were positive for P. vivax and 19% (17 of 91) were positive for P. falciparum. These results demonstrated that the OptiMAL test had sensitivities of 94 and 88% and specificities of 100 and 99%, respectively, when compared to traditional blood films for the detection of P. vivax and P. falciparum malaria. Blood samples not identified by OptiMAL as malaria positive normally contained parasites at concentrations of less than 100/μl of blood. Samples found to contain P. falciparum were further tested by two other commercially available rapid malaria diagnostic tests, ParaSight-F (Becton Dickinson, Cockeysville, Md.) and ICT Malaria P.f. (ICT Diagnostics, Sydney, Australia), both of which detect only P. falciparum. Only 11 of the 17 (65%) P. falciparum-positive blood samples were identified by the ICT and ParaSight-F tests. Thus, OptiMAL correctly identified P. falciparum malaria parasites in patient blood samples more often than did the other two commercially available diagnostic tests and showed an excellent correlation with traditional blood films in the identification of both P. vivax malaria and P. falciparum malaria. We conclude that the OptiMAL test is an effective tool for the rapid diagnosis of malaria.     

A review of dihydroartemisinin as another gift from traditional Chinese medicine not only for malaria control but also for schistosomiasis control

Artemisinin, also known as qinghaosu, is a sesquiterpene lactone endoperoxide extracted from the plant Artemisia annua L, an herb employed in traditional Chinese medicine. Artemisinin and its two main derivatives artemether and artesunate have been shown to be effective against both malaria and schistosomiasis, and therefore, they were described by Liu et al (Parasitol Res 110:2071–2074, 2012b) as the gifts from traditional Chinese medicine not only for malaria control but also for schistosomiasis control. However, another artemisinin derivative dihydroartemisinin (DHA) cannot be neglected. Dihydroartemisinin, a derivative of artemisinin with the C-10 lactone group replaced by hemiacetal and the active metabolite of all artemisinin compounds, was firstly identified as an antimalarial agent, and the dihydroartemisinin-piperaquine combination has been recommended as a first-line treatment of uncomplicated Plasmodium falciparum malaria by the WHO. It has been recently found that administration of dihydroartemisinin at a single dose of 300 mg/kg 2 h or 3, 5, 7, 10, 14, 18, 21, 28, or 35 days post-infection reduces total worm burdens by 1.1–64.8 % and female worm burden reductions by 11.9–90.5 %, and the in vivo activity of dihydroartemisinin against S. japonicum is enhanced by the use of multiple doses. However, a combination of praziquantel and dihydroartemisinin appears no more effective against S. japonicum schistosomulum than treatment with dihydroartemisinin alone. In mice experimentally infected with S. mansoni, administration with dihydroartemisinin at a single dose of 300 mg/kg on days 1, 7, 14, 21, 28, 35, 42, 49, or 56 post-infection results in total worm burden reductions of 13.8–82.1 % and female worm burden reductions of 13–82.8 %, and a clear-cut dose-response relationship of dihydroartemisinin against the schistosomula and adult worms of S. mansoni is observed. In addition, dihydroartemisinin was found to cause damages to the reproductive system of female S. mansoni worms, reduce the oviposition of survival worms, and inhibit the formation of granulomas around tissue-trapped eggs. More interestingly, no reduced sensitivity to dihydroartemisinin is detected in praziquantel non-susceptible S. japonicum, which provides a new option for the treatment of S. japonicum and S. mansoni infections, notably in endemic foci with praziquantel resistance or insensitivity detected. It is therefore considered that dihydroartemisinin is another gift from the traditional Chinese medicine not only for malaria control but also for schistosomiasis control.     

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.     

Longitudinal Survey of Plasmodium falciparum Infection in Vietnam: Characteristics of Antimalarial Resistance and Their Associated Factors

Plasmodium falciparum is the main cause of human malaria and is one of the important pathogens causing high rates of morbidity and mortality. The total number of malaria patients in Vietnam has gradually decreased over the last decade. However, the spread of pathogens with drug resistance remains a significant problem. Defining the trend in genotypes related to drug resistance is essential for the control of malaria in Vietnam. We undertook a longitudinal survey of Plasmodium falciparum malaria in 2001, 2002, and 2005 to 2007. The pfcrt, pfmdr1, pfdhfr, and pfdhps genes were analyzed by sequencing; and correlations by study year, age, gender, and genotype were identified statistically. The ratio of the chloroquine resistance genotype pfcrt 76T was found to have decreased rapidly after 2002. High numbers of mutations in the pfdhfr and pfdhps genes were observed only in 2001 and 2002, while the emergence of parasites with a new K540Y mutation in the P. falciparum dihydropteroate synthetase (PfDHPS) was observed in 2002. For males and those in younger age brackets, a correlation between vulnerability to P. falciparum infection and strains with pfcrt 76K or strains with decreased numbers of mutations in pfdhfr and pfdhps was demonstrated. The parasites with pfcrt 76T exhibited a greater number of mutations in pfdhfr and pfdhps.Plasmodium falciparum has long been one of the most important pathogens, causing severe illness and large numbers of deaths worldwide. In the 1990s, more than 1 million people living in Vietnam suffered from P. falciparum infections, resulting in thousands of deaths per year. Since then, the National Institute of Malariology, Parasitology, and Entomology (NIMPE) and the government of Vietnam have focused a great deal of time and effort on a malaria control program. As a result, the incidence of malaria reported in 2003 was only 12% of that reported in 1992 (2). However, the spread of drug-resistant isolates, including multidrug-resistant strains, has become a critical problem in Vietnam and has led to the significant failure of treatment. Thus, a further understanding of the incidence of malaria cases with detailed parasite genotype information and the identification of factors relating to the acquisition of drug-resistant isolates may prove important for the determination of effective and economical treatment choices in clinical settings.The pfcrt gene is located on chromosome 7 and encodes the vacuolar membrane transporter protein P. falciparum chloroquine-resistant transport (PfCRT) (21). While several point mutations associated with chloroquine resistance have been determined previously, substitution of K for T in codon 76 has been shown to be specifically related to resistance in vitro (21, 23). The allelic variation of pfcrt-related drug resistance differs among various geographical areas. Variants with the sequences CVIET, CVIDT, and SVMNT at pfcrt positions 72 to 76 are prevalent in the Indochinese Peninsula (13, 21, 24). The pfmdr1 gene is located on chromosome 5 and encodes P-glycoprotein homologue 1 (Pgh1). This protein is localized to the digestive vacuole membrane, where it is thought to function in the import of solutes, including some antimalarial drugs, into the digestive vacuole (21). pfmdr1 mutations in codons 86, 184, 1034, 1042, and 1246 have been reported previously and have been shown to correlate with susceptibility to chloroquine, quinine, and mefloquine (23). Sulfadoxine-pyrimethamine (SP) resistance is thought to be due to specific point mutations in the pfdhfr and the pfdhps genes. The pfdhfr gene encodes dihydrofolate reductase (DHFR), the target enzyme of pyrimethamine and trimethoprim. Conformational changes in this enzyme due to point mutations result in the prevention of adequate drug access. The codon positions in the pfdhfr gene that are related to resistance include 16, 50, 51, 59, 108, 140, and 164 (23). The deduced pathway for the resistant mutants suggested that all multiple mutants emerged through stepwise selection from the single mutant with the S108N mutation (18). The pfdhps gene encodes the enzyme dihydropteroate synthetase (DHPS). Point mutations in this gene also lead to conformational changes in DHPS and result in resistance to sulfadoxine and sulfamethoxazole. The loci responsible for resistance have been identified at positions 436, 437, 540, 581, and 613 (23).In Vietnam, chloroquine-resistant P. falciparum was reported for the first time in the 1960s (12, 14). Ngo et al. reported in 2003 that all of the isolates acquired from 18 adult rubber plantation workers residing in southern Vietnam demonstrated the pfcrt 76T mutation (14). In contrast, Phuc et al. reported that the prevalence of the mutant was only 38.5% when the strains from 39 malaria patients in the Quang Tri Province of central Vietnam were investigated (15). P. falciparum strains resistant to antifolates have also continued to increase in prevalence since the 1980s. Masimirembwa et al. analyzed 40 P. falciparum isolates obtained from malaria patients and reported that 97.5% of the isolates demonstrated a pfdhfr mutation that was related to pyrimethamine resistance, while 95.0% demonstrated a pfdhps mutation that was associated with sulfadoxine resistance (12).In the 1990s, the treatment for malaria in Vietnam mainly involved monotherapy with artemisinin or single-dose combinations of mefloquine with artemisinin or artesunate. However, the rates of recrudescence after the use of these treatment regimens were as high as 25%. As a result, the Vietnamese Ministry of Health introduced CV8 treatment, which consisted of dihydroartemisinin, piperaquine, trimethoprim, and primaquine, as part of the National Malaria Control Program (NMCP) (6, 20).Here we present the results of a longitudinal survey conducted in 2001, 2002, and 2005 to 2007 in the Binh Phuoc Province of Vietnam. The study was undertaken to investigate the incidence of malaria caused by P. falciparum and to document the changes in genotype that were related to drug resistance. From this study, we were able to identify the allelic and haplotype changes that occurred over the study years and deduce the factors associated with drug resistance.     

A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species

Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤5 parasites per μl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.     

Thioredoxin and glutathione systems in Plasmodium falciparum

Despite a 50% decrease in malaria infections between 2000 and 2010, malaria is still one of the three leading infectious diseases with an estimated 216 million cases worldwide in 2010. More than 90% of all malaria infections were caused by Plasmodium falciparum, a unicellular eukaryotic parasite that faces oxidative stress challenges while developing in Anopheles mosquitoes and humans. Reactive oxygen and nitrogen species threatening the parasite are either endogenously produced by heme derived from hemoglobin degradation or they are from exogenous sources such as the host immune defense. In order to maintain the intracellular redox balance, P. falciparum employs a complex thioredoxin and glutathione system based on the thioredoxin reductase/thioredoxin and glutathione reductase/glutathione couples. P. falciparum thioredoxin reductase reduces thioredoxin and a range of low molecular weight compounds, while glutathione reductase is highly specific for its substrate glutathione disulfide. Since Plasmodium spp. lack catalase and a classical glutathione peroxidase, their redox balance depends on a complex set of five peroxiredoxins differentially located in the cytosol, apicoplast, mitochondria, and nucleus with partially overlapping substrate preferences. Moreover, P. falciparum employs a set of members belonging to the thioredoxin superfamily such as three thioredoxins, two thioredoxin-like proteins, a dithiol and three monocysteine glutaredoxins, and a redox-active plasmoredoxin with largely redundant functions. This review aims at summarizing our current knowledge on the functional redox networks of the malaria parasite P. falciparum.     

An unusual case of Plasmodium vivax malaria monoinfection associated with crescentic glomerulonephritis: a need for vigilance

Plasmodium vivax infection is increasingly a major public health burden and the second most frequent human malaria. Higher levels of clinical severity and chloroquine resistance are major factors responsible for such increases. Malarial glomerular injury is uncommon and mainly observed in Plasmodium malariae-infected patients. Occasionally, transient immune complex-mediated glomerulonephritis is associated with Plasmodium falciparum infection. Coexistent crescentic glomerulonephritis and vivax malaria have not previously been reported. We report a fatal case of P. vivax malaria, who presented with acute renal failure. P. vivax monoinfection status was diagnosed with peripheral blood smear and rapid antigen test. Further evaluation for renal failure related to systemic illness and immunological markers were inconclusive. He was treated with antimalarial drugs, hemodialysis, and supportive therapy. Renal biopsy performed for nonrecovering renal failure reveled crescentic glomerulonephritis. This case highlights the need to thoroughly search for malaria-associated crescentic glomerulonephritis using renal biopsy after nonrecovering renal failure.     

Detecting mutations in PfCRT and PfMDR1 genes among Plasmodium falciparum isolates from Saudi Arabia by pyrosequencing

The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples (n = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.     

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.     

Genetic polymorphism at the C-terminal domain (region III) of knob-associated histidine-rich protein (KAHRP) of Plasmodium falciparum in isolates from Iran

The knob-associated histidine-rich protein (KAHRP) plays a major role in the virulence of Plasmodium falciparum and is one of the targets for molecular therapy. The primary structure of KAHRP of P. falciparum consists of three domains (regions I–III), of which the C-terminal domain (region III) is the most polymorphic segment of this protein. One of the main obstacles is genetic diversity in designing and developing of malaria control strategies such as molecular therapy and vaccines. The primary objective of the present study was to investigate and analyze the extent of genetic polymorphism at the region III of KAHRP of P. falciparum in isolates from Iran. A fragment of the kahrp gene spanning the C-terminal domain was amplified by nested PCR from 50 P. falciparum isolates collected from two malaria endemic areas of Iran during 2009 to August 2010 and sequenced. In this study, three allelic types were observed at the C-terminal domain of KAHRP on the basis of the molecular weight of nested PCR products and the obtained sequencing data. The presence of multiple alleles of the kahrp gene indicates that several P. falciparum strains exist in the malaria endemic areas of Iran. Our findings will be valuable in the design and the development of the molecular therapeutic reagents for falciparum malaria.     

Schistosoma mansoni Infection Impairs Antimalaria Treatment and Immune Responses of Rhesus Macaques Infected with Mosquito-Borne Plasmodium coatneyi

Malaria and schistosomiasis are the world''s two most important parasitic infections in terms of distribution, morbidity, and mortality. In areas where Plasmodium and Schistosoma species are both endemic, coinfections are commonplace. Mouse models demonstrate that schistosomiasis worsens a malaria infection; however, just as mice and humans differ greatly, the murine-infecting Plasmodium species differ as much from those that infect humans. Research into human coinfections (Schistosoma haematobium-Plasmodium falciparum versus Schistosoma mansoni-P. falciparum) has produced conflicting results. The rhesus macaque model provides a helpful tool for understanding the role of S. mansoni on malaria parasitemia and antimalarial immune responses using Plasmodium coatneyi, a malaria species that closely resembles P. falciparum infection in humans. Eight rhesus macaques were exposed to S. mansoni cercariae. Eight weeks later, these animals plus 8 additional macaques were exposed to malaria either through bites of infected mosquitos or intravenous inoculation. When malaria infection was initiated from mosquito bites, coinfected animals displayed increased malaria parasitemia, decreased hematocrit levels, and suppressed malaria-specific antibody responses compared to those of malaria infection alone. However, macaques infected by intravenous inoculation with erythrocytic-stage parasites did not display these same differences in parasitemia, hematocrit, or antibody responses between the two groups. Use of the macaque model provides information that begins to unravel differences in pathological and immunological outcomes observed between humans with P. falciparum that are coinfected with S. mansoni or S. haematobium. Our results suggest that migration of malaria parasites through livers harboring schistosome eggs may alter host immune responses and infection outcomes.     

Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites

Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC₅₀ in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.The World Health Organization (WHO) campaign to eradicate malaria in the 1950s and 1960s was initially largely successful in decreasing the burden of malaria. Drug failure eventually led to a resurgence in the number of malaria cases in the 1990s and caused vast numbers of deaths that could have been avoided through a better appreciation of the prevalence of drug-resistant malaria and more informed choices of first-line drugs (Greenwood et al. 2008). While malaria deaths are now likely to decline, primarily because of the introduction of artemisinin-based combination therapy (ACT) as well as increased insecticide spraying and the use of bed nets (Greenwood et al. 2008), this may only be temporary. Indeed, there has been a general increase in the parasite clearance times in ACT-treated Plasmodium falciparum malaria cases from near the Thai–Cambodian border, suggesting that case numbers may soon begin increasing (Dondorp et al. 2009).Remarkably, although artemisinin is used on tens of millions of individuals annually, we have little idea about how it acts or which genes contribute to resistance, confounding the community''s ability to monitor the spread of resistance using molecular markers and to deploy new therapies (Eastman and Fidock 2009). The fact that the genes involved in artemisinin resistance are not known is due to a variety of problems, including the fact that in vivo resistance has not been replicated in vitro (Dondorp et al. 2009). Additionally, the association of phenotypes with genotypes in P. falciparum is hampered by the difficulties in performing complementation studies due to extremely low transfection efficiencies and the fact that laboratory crosses of drug-sensitive and drug-resistant P. falciparum cannot be easily performed. Thus, it took more than 40 yr between the identification of chloroquine resistance in the field (Harinasuta et al. 1965) and confirmation that resistance is due to mutations in the chloroquine resistance transporter (pfcrt, MAL7P1.27) (Wellems et al. 1990; Fidock et al. 2000; Sidhu et al. 2002). While the recombinant progeny from one of the three extant crosses (Walliker et al. 1987; Wellems et al. 1990; Hayton et al. 2008) have most famously been used to map chloroquine resistance (Wellems et al. 1990), they have been used to map loci contributing to a wide variety of phenotypes that distinguish parental clones. For example, they have already been scored for a variety of different phenotypes that are related to drug sensitivity, including antifolate sensitivity (Wang et al. 2004b), quinine sensitivity (Ferdig et al. 2004), expression levels (Gonzales et al. 2008), and plasmodial surface ion channels (Alkhalil et al. 2009), but they could be scored for any phenotype that quantitatively distinguishes the parental strains Dd2 and HB3, such as propensity to mutate in the laboratory (Rathod et al. 1997). However, there are a limited number of phenotypes that distinguish these two laboratory strains that were derived from patients 40 yr ago. While more crosses would provide valuable data for many researchers interested in parasite genetics, there are ethical considerations associated with using primates in research. An alternative to creating new recombinant progeny is to find existing recombinant isolates that arose from recent meioses occurring in humans. Such parasites might be identified by performing full-genome analyses on parasites from a limited geographical area and could provide the malaria community with an unprecedented number of parasites differing in a variety of phenotypes for use in linkage or quantitative trait locus (QTL) studies.One attractive group of parasites for full genome investigation is from the Peruvian Amazon. Due to the low transmission rates it is expected that parasites isolated from an individual will be from a single clone infection. In addition, malaria was eradicated in the 1960s in this region but re-emerged with epidemics in the early 1990s (Aramburu Guarda et al. 1999; Branch et al. 2005), suggesting that the genomes might contain signatures of selective sweeps (Wootton et al. 2002; Roper et al. 2004). At first, malaria in this region was treated with chloroquine (first-line), sulfadoxine-pyrimethamine (second-line), and quinine with clindamycin or tetracycline (third-line) (Aramburu Guarda et al. 1999), but the emergence of resistance resulted in widespread chloroquine and antifolate treatment failure (Durand et al. 2007). Today, malaria remains hypoendemic with low levels of seasonal transmission of P. falciparum and P. vivax parasites in the region surrounding Iquitos, Peru (Roshanravan et al. 2003). Previous studies of parasites in the region describe only one or two independent haplotypes for important drug-resistance genes, suggesting a limited number of founders for this population (Bacon et al. 2009) and suggest that we might find recombinant progeny in this region.In this study, we performed genome scanning on 14 P. falciparum patient isolates from a limited geographical region. We show that the parasite population in the Peruvian Amazon is very closely related, with combinations of only two to four different genotypes for drug resistance genes, suggesting at most four parental haplotypes. Furthermore, some parasites taken from different patients who presented with symptoms were essentially clones of one another, while others were recent meiotic siblings that could be useful in linkage studies or eQTL analyses. Unexpectedly, genome scanning also revealed uncharacterized mutations in the apicoplast 23S rRNA that distinguished some Peruvian strains from the reference strain, 3D7. Because one of these mutations had been previously associated with lincosamide antibiotic resistance in the chloroplast and many bacterial species (Vester and Douthwaite 2001), sensitivity testing was performed revealing that the parasites harboring the mutation had indeed become resistant to clindamycin, a drug used in combination with quinine to treat pregnant women and infants for malaria in Peru. These are the first documented cases of resistance to this class of drugs in malaria and suggest that the use of lincosamide drugs in treating malaria should be reconsidered.     

Age-Dependent IgG Subclass Responses to Plasmodium falciparum EBA-175 Are Differentially Associated with Incidence of Malaria in Mozambican Children

Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-1₁₉) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.