The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates

Human chimeras are potentially invaluable models for hemoprotozoan parasites such as Plasmodium falciparum. The work presented assesses the susceptibility of immunomodulated NOD/LtSz-SCID mice to genetically distinct P. falciparum parasites. To this end, mice grafted with human erythrocytes were inoculated with two P. falciparum laboratory lines, 3D7 and Dd2 and four clinical isolates, ISCIII-230, ISCIII-231, ISCIII-381 and ISCIII-399. The results showed that, without a previous period of parasite adaptation, 100% of the inoculated mice developed an infection, generally self-limited, though some mice died. The parasitemias ranged from 0.05 to 8% and lasted an average of 19 days (15-26 days) depending on the line or isolate studied. Sexual forms of different maturity, stage II-IV and mature gametocytes were observed in the peripheral blood of mice in 22, 50, 25, 72 and 80% of the mice infected with Dd2, ISCIII-399, ISCIII-230, ISCIII-231 and ISCIII-381 isolates, respectively. The study of the clinical symptoms, the haematological parameters and the histopathological changes in the infected mice showed that most of the malaria features were present in the infected mice except that the sequestration of infected erythrocytes was absent or at most a minor phenomenon, as also indicated by the presence of mature forms of the parasites in the peripheral blood. This study shows that the human chimeras allow the complete asexual and sexual erythrocytic cycle of different P. falciparum lines and clinical isolates to be observed in vivo. It opens a new way to investigate any parasite population in terms of infectivity, transmission, and drug resistance.     

Genetic linkage and association analyses for trait mapping in Plasmodium falciparum

Genetic studies of Plasmodium falciparum laboratory crosses and field isolates have produced valuable insights into determinants of drug responses, antigenic variation, disease virulence, cellular development and population structures of these virulent human malaria parasites. Full-genome sequences and high-resolution haplotype maps of SNPs and microsatellites are now available for all 14 parasite chromosomes. Rapidly increasing genetic and genomic information on Plasmodium parasites, mosquitoes and humans will combine as a rich resource for new advances in our understanding of malaria, its transmission and its manifestations of disease.     

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.     

Genetic clonality of Plasmodium falciparum affects the outcome of infection in Anopheles gambiae

Mosquito infections with natural isolates of Plasmodium falciparum are notoriously variable and pose a problem for reliable evaluation of efficiency of transmission-blocking agents for malaria control interventions. Here, we show that monoclonal P. falciparum isolates produce higher parasite loads than mixed ones. Induction of the mosquito immune responses by wounding efficiently decreases Plasmodium numbers in monoclonal infections but fails to do so in infections with two or more parasite genotypes. Our results point to the parasites genetic complexity as a potentially crucial component of mosquito-parasite interactions.     

Enzyme Typing of Malaria Parasites

Analysis of various isolates of Plasmodium falciparum from East and West Africa and from South-east Asia showed that some of the parasite enzymes can exist in more than one electrophoretic form. At least one form of each enzyme was common to parasites from all three regions. The enzyme forms could be used to differentiate morphologically indistinguishable samples of P. falciparum.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

South American Plasmodium falciparum after the malaria eradication era: clonal population expansion and survival of the fittest hybrids

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.     

Genetic fingerprints of parasites causing severe malaria in a setting of low transmission in Sudan

In this study we intended to examine the extent of genetic diversity of Plasmodium falciparum parasites causing severe malaria (SM). For this purpose, 100 parasite isolates were obtained from patients with SM and uncomplicated malaria, from an area of low and unstable malaria transmission in Sudan. The diversity of infection (DOI) was estimated by relating the number of the different parasite genotypes that were detected to the total number of parasites that were genotyped (parasite population/subpopulation). We used different molecular markers individually (pfcrt-76, pfmr1-86, GLURP size and MSP2 family and size) and as a group to set a multilocus genetic profile for each parasite isolate. The DOI as estimated by MSP2 and GLURP was 0.553 and 0.435, respectively. However, combination of all four molecular markers (multilocus genetic profile) revealed a fingerprint pattern of genetic diversity with a DOI of 0.936, indicating that in SM infection, diversity is the rule and homogeny is the exception. Furthermore, our clinical data suggest that the virulence markers might also be more diverse than expected. In conclusion, the results are unexpected and overturn the assumption that parasites causing SM are a limited subpopulation of virulent parasites or of a clonal nature. However, it was more likely that there was a genetically unique parasite in each infection.     

Continuous cultivation and drug susceptibility testing of Plasmodium falciparum in a malaria endemic area.

Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum.(ABSTRACT TRUNCATED AT 250 WORDS)     

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

The gene coding for the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was isolated from Plasmodium falciparum. The gene contains 1 intron and the A+T content is characteristic for the codon usage of P. falciparum. The predicted open reading frame codes for 337 amino acids (36651Da) and is 63.5% identical to the human erythrocytic GAPDH. GAPDH sequences from several field isolates of P. falciparum displayed 100% conservation. Phylogenetic analysis supports the hypothesis that dinoflagellates and Plasmodium are closely related. The protein encoded by the pfGAPDH was expressed recombinantly in Escherichia coli and exhibited enzymatic activity with NAD(+) but not with NADP(+) as cofactor. Antiserum raised against the recombinantly expressed enzyme detected specifically all developmental stages of cultured P. falciparum blood-stage parasites.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Variation of incubation time in an in vitro drug susceptibility test of Plasmodium falciparum isolates studied in the Solomon Islands

A study on chloroquine resistance of falciparum malaria was conducted in the Solomon Islands. Both in vitro and clinical tests were performed. In our regular studies of in vitro chloroquine susceptibility tests on Plasmodium falciparum from non-immuners in Japan, the threshold point to differentiate resistant and susceptible isolates was set at a 0. 114 microM chloroquine in the semi-micro culture system, and this point was also applicable in the study of the malaria parasite taken in the highly endemic malarious area with good coincidence with clinical observation. Variation in the incubation time (24-63) to reach the schizont stage of the isolated parasites were noted. It appeared that chloroquine resistant P. falciparum showed traits to reach the schizont stage within a shorter incubation period.     

Site-based study on polymorphism of Plasmodium falciparum MSP-1 and MSP-2 genes in isolates from two villages in Central Africa.

We investigated Plasmodium falciparum genetic diversity in isolates collected from school-going residents aged from 5 to 15 years in the village of Pouma (Cameroon, Central Africa). Seventy-six children were grouped according to the clinical status. Asymptomatic status was defined as parasite carriage in the absence of any clinical symptom and malaria symptomatic status with patent parasitemia over 5000 parasites/microliter of blood and an axillary temperature > 37.5 degrees C. Parasite DNA was analysed prior to malaria treatment. Genotyping of the P. falciparum merozoite surface proteins (MSP) 1 and 2 was performed by polymerase chain reaction using allele-specific primers. K1, MAD20, Ro33 and 3D7/CAMP, FC27 allelic families were attributed to MSP-1 and MSP-2 genes, respectively. No association was found between P. falciparum MSP-1 and MSP-2 genotypes and the clinical status of children. Mixed P. falciparum infections were detected in 78% of overall samples and all isolates from symptomatic children contained more than 1 clone. The results obtained in the village of Pouma were compared to those of the village of Dienga in Gabon where a similar study, using the same genotyping methods, had been carried out in the same age group of schoolchildren. Data are interpreted in the context of malaria epidemiology in both settings.     

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.     

Cytoadherence characteristics to endothelial receptors ICAM-1 and CD36 of Plasmodium falciparum populations from severe and uncomplicated malaria cases

The adhesion of infected red blood cells (IRBCs) to the cell lining of microvasculature is thought to play a central role in the pathogenesis of severe malaria. Individual IRBC can bind to more than one host receptor and parasites with multiple binding phenotypes may cause severe disease more frequently. However, as most clinical isolates are multiclonal, previous studies were hampered by the difficulty to distinguish whether a multiadherent phenotype was due to one or more parasite population(s). We have developed a tool, based on cytoadhesion assay and GeneScan genotyping technology, which enabled us to assess on fresh isolates the capacity of adherence of individual P. falciparum genotypes to human receptors expressed on CHO transfected cells. The cytoadhesion to ICAM-1 and CD36 of IRBCs from uncomplicated and severe malaria attacks was evaluated using this methodology. In this preliminary series conducted in non immune travelers, IRBCs from severe malaria appeared to adhere more frequently and/or strongly to ICAM-1 and CD36 in comparison with uncomplicated cases. In addition, a majority genotype able to strongly adhere to CD36 was found more frequently in isolates from severe malaria cases. Further investigations are needed to confirm the clinical relevance of these data.     

Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros

We report the outcome of chloroquine treatment and the prevalence of mutations at codon 86 of the pfmdr1 gene, at codon 76 of the pfcrt gene, and at codon 108 of the pfdhfr gene in clinical isolates of Plasmodium falciparum collected from 30 children under 10 years of age living in the Comoros Union. This in vivo study was carried out in February and March 2001 in Moroni. Chloroquine treatment failed in 23 children (76.6%; 95% confidence interval: 57.7 to 90.1%). Subsequent genotyping showed that all P. falciparum isolates (100%) harboured a tyrosine residue at position 86 in pfMDR1. 83.3% (25/30) of these isolates harboured a mutation at position 76 in pfCRT and half (15/30) of these isolates also harboured a mutation at position 108 in pfDHFR. Chloroquine resistance is a real concern in the Comoros Union. The prevalence of pfDHFR mutant parasites is alarming. The alternative drugs proposed as a replacement for chloroquine as first-line treatment in Comoros, and the strategy to monitor the drug susceptibility of Plasmodium sp in this part of the Indian Ocean sub-region are discussed.     

Continuous Cultivation and Drug Susceptibility Testing of Plasmodium falciparum in a Malaria Endemic Area

ABSTRACT Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum. This finding will have practical implications in malaria endemic areas where difficulties in obtaining non-immune human plasma or serum limits establishment of continuous culture of P. falciparum and its application in studies on malaria.