Genetic polymorphism of circumsporozoite protein (CSP) in Plasmodium malariae isolates from Malaysia.

Information about Plasmodium malariae is scanty worldwide due to its "benign" nature and low infection rates. Consequently, studies on the genetic polymorphisms of P. malariae are lacking. Here, we report genetic polymorphisms of 28 P. malariae circumsporozoite protein (Pmcsp) isolates from Malaysia which were compared with those in other regions in Asia as well as those from Africa. Phylogenetic analysis revealed that most Malaysian P. malariae isolates clustered together but independently from other Asian isolates. Low nucleotide diversity was observed in Pmcsp non-repeat regions in contrast to high nucleotide diversity observed in non-repeat regions of Plasmodium knowlesi CSP gene, the current major cause of malaria in Malaysia. This study contributes to the characterisation of naturally occurring polymorphisms in the P. malariae CSP gene.

Rapid Screening for Non-falciparum Malaria in Elimination Settings Using Multiplex Antigen and Antibody Detection: Post Hoc Identification of Plasmodium malariae in an Infant in Haiti.

Haiti is targeting malaria elimination by 2025. The Grand'Anse department in southwestern Haiti experiences one-third to half of all nationally reported Plasmodium falciparum cases. Although there are historical reports of Plasmodium vivax and Plasmodium malariae, today, non-falciparum infections would remain undetected because of extensive use of falciparum-specific histidine-rich protein 2 (HRP2) rapid diagnostic tests (RDT) at health facilities. A recent case-control study was conducted in Grand'Anse to identify risk factors for P. falciparum infection using HRP2-based RDTs (n = 1,107). Post hoc multiplex Plasmodium antigenemia and antibody (IgG) detection by multiplex bead assay revealed one blood sample positive for pan-Plasmodium aldolase, negative for P. falciparum HRP2, and positive for IgG antibodies to P. malariae. Based on this finding, we selected 52 samples with possible P. malariae infection using IgG and antigenemia data and confirmed infection status by species-specific PCR. We confirmed one P. malariae infection in a 6-month-old infant without travel history. Congenital P. malariae could not be excluded. However, our finding-in combination with historical reports of P. malariae-warrants further investigation into the presence and possible extent of non-falciparum malaria in Haiti. Furthermore, we showed the use of multiplex Plasmodium antigen and IgG detection in selecting samples of interest for subsequent PCR analysis, thereby reducing costs as opposed to testing all available samples by PCR. This is of specific use in low-transmission or eliminating settings where infections are rare.

Incrimination of Anopheles (Anopheles) intermedius Peryassú, An. (Nyssorhynchus) nuneztovari Gabaldón, An. (Nys.) oswaldoi Peryassú as natural vectors of Plasmodium falciparum in French Guiana

Anopheles darlingi Root is the major vector of human malaria in the Neotropics and has been considered to be the sole malaria vector in French Guiana. The presence of other potential vectors suggests that malaria may be transmitted by other species under certain conditions. From 2006-2011, all anopheline specimens collected from 11 localities were assayed to determine if the Plasmodium circumsporozoite protein was present. In addition to An. darlingi, we found Anopheles oswaldoi, Anopheles intermedius and Anopheles nuneztovari specimens that were infected with Plasmodium sp. Further investigations on the behaviour and ecology of An. oswaldoi, An. intermedius and An. nuneztovari are necessary to determine their role in malaria transmission in French Guiana.

Incrimination of Anopheles (Anopheles) intermedius Peryassú, An. (Nyssorhynchus) nuneztovari Gabaldón, An. (Nys.) oswaldoi Peryassú as natural vectors of Plasmodium falciparum in French Guiana.

Anopheles darlingi Root is the major vector of human malaria in the Neotropics and has been considered to be the sole malaria vector in French Guiana. The presence of other potential vectors suggests that malaria may be transmitted by other species under certain conditions. From 2006-2011, all anopheline specimens collected from 11 localities were assayed to determine if the Plasmodium circumsporozoite protein was present. In addition to An. darlingi, we found Anopheles oswaldoi, Anopheles intermedius and Anopheles nuneztovari specimens that were infected with Plasmodium sp. Further investigations on the behaviour and ecology of An. oswaldoi, An. intermedius and An. nuneztovari are necessary to determine their role in malaria transmission in French Guiana.

Structure of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae bound to an allophenylnorstatine-based inhibitor.

The malarial parasite continues to be one of the leading causes of death in many developing countries. With the development of resistance to the currently available treatments, the discovery of new therapeutics is imperative. Currently, the plasmepsin enzymes found in the food vacuole of the parasite are a chief target for drug development. Allophenylnorstatine-based compounds originally designed to inhibit HIV-1 protease have shown efficacy against all four plasmepsin enzymes found in the food vacuole of Plasmodium falciparum. In this study, the first crystal structure of P. malariae plasmepsin 4 (PmPM4) bound to the allophenylnorstatine-based compound KNI-764 is described at 3.3 Angstroms resolution. The PmPM4-inhibitor complex crystallized in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.9, b = 112.6, c = 90.4 Angstroms, with two molecules in the asymmetric unit related by a non-crystallographic symmetry operator. The structure was refined to a final R factor of 24.7%. The complex showed the inhibitor in an unexpected binding orientation with allophenylnorstatine occupying the S1' pocket. The P2 group was found outside the S2 pocket, wedged between the flap and a juxtaposed loop. Inhibition analysis of PmPM4 also suggests the potential for allophenylnorstatine-based compounds to be effective against all species of malaria infecting humans and for the future development of a broad-based inhibitor.

Monkeys of the rainforest in French Guiana are natural reservoirs for P. brasilianum/P. malariae malaria.

Monkey blood samples were collected from 214 monkeys relocated as part of the wildlife rescue organized in French Guiana during the filling of the Petit Saut Dam on the Sinnamary River. These samples were tested for malaria parasites by microscopy of thick blood filsm and by nested PCR for small subunit rRNA genes (SSUrRNA). Parasitic blood forms similar to Plasmodium brasilianum were detected in 4 monkey species: Alouatta seniculus macconnelli, Saguinus midas midas, Pithecia pithecia and Ateles paniscus paniscus, with the highest prevalence in Alouatta monkeys. PCR was more sensitive than the conventional method for detecting low-grade parasitaemia in positive monkeys. The examination of blood films indicated that 5.6% of the animals carried parasites whereas the nested PCR for ribosomal DNA indicated a prevalence of 11.3%. The P. brasilianum SSUrRNA gene sequence was analysed and aligned with those from P. malariae, P. falciparum and P. vivax. This suggested that P. brasilianum and P. malariae are very closely related. Similar results were obtained from analysis of the sequences in P. malariae and P. brasilianum isolates of a polymorphic gene fragment analogous to the merozoite surface protein-1 (MSP-1) gene of P. falciparum. The P. brasilianum/P. malariae sequences were more similar to those of P. vivax than to those of P. falciparum, at least in the gene region examined. The high degree of DNA homology in the sequences of the SSUrRNA and msp1-like genes is consistent with other characterizations demonstrating a taxonomic relationship between P. brasilianum and P. malariae species. Our results provide further evidence that P. brasilianum and P. malariae are virtually identical and should probably be considered to be a single malaria species. This raises the question as to whether monkeys living in the rainforest are natural reservoirs for both simian and human malaria. These results have implications for the interpretation of the current epidemiological situation in French Guiana.

A study of polymorphism in the circumsporozoite protein of human malaria parasites.

We have characterized the circumsporozoite (CS) gene sequences of Plasmodium malariae China-1 CDC, isolated recently from a person who was infected 50 years ago in China, and P. vivax Chesson, isolated 48 years ago from a patient who had returned from New Guinea. These protein sequences were compared with the CS protein sequences of recently isolated P. vivax and P. malariae parasites. In a similar manner, we compared the previously characterized CS protein gene of P. falciparum clone 7G8, derived from a Brazilian isolate collected in 1980, with the CS protein genes of recent P. falciparum field isolates. In the case of the P. malariae CS protein gene, with the exception of an additional copy of major (NAAG) and minor (NDAG) repeat sequences and the presence of one copy of NDEG sequence, the China-1 CDC P. malariae parasite is similar to the Uganda-1 CDC isolate of 1982. In the nonrepeat region, changes were noted in two amino acid residues, one of which is also seen in a closely related monkey malaria parasite, P. brasilianum. In the case of P. vivax CS proteins, the nonrepeat region of the protein in Chesson strain shares identity with nearly 71% of the CS clones characterized from field isolates. In the P. falciparum CS proteins, the 7G8 CS protein sequence is identical to 75% of the genes of recent field isolates in the Th1R-N1 region. In the Th2R and Th3R regions, 34% and 55% of the CS clones analyzed, respectively, had changes at two amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of clinical grade proteins produced by recombinant DNA technologies.

Methods were developed for the production of clinical grade malaria vaccine candidates expressed in E. coli by recombinant DNA technologies. The essential features of the purification protocol consist of (1) mechanical breakage of host cells and solubilization of the recombinant proteins in 6 M guanidine hydrochloride; (2) ammonium sulfate fractionation; (3) affinity chromatography on a Ni(2+)-chelate gel in the presence of 6 M guanidine hydrochloride; and (4) ion exchange chromatograph on a Phospho Ultrogel column in the presence of 6 M urea. The use of undesirable chemicals (PMSF, DFP, TFA, acetonitrile, etc.) was avoided rather than demonstrating their complete removal after the purification steps. Testing of chromatographic fractions for host-cell proteins and the elimination of fractions with E. coli protein content was found necessary to obtain a final product that contained less than 0.01% of host derived proteins. The recombinant proteins were renatured either from 8 M urea or from 6 M guanidine hydrochloride by increasing the pH to 10.5 in the presence of glycine and EDTA, reduction with DTT, dilution to a protein concentration below 1 mg.ml-1, and dialysis against 0.9% NaCl. The method presented here can be tailor-fit, with minor modification, for the purification of almost any recombinant protein and the final product satisfies current regulations concerning the production of clinically acceptable therapeutic products.