Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America / Variación molecular de la dehidrofolato reductasa de Plasmodium vivax en México y Nicaragua contrasta con la que ocurre en Sudamérica

Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Genetic Polymorphisms in Plasmodium vivax Dihydrofolate Reductase and Dihydropteroate Synthase in Isolates from the Philippines, Bangladesh, and Nepal

Genetic polymorphisms of pvdhfr and pvdhps genes of Plasmodium vivax were investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal. The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP. Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia. All samples carried mutant combination alleles of pvdhfr and pvdhps. The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively. Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles. New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.

Diagnosis of Malaria by Detection of Parasite Lactate Dehydrogenase with Advantage Mal Card Test.

Background : Malaria is a global problem. Rapid diagnosis is essential for effective treatment and reducing mortality and morbidity of malaria. Diagnosis of malariaby peripheral smear is labor-intensive and requires considerable expertise for its interpretation. A rapid test , Advantage MAL card test is based on detection of parasite lactate dehydrogenase (pLDH) and has the ability to differentiate the four major Plasmodium species in 20 minutes. Objectives: 1) To evaluate utility of parasite lactate dehydrogenase for diagnosis of malaria with Advantage Mal card test.2) To compare the results of Advantage Mal card test with peripheral smear findings. Materials and Methods: In this retrospective study, total 5242 patients with malaria like symptoms attending OPD and admitted in wards at Acharya Vinoba Bhave Rural Hospital (AVBRH) from January 2008 to August 2011 were studied. Result: The age of patients ranged from < 1 year- >80 years. The commonest age group affected was 21-30 years. Male to female ratio was 1.04: 1. Prevalence rate of malaria was 101/1000 population in AVBRH. Malarial parasites were detected in PS in10.11% patients (P.falciparum 27.73% , P.vivax 71.32% , mixed infection 0.94%) and in 10.07% patients with Advantage Mal test (P. falciparum 28.03%, P.vivax 71.02%, mixed infection 0.95%). 3 cases of P.vivax and 1case of P.falciparum detected by PS were not detected by Advantage Mal test. 2 cases of P.falciparum detected by Advantage Mal and not by PS. Compared to PS, the Advantage Mal had sensitivity 99.24%, specificity 100%, positive predictive value 100%, negative predictive value was 89.92%. Conclusion: Diagnosis of malaria by detection of pLDH with Advantage Mal card test is simple ,rapid, reliable and cheap method. Results are comparable to blood films. It can detect P.flciparum infection when parasites are sequestered.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99 percent). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5 percent), followed by the wild-type A383/A553 (17.5 percent) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Plasmodium vivax dhfr Mutations among Isolates from Malarious Areas of Iran

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.

Aspartic proteases of Plasmodium vivax are highly conserved in wild isolates

The plasmepsins are the aspartic proteases of malaria parasites. Treatment of aspartic protease inhibitor inhibits hemoglobin hydrolysis and blocks the parasite development in vitro suggesting that these proteases might be exploited their potentials as antimalarial drug targets. In this study, we determined the genetic variations of the aspartic proteases of Plasmodium vivax (PvPMs) of wild isolates. Two plasmepsins (PvPM4 and PvPM5) were cloned and sequenced from 20 P. vivax Korean isolates and two imported isolates. The sequences of the enzymes were highly conserved except a small number of amino acid substitutions did not modify key residues for the function or the structure of the enzymes. The high sequence conservations between the plasmepsins from the isolates support the notion that the enzymes could be reliable targets for new antimalarial chemotherapeutics.

Markers for population genetic analysis of human plasmodia species, P. falciparum and P. vivax.

Present report deals with the genetic diversity existing among the field isolates of Plasmodium falciparum and P. vivax in India. Isoenzymes and molecular markers were used to analyse field isolates of P. falciparum and P. vivax. High level of length polymorphism was observed in repeat nucleotide sequences of MSP-1, MSP-2 and GLURP in P. falciparum isolates and CSP, GAM-1 and MSP-3 alpha in P. vivax isolates. In study populations a high proportion of isolates (up to 60%) were comprised of more than one genetically distinct parasite type--multiclonal. Presence of identical allelic forms of enzyme and DNA variations in different geographical areas and in different years suggest that isolates belong to a single random mating population of P. vivax and P. falciparum. Observed random combination of alleles in the field isolates suggest the unlinked nature of loci studied. Study supports the feasibility of using molecular markers for the identification of recrudescence in P. falciparum from fresh infection.

Diagnostic performance characteristics of rapid dipstick test for Plasmodium vivax malaria

We compared the diagnostic performance characteristics of newly developed method, the rapid dipstick test, which provides colorimetric determination by developing antibody to the lactate dehydrogenase enzyme of parasites, with conventional standard thick-blood film examination. For the rapid test, OptiMAL commercial kits were used. The results were also evaluated with clinical findings from patients. The parasites were determined by microscopic examination of thick-blood films from 81 patients with vivax malaria from southeastern Anatolia, Turkey. The OptiMAL test results were found to be negative in five patients who were diagnosed clinically and through thick-film testing as having vivax malaria. There was no false positivity observed with the OptiMAL test. We concluded that this rapid malaria test has a lower level of sensitivity than the classical thick-blood-film test for malaria, but that these methods have equal specificity

Comparison of the OptiMAL rapid test with routine microscopic examination of Giemsa-Stained Thick Blood Film for diagnosis of malaria.

The OptiMAL is a rapid immunodiagnostic test developed by Flow Inc., Portland, Oreg. for diagnosis and differentiation of P. falciparum and non P. falciparum malaria infection. It has been based on detection of circulating parasite lactate dehydrogenase enzyme (pLDH), produced by live Plasmodium parasites. The purpose of this study was to compare the efficacy of the OptiMAL test with routine microscopic examination of Giemsa-Stained Thick Blood Film (routine GS-TBF) for the diagnosis of malaria at a local malaria clinic in a hyperendemic area of Thailand by using a standard GS-TBF (standard GS-TBF) as reference. One hundred and seventy five patients attending the clinic were recruited; 50, 42 and 83 were falciparum malaria, vivax malaria and non-malaria patients, respectively. Compared with the reference, the OptiMAL test had sensitivities of 92 per cent and 97.6 per cent, whereas, the routine GS-TBF had sensitivities of 81.3 per cent and 81 per cent for the detection of P. falciparum and P. vivax, respectively. Both tests showed no false positive resulting in 100 per cent specificities. However, the OptiMAL test was able to detect only 20 per cent of infection with less than 200 parasitaemia/microlitre. It was also shown in our study that the OptiMAL test was advantageous in follow-up of the treatment outcome. No false positive occurred among 40 follow-up cases. The OptiMAL test detected malaria infection more accurately than the routine GS-TBF (p < 0.05) and was simple, easy to perform and rapid. It is an alternative tool for the diagnosis of malaria in a hyperendemic area where experienced microscopists are not available.

Partial sequence analysis of a Plasmodium vivax cation transporting ATPase gene homologue & a putative pseudohomologue.

Molecular characterization of P. vivax is essential to develop suitable antimalarial drugs and vaccines. We describe here isolation and sequence analysis of a partial cDNA of a calcium ATPase as well as a putative pseudogene from this parasite. The immunoscreening of lambda gtll- P. vivax DNA library with patients serum has earlier resulted in the isolation of several seroreactive clones including Pv14. This clone contains a 299 bp insert having 18 amino acids (aa) reading frame fused with beta galactosidase. A larger fragment of approximately 15 kb was isolated from the EMBL3 library for Pv14 but it had only 2 extra aa in its reading frame. The far upstream region of Pv14 revealed a 101aa long putative open reading frame (ORF) showing homology to a variety of calcium ATPases in the M8 and M9 transmembrane region. But in the absence of a transcript in the parasite could indicate that it represents a pseudogene. However, the real gene for calcium ATPase in P. vivax was detected by RT-PCR using degenerate primers, designed from the conserved sequences of energy transduction and phosphorylation domains. The amplified cDNA-PCR product of 550 bp was cloned and sequenced which showed a significant aa homology to the calcium ATPase4 of P. falciparum. The present study, therefore, establishes the existence of calcium ion pumps in P. vivax which will be useful in drug development.

Subcellular distribution of superoxide dismutase and catalase in human malarial parasite Plasmodium vivax.

Endogenous superoxide dismutase (SOD) has not been found to be present in P. vivax, a human malarial parasite and therefore it adopts and concentrates SOD from the host cell erythrocytes. It is demonstrated here that this adopted SOD from the host gets localized in lysosomes (10 k and 100 k fractions) of the malarial merozoites. P. vivax parasites were also found to contain very low levels of catalase, presumably as a result of contamination or adoption from the host red cell materials. It is therefore suggested that P. vivax merozoites are deficient in enzymes which are protective against the reactive oxygen species.