Safety and tolerability of elubaquine (bulaquine, CDRI 80/53) for treatment of Plasmidium vivax malaria in Thailand

We conducted a study to compare the safety and tolerability of anti-relapse drugs elubaquine and primaquine against Plasmodium vivax malaria. After standard therapy with chloroquine, 30 mg/kg given over 3 days, 141 patients with P. vivax infection were randomized to receive primaquine or elubaquine. The 2 treatment regimens were primaquine 30 mg once daily for 7 days (group A, n = 71), and elubaquine 25 mg once daily for 7 days (group B, n = 70). All patients cleared parasitemia within 7 days after chloroquine treatment. Among patients treated with primaquine, one patient relapsed on day 26; no relapse occurred with elubaquine treatement. Both drugs were well tolerated. Adverse effects occurred only in patients with G6PD deficiency who were treated with primaquine (group A, n = 4), whose mean hematocrit fell significantly on days 7, 8 and 9 (P = 0.015, 0.027, and 0.048, respectively). No significant change in hematocrit was observed in patients with G6PD deficiency who were treated with elubaquine (group B, n = 3) or in patients with normal G6PD. In conclusion, elubaquine, as anti-relapse therapy for P. vivax malaria, was as safe and well tolerated as primaquine and did not cause clinically significant hemolysis.     

Evaluation of rapid diagnostics for Plasmodium falciparum and P. vivax in Mae Sot Malaria endemic area, Thailand

Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/Pan™, Malaria Ag-Pf™, and Malaria Ag-Pv™ tests, in comparison with the microscopic and PCR methods. A total of 460 blood samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P. vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collected. The sensitivities of Malaria Ag-Pf™ and Malaria Antigen Pf/Pan™ compared with the microscopic method for P. falciparum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-Pf™, Malaria Ag-Pv™, and Malaria Antigen Pf/Pan™ were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria Ag-Pf™, Malaria Antigen Pf/Pan™, and microscopic method, when PCR was used as a reference method for P. falciparum or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Malaria Ag-Pf™, Malaria Ag-Pv™, Malaria Antigen Pf/Pan™, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%, respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for application to malaria diagnosis.     

A case of symptomatic splenic infarction in vivax malaria

Splenic infarction is a rare complication in malaria cases, and is caused primarily by Plasmodium falciparum. Recently in South Korea, only P. vivax has prevailed since 1993. Although the probability that symptomatic splenic infarction may occur in vivax malaria cases is considered relatively high, there have never been any case reports describing the occurrence of symptomatic splenic infarction in cases of vivax malaria. A 34-year-old man presented with fever that had persisted for 5 days. P. vivax infection was verified using a peripheral blood smear, and chloroquine was utilized to treat the fever successfully. Six days later, the patient developed pain in the left upper abdomen, which was diagnosed as splenic infarction by computed tomography.     

Incidence patterns of vivax malaria in civilians residing in a high-risk county of Kyonggi-do (province), Republic of Korea.

The characteristics of vivax malaria epidemics along the demilitarized zone (DMZ) in the Republic of Korea has been established by the early surveillance data. To further characterize the epidemic, data of civilian patients microscopically diagnosed with malaria from 1995 through 2000 were analyzed in Yonchon-gun (county). Malaria incidence was greater in male civilians > 30 years of age (p < 0.05). The annual parasite index was significantly higher in those living in the administrative areas (Myeon) traversed by DMZ than those living in Myons not traversed by DMZ (p < 0.05). Analysis according to the distance (4 to 14 km) from DMZ showed that people living in villages close to DMZ had higher annual parasite indices than those living in villages remote from DMZ (p for trend < 0.05). Civilians living in Myeons with plains and located in northwestern part of the county had higher annual parasite indices than those living in hilly Myeons located in southeastern part of the county (p for trend < 0.05). These findings suggest that the contraction of vivax malaria is related with night-time outdoor activities, and that the distance from DMZ is a risk factor. In this area, the flying distance of infected vector mosquitos can explain the annually repeating occurrence of civilian cases.     

Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.     

Variation in relapse frequency and the transmission potential of Plasmodium vivax malaria

There is substantial variation in the relapse frequency of Plasmodium vivax malaria, with fast-relapsing strains in tropical areas, and slow-relapsing strains in temperate areas with seasonal transmission. We hypothesize that much of the phenotypic diversity in P. vivax relapses arises from selection of relapse frequency to optimize transmission potential in a given environment, in a process similar to the virulence trade-off hypothesis. We develop mathematical models of P. vivax transmission and calculate the basic reproduction number R₀ to investigate how transmission potential varies with relapse frequency and seasonality. In tropical zones with year-round transmission, transmission potential is optimized at intermediate relapse frequencies of two to three months: slower-relapsing strains increase the opportunity for onward transmission to mosquitoes, but also increase the risk of being outcompeted by faster-relapsing strains. Seasonality is an important driver of relapse frequency for temperate strains, with the time to first relapse predicted to be six to nine months, coinciding with the duration between seasonal transmission peaks. We predict that there is a threshold degree of seasonality, below which fast-relapsing tropical strains are selected for, and above which slow-relapsing temperate strains dominate, providing an explanation for the observed global distribution of relapse phenotypes.     

Unstable vivax malaria in Korea

Korean vivax malaria had been prevalent for longtime throughout the country with low endemicity. As a result of the Korean war (1950-1953), malaria became epidemic. In 1959-1969 when the National Malaria Eradication Service (NMES) was implemented, malaria rates declined, with low endemicity in the south-west and south plain areas and high endemic foci in north Kyongsangbuk-do (province) and north and east Kyonggi-do. NMES activities greatly contributed in accelerating the control and later eradication of malaria. The Republic of Korea (South Korea) was designated malaria free in 1979. However, malaria re-emerged in 1993 and an outbreak occurred in north Kyonggi-do and north-west Kangwon-do (in and/or near the Demilitarized Zone, DMZ), bordering North Korea. It has been postulated that most of the malaria cases resulted from bites of sporozoite-infected females of An. sinensis dispersed from North Korea across the DMZ. Judging from epidemiological and socio-ecological factors, vivax malaria would not be possible to be endemic in South Korea. Historical data show that vivax malaria in Korea is a typical unstable malaria. Epidemics may occur when environmental, socio-economical, and/or political factors change in favor to malaria transmission, and when such factors change to normal conditions malaria rates become low and may disappear. Passive case detection is a most feasible and recommendable control measure against the unstable vivax malaria in Korea in cost-effect point of view.     

Plasmodium vivax Drug Resistance Genes; Pvmdr1 and Pvcrt-o Polymorphisms in Relation to Chloroquine Sensitivity from a Malaria Endemic Area of Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 μl) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC₅₀ of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.     

Minor liver profile dysfunctions in Plasmodium vivax, P. malaria and P. ovale patients and normalization after treatment

Liver function tests were performed in 61 vivax, 54 malariae and 15 ovale malaria patients who were admitted to Bangkok Hospital for Tropical Diseases between 2001 and 2004. The objective of the study was to evaluate changes in hepatic biochemical indices before and after treatment with artemisinin derivatives. On admission and prior to treatment, hepatic dysfunction was found among the 3 groups. Serum liver function tests and physical examinations were performed weekly during the 28-day follow-up period. Initially elevated serum bilirubin and diminished albumin returned to normal within 2 weeks of treatment. Serum alkaline phosphatase and aminotransferases returned to within normal limits within 3 weeks. We conclude that patients with Plasmodium vivax, P. malariae and P. ovale infections had slightly elevated serum bilirubin, aminotransferase and alkaline phosphatase levels, and hypoalbuminemia. These minor abnormalities returned to normal within a few weeks after treatment with therapies based on artemisinin derivatives.     

Emerging Plasmodium vivax resistance to chloroquine in South America: an overview

The global emergence of Plasmodium vivax strains resistant to chloroquine (CQ) since the late 1980s is complicating the current international efforts for malaria control and elimination. Furthermore, CQ-resistant vivax malaria has already reached an alarming prevalence in Indonesia, East Timor and Papua New Guinea. More recently, in vivo studies have documented CQ-resistant P. vivax infections in Guyana, Peru and Brazil. Here, we summarise the available data on CQ resistance across P. vivax-endemic areas of Latin America by combining published in vivo and in vitro studies. We also review the current knowledge regarding the molecular mechanisms of CQ resistance in P. vivax and the prospects for developing and standardising reliable molecular markers of drug resistance. Finally, we discuss how the Worldwide Antimalarial Resistance Network, an international collaborative effort involving malaria experts from all continents, might contribute to the current regional efforts to map CQ-resistant vivax malaria in South America.     

Slow clearance of Plasmodium vivax with chloroquine amongst children younger than six months of age in the Brazilian Amazon

Plasmodium vivax is the most widespread parasite causing malaria, being especially prevalent in the Americas and Southeast Asia. Children are one of the most affected populations, especially in highly endemic areas. However, there are few studies evaluating the therapeutic response of infants with vivax malaria. This study retrospectively evaluated the parasitaemia clearance in children diagnosed with vivax malaria during the first five days of exclusive treatment with chloroquine (CQ). Infants aged less than six months old had a significantly slower parasitaemia clearance time compared to the group of infants and children between six months and 12 years old (Kaplan-Meier survival analysis; Wilcoxon test; p = 0.004). The impaired clearance of parasitaemia in younger children with vivax malaria is shown for the first time in Latin America. It is speculated that CQ pharmacokinetics in young children with vivax malaria is distinct, but this specific population may also allow the detection of CQ-resistant parasites during follow-up, due to the lack of previous immunity.     

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.     

Prevalence of Drug Resistance-Associated Gene Mutations in Plasmodium vivax in Central China

Resistance of Plasmodium spp. to anti-malarial drugs is the primary obstacle in the fight against malaria, and molecular markers for the drug resistance have been applied as an adjunct in the surveillance of the resistance. In this study, we investigated the prevalence of mutations in pvmdr1, pvcrt-o, pvdhfr, and pvdhps genes in temperate-zone P. vivax parasites from central China. A total of 26 isolates were selected, including 8 which were previously shown to have a lower susceptibility to chloroquine in vitro. For pvmdr1, pvcrt-o, and pvdhps genes, no resistance-conferring mutations were discovered. However, a highly prevalent (69.2%), single-point mutation (S117N) was found in pvdhfr gene. In addition, tandem repeat polymorphisms existed in pvdhfr and pvdhps genes, which warranted further studies in relation to the parasite resistance to antifolate drugs. The study further suggests that P. vivax populations in central China may still be relatively susceptible to chloroquine and sulfadoxine-pyrimethamine.     

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.     

Reemerging vivax malaria: changing patterns of annual incidence and control programs in the Republic of Korea

Changing patterns of the reemerging Plasmodium vivax malaria in the Republic of Korea (South Korea) during the period 1993 to 2005 are briefly analyzed with emphasis on the control measures used and the effects of meteorological and entomological factors. Data were obtained from the Communicable Diseases Monthly Reports published by the Korea Center for Disease Control and Prevention, and webpages of World Health Organization and United Nations. Meteorological data of Kangwon-do (Province) were obtained from local weather stations. After its first reemergence in 1993, the prevalence of malaria increased exponentially, peaking in 2000, and then decreased. In total, 21,419 cases were reported between 1993 and 2005 in South Korea. In North Korea, a total of 916,225 cases were reported between 1999 and 2004. The occurrence of malaria in high risk areas of South Korea was significantly (P > 0.05) correlated with the mosquito population but not with temperature and rainfall. Control programs, including early case detection and treatment, mass chemoprophylaxis of soldiers, and international financial aids to North Korea for malaria control have been instituted. The situation of the reemerging vivax malaria in the Republic of Korea is remarkably improving during the recent years, at least in part, due to the control activities undertaken in South and North Korea.     

Travelers' malaria among foreigners at the Hospital for Tropical Diseases, Bangkok, Thailand - a 6-year review (2000-2005)

We retrospectively examined the charts of travelers admitted to the Hospital for Tropical Diseases, Bangkok, Thailand, with malaria during the years 2000-2005. Twenty-one cases of malaria were identified, of which 12 (57%) were Plasmodium vivax infections and 9 (43%) were P. falciparum infections. There was one mixed case with vivax and falciparum infection. Only 1 P. falciparum case had complications. All cases were successfully treated with standard antimalarial drugs. Only 3 of the 21 cases were thought to be acquired in Thailand, the rest were regarded to be imported.     

Seroprevalence of Plasmodium vivax in the Republic of Korea (2003-2005) using Indirect Fluorescent Antibody Test

Plasmodium vivax reemerged in the Republic of Korea (ROK) in 1993, and is likely to continue to affect public health. The purpose of this study was to measure levels of anti-P. vivax antibodies using indirect fluorescent antibody test (IFAT) in border areas of ROK, to determine the seroprevalence of malaria (2003-2005) and to plan effective control strategies. Blood samples of the inhabitants in Gimpo-si, Paju-si, and Yeoncheon-gun (Gyeonggi-do), and Cheorwon-gun (Gangwon-do) were collected and kept in Korea Centers for Disease Control and Prevention (KCDC). Out of a total of 1,774 serum samples tested, the overall seropositivity was 0.94% (n=17). The seropositivity was the highest in Paju-si (1.9%, 7/372), followed by Gimpo-si (1.4%, 6/425), Yeoncheon-gun (0.67%, 3/451), and Cheorwon-gun (0.19%, 1/526). The annual parasite incidence (API) in these areas gradually decreased from 2003 to 2005 (1.69, 1.09, and 0.80 in 2003, 2004, and 2005, respectively). The highest API was found in Yeoncheon-gun, followed by Cheorwon-gun, Paju-si, and Gimpo-si. The API ranking in these areas did not change over the 3 years. The seropositivity of Gimpo-si showed a strong linear relationship with the API of 2005 (r=0.9983, P=0.036). Seropositivity data obtained using IFAT may be useful for understanding malaria prevalence of relevant years, predicting future transmission of malaria, and for establishing and evaluating malaria control programs in affected areas.     

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.     

Genetic Polymorphisms in VIR Genes among Indian Plasmodium vivax Populations

The vir genes are antigenic genes and are considered to be possible vaccine targets. Since India is highly endemic to Plasmodium vivax, we sequenced 5 different vir genes and investigated DNA sequence variations in 93 single-clonal P. vivax isolates. High variability was observed in all the 5 vir genes; the vir 1/9 gene was highly diverged across Indian populations. The patterns of genetic diversity do not follow geographical locations, as geographically distant populations were found to be genetically similar. The results in general present complex genetic diversity patterns in India, requiring further in-depth population genetic and functional studies.     

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.