Border malaria characters of reemerging vivax malaria in the Republic of Korea.

Since 1993, the number of vivax malaria cases has increased every year in the northern part of the Republic of Korea (ROK). This study was designed to characterize factors related to the reemergence of malaria in the ROK. A total of 21 cases diagnosed in 1993 and 1994 distributed sporadically in the narrow zone along the demilitarized zone (DMZ). Of total 317 civilian inhabitant cases reported in 1994-1997, 287 cases were studied and 80.8% of them resided within 10 km from the southern border of the DMZ. The frequency distribution of anti-Plasmodium vivax antibody titers using indirect fluorescent antibody test was compared in three villages in relation with distance from the DMZ. The number of inhabitants with high antibody titers was larger in the village nearest to the border than that in more distant villages. The present results highly suggested that the reemerging vivax malaria start in the border area, most possibly caused by infected mosquitoes which flew across the border. This pattern of transmission repeated year after year.     

First Evaluation of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Vivax Malaria Endemic Regions in the Republic of Korea

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).

Methods

Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.

Results

Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.

Conclusions

No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

BACKGROUND: Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. METHODS: Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. RESULTS: Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). CONCLUSIONS: Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.     

Seroprevalence to the circumsporozoite protein peptide antigen of Plasmodium vivax in Korean children

Recently, malaria re-emerged in the Republic of Korea (ROK), but there have been only a few reports on malaria seroprevalence. For the epidemiological study in children, a total of 1,176 serum samples were obtained from children and adolescent inhabitants from the three different regions, Pajoo, the Guro district in the western part of Seoul (Guro), and Ansan, from June to September 2002, when the transmission rate was high. Anti-circumsporozoite protein (CSP) antibody levels were assessed in samples using an indirect enzyme-linked immunosorbent assay (ELISA) method. Among the three regions, the highest IgG seroreactivity against the CSP antigen of Plasmodium vivax was found in the children from Pajoo (8.0%), followed by the children from Guro (3.2%), and those in Ansan (0.02%) had the lowest seroreactivity. In Pajoo, the profile of antibody response showed the highest in age 9, but decreased with age towards 12 years old. We found significant correlation between the seroprevalence and annual incidence in the investigated areas, suggesting that the area-related patterns reflected the differences of inoculation rates in children.     

A new survey of the serology of human Trypanosoma cruzi infection in the Rio Negro microregion,Brazilian Amazon: a critical analysis

The serology of human Trypanosoma cruzi infection in the Rio Negro microregion is very complex because of the large numbers of false-positive cases that result from low antibody titres and cross-reactions with other infections. In the present study, we collected 4,880 blood samples on filter paper; of these, indirect immunofluorescence (IIF) was strongly reactive in 221 (4.5%), which were considered to be positive (IIF strongly reactive; high intensity of fluorescence) and weakly reactive in 302 (6.2%), which were considered to be doubtful (IIF weakly reactive; low intensity of fluorescence). The confirmatory test on the serum using at least two of three techniques (IIF, conventional ELISA and recombinant ELISA) on 137 samples that were positive in the screening test only confirmed 33 cases (24.1%). Of the 178 samples that were considered doubtful in the screening test, only 10 (5.6%) were considered to be positive in the confirmatory test. Finally, we recommend that the serological diagnosis of T. cruzi infection in the Amazon region be made using at least two different techniques, for example immunofluorescence and ELISA and confirmed by Western blot analysis when possible.     

Comparison of immunological responses to the various types circumsporozoite proteins of Plasmodium vivax in malaria patients of Korea

We compared the seroreactivities against four synthetic peptide antigens (VK210, VK247, Korean type 1, and type 2) and a full length recombinant circumsporozoite protein (CSP) antigen of Plasmodium vivax (P. vivax ) in samples of sixty-three tertian malaria patients in Korea. Among the various CSP antigens, the full-length recombinant CSP showed the highest reactivity in malaria-exposed groups (85.7%, 54/63). No significant difference was found in the percentage of malaria patients with antibodies among four peptides examined, except a full-length recombinant CSP. Absorbance values from the peptide-based ELISA showed high correlations (r > 0.9, P < 0.05) at significant values. Five sera without the immunoaffinity against peptides were reactive towards the full-length recombinant CSP in ELISA. Sera, which were not reactive to a full length recombinant CSP antigen, were not recognized by any of peptide based ELISA. These data suggested that peptide structures included in Korean isolates, GNGAGGQAA, and VK247 peptides had immune reactivity and recognition epitopes. Among the antigens, GNGAGGQAA was less recognized by patients exposed to Korean strains of P. vivax in comparison to the VK210 structures.     

Malaria seroepidemiology: comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates.

Blood sampling on filter paper is a current practice in malaria seroepidemiological studies by indirect fluorescent antibody test (IFAT). There is, however, scant comparative information about the use of bloodspot eluates for detection of malarial IgG antibodies simultaneously by IFAT and enzyme immunoassay (ELISA). Here we report data obtained by both serological methods done on 219 bloodspot eluate samples collected in a rural community in Brazilian Amazon Basin (Alto Paraíso, Ariquemes municipality) where malaria is endemic. Plasmodium falciparum and P. vivax thick smear antigens were used in the IFAT; a detergent-soluble P. falciparum antigen was prepared for ELISA. Substantial agreement of results (Kappa coefficient k = 0.686) was observed when P. falciparum antigen was used in both tests, and IFAT titers were found to be strongly correlated to ELISA antibody units (Spearman correlation coefficient rs = 0.818, p < 0.001). Only moderate agreement (k = 0.467) between IFAT with P. vivax antigen and ELISA with P. falciparum antigen was observed. Spearman correlation coefficient value between quantitative results (IFAT titers and ELISA antibody units) in this case was numerically lower (rs = 0.540, p < 0.0001). Our results suggest that, with P. falciparum antigen, both IFAT and ELISA performed on bloodspot eluates are equivalent for seroepidemiological purposes.     

ELISA detection of vivax malaria with recombinant multiple stage-specific antigens and its application to survey of residents in endemic areas

An ELISA was developed for the diagnosis of vivax malaria using multiple stage-specific recombinant antigens of Plasmodium vivax. The DNA from the whole blood of a malaria patient was used as template to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1). Each amplified DNA fragment was inserted into pQE30 plasmid to induce the expression of His-tagged protein in Escherichia coli (M15 strain) by IPTG. His-tagged proteins were purified by Ni-NTA metal-affinity chromatography and used as antigens for ELISA with patient sera that were confirmed previously by blood smear examinations. When applied to patient sera, 122 (80.3%) out of 152 vivax malaria cases reacted to at least one antigen, while no reactions were observed with 128 uninfected serum samples. We applied this ELISA to the screening of 3,262 civilian residents in endemic regions near the DMZ, which resulted in 236 positively detected (7.2%) cases. This method can be applied to serological diagnosis and mass screening in endemic regions, or can be used as a safety test for transfusion blood in endemic areas.     

Estimating the malaria transmission of Plasmodium vivax based on serodiagnosis

ABSTRACT: BACKGROUND: Plasmodium vivax re-emerged in 1993 and has now become a major public health problem during the summer season in South Korea. The aim of this study was to interpret and understand the meaning of seroepidemiological studies for developing the best malaria control programme in South Korea. METHODS: Blood samples were collected in Gimpo city, Paju city, Yeoncheon County, Cheorwon County and Goseong County of high risk area in South Korea. Microscopy was performed to identify patients infected with P. vivax. Antibody detection for P. vivax was performed using indirect fluorescent antibody test (IFAT). RESULTS: A total of 1,574 blood samples was collected from participants in the study areas and evaluated against three parameters: IFAT positive rate, annual antibody positive index (AAPI), and annual parasite index (API). The IFAT positive rate was 7.24% (n = 114). Of the five study areas, Gimpo had the highest IFAT positive rate (13.68%) and AAPI (4.63). Yeongcheon had the highest API in 2005 (2.06) while Gimpo had the highest API in 2006 (5.00). No correlation was observed between any of the three parameters and study sites' distance from the demilitarized zone (DMZ). CONCLUSIONS: These results showed that P. vivax antibody levels could provide useful information about the prevalence of malaria in endemic areas. Furthermore, AAPI results for each year showed a closer relationship to API the following year than the API of the same year and thus could be helpful in predicting malaria transmission risks.     

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.     

Susceptibility of Anopheles aquasalis and an. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F3 generation) and An. darlingi (F1 generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.     

Evaluation of the efficacy of chloroquine chemoprophylaxis for vivax malaria among Republic of Korea military personnel

Chloroquine has been used massively for vivax malaria prophylaxis and treatment in the Republic of Korea (ROK) military personnel from 1997. Although prophylaxis is generally regarded as successful among ROK military, prophylaxis failure has been repeatedly reported. Before the prophylaxis program was started on July 4th 2011, which was completed on October 16th 2011, by the ROK military, more than 60% of malaria cases were attributed to new infection or long-latency relapse. During the prophylaxis program, the authors re-examined the efficiency of chloroquine chemoprophylaxis in ROK military during the last 6 months of 2011 by measuring compliance and whole blood chloroquine levels in 41 malaria patients immediately before instituting antimalarial therapy between July and December. Three patients (7.3%) showed good compliance, and had whole blood total chloroquine levels above the minimally inhibitory concentration (100 ng/mL). However, 28 (69.3%) of these 41 patients when admitted to hospital showed poor or no compliance with prophylaxis; 4 of the 28 (14.3%) were stationed outside the mass prophylaxis region, and 5 (17.9%) subjects were infected after the prophylaxis program had finished. These findings indicate that the current malaria control program should be carefully reconsidered, in terms of, individual instruction, current chemoprophylaxis program regimens, and schedules to improve the efficacy of prophylaxis in the ROK military.     

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.     

The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia

ABSTRACT: BACKGROUND: Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island's readiness to begin the elimination process. METHODS: The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households. Results and Discussion Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 - 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 - 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance. CONCLUSION: Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages.     

A dot-blot ELISA comparable to immunoblot for the specific diagnosis of human parastrongyliasis

A dot-blot enzyme-linked immunosorbent assay (dot-blot ELISA) using an electroeluted 31-kDa glycoprotein from adult worms of Parastrongylus cantonensis as the specific antigen was evaluated for the immunological diagnosis of patients infected with P. cantonensis. The sensitivity and specificity for the detection of serum antibody to P. cantonensis in dot-blot ELISA were both 100%, as determined with serum samples of ten P. cantonensis-infected patients, 60 patients with other related parasitic infections, and 20 uninfected controls. The test was as sensitive and specific as the immunoblot test which revealed a reactive band of 31 kDa. Both the dot-blot ELISA and immunoblot detected all sera from ten P. cantonensis-infected individuals, but not with those of other heterologous parasitoses (gnathostomiasis, toxocariasis, filariasis, paragonimiasis, cysticercosis and malaria) or sera from healthy controls. The dot-blot ELISA is much simpler to perform than the immunoblot technique, and the test can be applied under field conditions where sophisticated facilities are lacking.     

Search for malaria parasites by PCR and Southern blot in patients with imported malaria in Italy

The present study evaluates the sensitivity, specificity and usefulness of a PCR method with Southern blot hybridization to detect malaria parasites in blood samples from subjects with a suspect clinical diagnosis of malaria imported to Italy. Plasmodia were detected by PCR using a genus-specific primer-set corresponding to the sequences common to P. falciparum, P. vivax, P. malariae and P. ovale, as described by Arai (Arai et al., Nucleosides Nucleotides, 1994, 13, 1363-1364) and Kimura (Kimura et al., Journal of Clinical Microbiology, 1995, 33, 2342-2346). In addition, four distinct tandemly repetitive species-specific probes, described by Kawai (Kawai et al., Analytical Biochimestry, 1993, 209, 63-69), were synthesized to specifically detect the four malaria parasites species by Southern blot hybridization. Fifteen blood samples from 12 patients (7 with malaria) were tested and the genus-specific PCR method showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy, in detecting malaria parasites in the tested blood samples. Fourteen samples (nine were positive and five negative by PCR) were confirmed by Southern blot, whereas only one P. vivax positive sample was not hybridized with the species-specific probes. We conclude that this PCR method with Southern blot hybridization may be useful in detecting malaria parasites in patients with malaria imported to Italy.     

Multiplicity and diversity of Plasmodium vivax infections in a highly endemic region in Papua New Guinea

Plasmodium vivax is highly endemic in the lowlands of Papua New Guinea and accounts for a large proportion of the malaria cases in children less than 5 years of age. We collected 2117 blood samples at 2-monthly intervals from a cohort of 268 children aged 1 to 4.5 years and estimated the diversity and multiplicity of P. vivax infection. All P. vivax clones were genotyped using the merozoite surface protein 1 F3 fragment (msp1F3) and the microsatellite MS16 as molecular markers. High diversity was observed with msp1F3 (H(E) = 88.1%) and MS16 (H(E) = 97.8%). Of the 1162 P. vivax positive samples, 74% harbored multi-clone infections with a mean multiplicity of 2.7 (IQR = 1-3). The multiplicity of P. vivax infection increased slightly with age (P = 0.02), with the strongest increase in very young children. Intensified efforts to control malaria can benefit from knowledge of the diversity and MOI both for assessing the endemic situation and monitoring the effects of interventions.