Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea.

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.

Evaluation of four rapid diagnostic tests for the diagnosis of Plasmodium vivax in Korea.

OBJECTIVE: To evaluate 4 rapid malaria diagnostic kits (RDTs) in Korea: OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, Humasis Malaria P.f/Pan antigen test and CareStart Malaria Pf/Pv Combo test. METHODS: Hundred malaria patients with Plasmodium vivax (P. vivax) and 100 healthy volunteers were recruited. The results from earlier four RDTs were compared with the reference standard, the Giemsa-stained traditional microscopic diagnosis. RESULTS: Compared with the reference standard, the sensitivity and specificity for Plasmodium vivax were 92.7 and 100% for SD BIOLINE Malaria Ag P.f/Pan; and 94.6% and 100% for OptiMAL; 95.5% and 100% for both Humasis Malaria P.f/Pan antigen test and CareStart Malaria Pf/Pv Combo test. CONCLUSION: The performances of all four malaria RDT kits were acceptable, although Humasis Malaria P.f/Pan antigen test and CareStart Malaria Pf/Pv Combo test gave superior performances with ROK isolates.

Genetic variability in Plasmodium vivax aldolase gene in Korean isolates and the sensitivity of the Binax Now malaria test.

Introduction of rapid malaria diagnostic tests (RDT) initiated numerous field evaluations in various epidemiologic settings. But the efficiency of some RTD kits based on aldolase raised reservations for direct implementation of RDT into clinical settings. We performed Binax Now malaria test in 84 Korean Plasmodium vivax isolates and compared it with the traditional Giemsa stain microscopy test as the reference standard. The sensitivity of Binax Now was 62.0% for P. vivax cases (52/84, 95% CI 51.2-71.6%) with 100.0% specificity (50/50, 95% confidence interval 92.9-100%). After the aldolase gene sequence analysis of 84 isolates, two synonymous mutations in aldolase gene were identified in both Binax Now positive and negative samples. No significant association between the mutations and Binax Now malaria tests was found. Thus, the genetic variability would not explain the poor performance of P. vivax RDTs by detecting aldolase in ROK isolates.

Emergence of new alleles of the MSP-3alpha gene in Plasmodium vivax isolates from Korea.

Nucleotide sequence analysis of the Plasmodium vivax PvMSP-3alpha gene was conducted on blood from 143 malaria patients admitted to Korea University Medical Center from 1996 to 2007 in the Republic of Korea (ROK). From 1996 to 2002, the PvMSP-3alpha alleles were of two types, SKOR-67 (2.53 kb) and SKOR-69 (1.78 kb), which differed in length and amino acid sequence. Two new variants with similar size to SKOR-67 were first observed in 2002 and in 2006-2007 accounted for nearly 50% (25/51) of the sampled isolates. The new variants had the same amino acid sequence as SKOR-69 in the N-terminal region, but in Blocks I and II and in the C-terminal region, they were similar to previously reported isolates from Thailand, Papua New Guinea, India, Brazil, and Ecuador strains.

Absolute CD4+ cell count using a plastic microchip and a microscopic cell counter.

We have designed and evaluated the performance of a simple, rapid, and affordable method for counting CD4(+) T-cells with the use of plastic microchips. This new system is an adaptation of a "no-lyse, no-wash," volumetric single platform assay, and absolute CD4(+) counts are determined with the use of a microscopic scanning cell counter. To assess the CD4(+) count test precision and linearity of the system, measured CD4(+) counts were compared with two other reference assays (single and dual platform flow cytometry) with the use of 123 clinical samples including samples obtained from 35 HIV-infected patients, and artificially diluted samples. A correlation between the results from the use of the new method and from the use of the two other reference assays was r = 0.98 for the clinical samples. A dilution test of the new method demonstrated a linearity of r >or= 0.99, with coefficients of variation

Gamma-irradiation enhances RECK protein levels in Panc-1 pancreatic cancer cells.

Radiotherapy is an important treatment for many malignant tumors, but there are recent reports that radiation may increase the malignancy of cancer cells by stimulating expression of type IV collagenases. In this study, we examined changes in matrix metalloproteinase (MMP) inhibitors, such as the tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and RECK, in response to irradiation in Panc-1 pancreatic cancer cells. Irradiation increased RECK protein levels but not mRNA levels, whereas no significant changes were found in TIMP-1 and TIMP-2. The enhanced RECK protein levels were associated with an increase in MMP inhibitory activity. However, irradiation slightly but reproducibly increased the invasiveness of the Panc-1 cells. Like irradiation, treatment of Panc-1 cells with transforming growth factor (TGF)-Beta1 led to a 2-fold increase in RECK protein levels. Transient transfection with Smad3 also increased RECK protein levels, but transfection with Smad7 markedly reduced them. Stable expression of Smad7 and treatment with SB431542, an inhibitor of TGF-Beta receptor I kinase, abolished TGF-Beta1- and radiation-mediated effects on RECK. Furthermore, irradiation increased levels of phosphorylated Smad3. We conclude that radiation post-transciptionally enhances RECK protein levels in Panc-1 cells, at least in part, via TGF-Beta signaling, and that irradiation increases Panc-1 invasiveness via a mechanism that may not be linked to MMP-2 activity.

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening.

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

Alteration of platelet counts and lipid profiles after treatment of acute Plasmodium vivax.

During malaria infections, thrombocytopenia and low cholesterol levels are frequently observed changes. We compared these changes in patients admitted with fevers and infected with Plasmodium vivax, patients admitted with fevers with respiratory/urinary infections and afebrile normal (control) non-infected volunteers. Changes in the platelet count and lipid parameters are reported for malaria patients after treatment with hydroxychloroquine and primaquine for acute P. vivax malaria. Of a total 141 participants, 55 patients were diagnosed with malaria (positive blood smear) prior to treatment. Compared to the normal (n=52) and non-malaria fever groups (n=34), there was a significant decrease in five hematologic indices (white blood cell, red blood cell, hemoglobin, hematocrit and platelet) and three lipid parameters (total cholesterol, HDL-c and LDL-c) in the vivax malaria group at day 0 (pre-treatment). Following treatment, the platelet counts returned to normal limits (P<0.05) from 91,058/microL on day 0 to 246,833/microL by day 17 after treatment. However, changes in the lipid parameters of malaria patients showed a slow recovery to normal limits compared to the platelet counts. The HDL-c and LDL-c remained low for 1 month after treatment but increased at 3 and 6 months post-treatment. At 12 months after treatment, the levels of two lipid parameters had fully recovered to the normal limits. Thus, special attention should be applied when interpreting laboratory blood profiles of malaria patients, especially platelet and lipid based tests, until full recovery after treatment.

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0 percent and a clinical specificity of 94.0 percent for P.vivax. Twenty out of 325 domestic travelers (6.2 percent) were reactive and 28 cases (8.6 percent) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.