An Evaluation of Active Case Detection in Malaria Control Program in Kiyuni Parish of Kyankwanzi District, Uganda.

Malaria remains one of the leading health burdens in the developing world, especially in several sub-Saharan Africa countries; and Uganda has some of the highest recorded measures of malaria transmission intensity in the world. It is evident that the prevalence of malaria infection, the incidence of disease, and mortality from severe malaria remain very high in Uganda. Although the recent stable political and economic situation in the last few decades in Uganda supported for a fairly good appreciation of malaria control, the declines in infection, morbidity, and mortality are not sufficient to interrupt transmission and this country is among the top 4 countries with cases of malaria, especially among children under 5 years of age. In fact, Uganda, which is endemic in over 95% of the country, is a representative of challenges facing malaria control in Africa. In this study, we evaluated an active case detection program in 6 randomly selected villages, Uganda. This program covered a potential target population of 5,017 individuals. Our team screened 12,257 samples of malaria by active case detection, every 4 months, from February 2015 to January 2017 in the 6 villages (a total of 6 times). This study assessed the perceptions and practices on malaria control in Kiyuni Parish of Kyankwanzi district, Uganda. Our study presents that the incidence of malaria is sustained high despite efforts to scale-up and improve the use of LLINs and access to ACDs, based on the average incidence confirmed by RDTs.

Monitoring of Noxious Protozoa for Management of Natural Water Resources.

Waterborne parasitic protozoa, particularly Giardia lamblia and Cryptosporidium spp., are common causes of diarrhea and gastroenteritis worldwide. The most frequently identified source of infestation is water, and exposure involves either drinking water or recreation in swimming pools or natural bodies of water. In practice, studies on Cryptosporidium oocysts and Giardia cysts in surface water are challenging owing to the low concentrations of these microorganisms because of dilution. In this study, a 3-year monitoring of Cryptosporidium parvum, Giardia lamblia, and Naegleria fowleri was conducted from August 2014 to June 2016 at 5 surface water sites including 2 lakes, 1 river, and 2 water intake plants. A total of 50 water samples of 40 L were examined. Cryptosporidium oocysts were detected in 22% of samples and Giardia cysts in 32%. Water at the 5 sampling sites was all contaminated with Cryptosporidium oocysts (0-36/L), Giardia cysts (0-39/L), or both. The geometric mean concentrations of Cryptosporidium and Giardia were 1.14 oocysts/L and 4.62 cysts/L, respectively. Thus, effective monitoring plans must take into account the spatial and temporal parameters of contamination because they affect the prevalence and distribution of these protozoan cysts in local water resources.

Comparison of the diagnostic performance of microscopic examination with nested polymerase chain reaction for optimum malaria diagnosis in Upper Myanmar.

BACKGROUND: Accurate diagnosis of Plasmodium infection is crucial for prompt malaria treatment and surveillance. Microscopic examination has been widely applied as the gold standard for malaria diagnosis in most part of malaria endemic areas, but its diagnostic value has been questioned, particularly in submicroscopic malaria. In this study, the diagnostic performance of microscopic examination and nested polymerase chain reaction (PCR) was evaluated to establish optimal malaria diagnosis method in Myanmar. METHODS: A total of 1125 blood samples collected from residents in the villages and towns located in Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships and Mandalay of Upper Myanmar were screened by microscopic examination and species-specific nested PCR method. RESULTS: Among the 1125 blood samples, 261 samples were confirmed to be infected with malaria by microscopic examination. Evaluation of the 1125 samples by species-specific nested PCR analysis revealed that the agreement between microscopic examination and nested PCR was 87.3% (261/299). Nested PCR successfully detected 38 Plasmodium falciparum or Plasmodium vivax infections, which were missed in microscopic examination. Microscopic examinations also either misdiagnosed the infected Plasmodium species, or did not detect mixed infections with different Plasmodium species in 31 cases. CONCLUSIONS: The nested PCR method is more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections. Given the observed higher sensitivity and specificity of nested PCR, the molecular method holds enormous promise in malaria diagnosis and species differentiation, and can be applied as an effective monitoring tool for malaria surveillance, control and elimination in Myanmar.

Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 µM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 µM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

Population genetic structure and natural selection of apical membrane antigen-1 in Plasmodium vivax Korean isolates.

BACKGROUND: Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a leading candidate antigen for blood stage malaria vaccine. However, antigenic variation is a major obstacle in the development of an effective vaccine based on this antigen. In this study, the genetic structure and the effect of natural selection of PvAMA-1 among Korean P. vivax isolates were analysed. METHODS: Blood samples were collected from 66 Korean patients with vivax malaria. The entire PvAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvAMA-1 sequence of each isolate was sequenced and the polymorphic characteristics and effect of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Thirty haplotypes of PvAMA-1, which were further classified into seven different clusters, were identified in the 66 Korean P. vivax isolates. Domain II was highly conserved among the sequences, but substantial nucleotide diversity was observed in domains I and III. The difference between the rates of non-synonymous and synonymous mutations suggested that the gene has evolved under natural selection. No strong evidence indicating balancing or positive selection on PvAMA-1 was identified. Recombination may also play a role in the resulting genetic diversity of PvAMA-1. CONCLUSIONS: This study is the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Korea. Korean PvAMA-1 had limited genetic diversity compared to PvAMA-1 in global isolates. The overall pattern of genetic polymorphism of Korean PvAMA-1 differed from other global isolates and novel amino acid changes were also identified in Korean PvAMA-1. Evidences for natural selection and recombination event were observed, which is likely to play an important role in generating genetic diversity across the PvAMA-1. These results provide useful information for the understanding the population structure of P. vivax circulating in Korea and have important implications for the design of a vaccine incorporating PvAMA-1.

Polymorphic patterns of the merozoite surface protein-3ß in Korean isolates of Plasmodium vivax.

BACKGROUND: The merozoite surface protein-3ß of Plasmodium vivax (PvMSP-3ß) is one of the candidate antigens for blood stage malaria vaccine development. The polymorphisms in PvMSP-3ß have been reported in certain P. vivax isolates. However, the diversity of PvMSP-3ß throughout its global distribution has not been well understood. In this study, the genetic diversity and the effects of natural selection in PvMSP-3ß among P. vivax Korean isolates were analysed. METHODS: Blood samples were collected from 95 patients with vivax malaria in Korea. The region flanking full-length PvMSP-3ß was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvMSP-3ß sequence of each isolate was determined and the polymorphic characteristics and effects of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Five different subtypes of PvMSP-3ß were identified based on single nucleotide polymorphisms (SNPs), insertions, and deletions. Although a high level of sequence diversity was observed in the PvMSP-3ß gene, the coiled-coil tertiary structure of the PvMSP-3ß protein was well conserved in all of the sequences. The PvMSP-3ß of Korean isolates is under natural selection. DNA polymerase slippage and intragenic recombination likely contributed to PvMSP-3ß diversity in Korean P. vivax isolates. CONCLUSIONS: The PvMSP-3ß of Korean P. vivax isolates displayed polymorphisms, with SNPs, insertions and deletions scattered throughout of the gene. These results of parasite heterogeneity are relevant to the development of a PvMSP-3ß based vaccine against P. vivax and the implementation of malaria control programmes in Korea.

Molecular characterization of voltage-gated calcium channel ß-subunits of Clonorchis sinensis.

The voltage-gated Ca(2+) channel ß-subunit is a member of the membrane-associated guanylate kinase family and modulates kinetic properties of the Ca(2+) channels, such as their voltage-dependent activation and inactivation rates. Two cDNA clones were identified to encode each ß-subunit isotype of the voltage-gated Ca(2+) channel of Clonorchis sinensis, CsCavß1 and CsCavß2, which consist of 606 and 887 amino acids, respectively. CsCavß1 was found to be similar to the ß-subunit containing two conserved serine residues that constitute the consensus protein kinase C phosphorylation site in the ß-interaction domain (BID). CsCavß2 had cysteine and alanine residues instead of the two serine residues conserved in BID and was homologous to variant ß-subunit of Schistosoma mansoni and Schistosoma japonicum. CsCavß1 and CsCavß2 were almost equally expressed in the adults and metacercariae, but were more expressed in adult C. sinensis than in metacercariae. Collectively, our findings suggest that substitution of the two serine residues in BID of CsCavß2 may render C. sinensis sensitive to praziquantel.

Probability of antibody formation against circumsporozoite protein of Plasmodium vivax among Korean malaria patients.

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.

Evaluation of the accuracy of the EasyTest™ malaria Pf/Pan Ag, a rapid diagnostic test, in Uganda.

In recent years, rapid diagnostic tests (RDTs) have been widely used for malaria detection, primarily because of their simple operation, fast results, and straightforward interpretation. The Asan EasyTest™ Malaria Pf/Pan Ag is one of the most commonly used malaria RDTs in several countries, including Korea and India. In this study, we tested the diagnostic performance of this RDT in Uganda to evaluate its usefulness for field diagnosis of malaria in this country. Microscopic and PCR analyses, and the Asan EasyTest™ Malaria Pf/Pan Ag rapid diagnostic test, were performed on blood samples from 185 individuals with suspected malaria in several villages in Uganda. Compared to the microscopic analysis, the sensitivity of the RDT to detect malaria infection was 95.8% and 83.3% for Plasmodium falciparum and non-P. falciparum, respectively. Although the diagnostic sensitivity of the RDT decreased when parasitemia was ≤500 parasites/µl, it showed 96.8% sensitivity (98.4% for P. falciparum and 93.8% for non-P. falciparum) in blood samples with parasitemia ≥100 parasites/µl. The specificity of the RDT was 97.3% for P. falciparum and 97.3% for non-P. falciparum. These results collectively suggest that the accuracy of the Asan EasyTest™ Malaria Pf/Pan Ag makes it an effective point-of-care diagnostic tool for malaria in Uganda.

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.

Limited sequence polymorphisms of four transmission-blocking vaccine candidate antigens in Plasmodium vivax Korean isolates.

BACKGROUND: Transmission-blocking vaccines (TBVs), which target the sexual stages of malaria parasites to interfere with and/or inhibit the parasite's development within mosquitoes, have been regarded as promising targets for disrupting the malaria transmission cycle. In this study, genetic diversity of four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, among Plasmodium vivax Korean isolates was analysed. METHODS: A total of 86 P. vivax-infected blood samples collected from patients in Korea were used for analyses. Each of the full-length genes encoding four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, were amplified by PCR, cloned into T&A vector, and then sequenced. Polymorphic characteristics of the genes were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Polymorphism analyses of the 86 Korean P. vivax isolates revealed two distinct haplotypes in Pvs25 and Pvs48/45, and three different haplotypes in PvWARP. In contrast, Pvs28 showed only a single haplotype. Most of the nucleotide substitutions and amino acid changes identified in all four TBV candidate antigens were commonly found in P. vivax isolates from other geographic areas. The overall nucleotide diversities of the TBV candidates were much lower than those of blood stage antigens. CONCLUSIONS: Limited sequence polymorphisms of TBV candidate antigens were identified in the Korean P. vivax population. These results provide baseline information for developing an effective TBV based on these antigens, and offer great promise for applications of a TBV against P. vivax infection in regions where the parasite is most prevalent.

Evaluation of circumsporozoite protein of Plasmodium vivax to estimate its prevalence in the Republic of Korea: an observational study of incidence.

BACKGROUND: Plasmodium vivax re-emerged in 1993. Although the number of infections has been steadily decreasing, it is likely to continue to affect public health until it is eradicated. The aim of this study is to measure anti-circumsporozoite protein (CSP) antibody and compare malaria prevalence. As to understand the prevalence, an epidemiology study has to be conducted in the Republic of Korea. METHODS: A total of 1,825 and 1,959 blood samples were collected in 2010 and 2011, respectively, from the inhabitants of Ganghwa and Cheorwon counties. The antibody titers of the inhabitants were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant protein purified from Escherichia coli transformed with a CSP gene-inserted pET-28a(+) expression vector. Microscopic examination was performed to identify malaria parasites. RESULTS: The annual parasite incidence (API) in Ganghwa decreased from 4.28 in 2010 to 2.23 in 2011, and that in Cheorwon decreased from 1.88 in 2010 to 1.15 in 2011. The antibody-positive CSP rate in these areas also decreased from 18.14% (331/1825) in 2010 to 15.36% (301/1959) in 2011. Pearson analysis showed a strong correlation between the API and the antibody-positive CSP rate in these areas (r = 1.000, P < 0.01). The intensity of the immune responses of the inhabitants of Cheorwon, as measured by the mean optical density, decreased from 0.9186 ± 0.0472 in 2010 to 0.7035 ± 0.0457 in 2011 (P = 0.034), but increased in Ganghwa from 0.7649 ± 0.0192 in 2010 to 0.8237 ± 0.1970 in 2011 (P = 0.006). The immune response increased according to age (r = 0.686, P = 0.041). CONCLUSIONS: The positive CSP-ELISA rate was closely related to the API in the study areas. This suggests that seroepidemiological studies based on CSP-ELISA may be helpful in estimating the malaria prevalence. Moreover, such studies can be used to establish and evaluate malaria control and eradication programmes in high-risk areas in Korea.

A surge in the seroprevalence of toxoplasmosis among the residents of islands in Gangwha-gun, Incheon, Korea.

Seroepidemiological changes of Toxoplasma gondii infection among the residents of the islands of Gangwha-gun, Incheon for 2 years were surveyed and evaluated by ELISA using a crude extract antigen. In 2010, sera of 919 adult residents in Gyodong-myeon and 313 adults in Samsan-myeon were collected and checked for IgG antibody titers, which showed 14.5% (133 sera) and 19.8% (62 sera) positive rates, respectively. In 2011, sera of 955 adults in Gyodong-myeon and 341 adults in Samsan-myeon were examined, which showed an increase of positive rates to 23.8% (227 sera) and 31.7% (108 sera), respectively. Totally, the seroprevalence of the first year was 15.8% and it increased rapidly to 25.8% in the second year. The positive rates of both sexes increased simultaneously with the significant ratio of males to females by 1.7-2.2 fold (P<0.05). In both myeons, 661 sera were collected every year and showed changes in optical density (OD) in 177 sera; newly found as positives in 73 persons (11.0%), negative conversion in 10 persons (1.5%), and maintained or increased in 94 persons (14.2%). This rapid increase in the prevalence of toxoplasmosis in Gangwha islands may be due to in part peculiar changes in the toxoplasmic environment of the islands and presumably the consumption of the pork bred domestically within the islands or imported from high endemic nations. It is necessary to find out symptomatic toxoplasmic patients and confirm the risk factors for further infection in the islands of Gangwha-gun.

Seroprevalence of toxoplasmosis in the residents of Cheorwon-gun, Gangwon-do, Korea.

The seroepidemiological status of toxoplasmosis was surveyed among the residents of Cheorwon-gun, Gangwon-do by means of ELISA using a crude extract antigen of Toxoplasma gondii. The sera of 1,661 adult residents (866 males and 795 females) were collected and checked for IgG antibody titers, which showed 17.0% positive rate (282 sera). The positive rate was significantly different between the sex; 20.6% for males and 13.1% for females (P<0.05). The positive rates were higher in fifties of males (28.7%) and forties of females (20.0%). This positive rate of toxoplasmosis in Cheorwon-gun residents is regarded as the highest among the surveys of different geographical regions of Korea. This high positive rate may due in part to peculiar geographical locality of the surveyed area near the naturally well preserved demilitarized zone (DMZ) or presumably consumption of the pork imported from high endemic nations. Therefore, it is necessary to study further the epidemiology of toxoplasmosis in Cheorwon-gun.

Phragmites australis (Cav.) Trin. ex Steud. Extract Induces Apoptosis-like Programmed Cell Death in Acanthamoeba castellanii Trophozoites.

Acanthamoeba keratitis (AK) is an infectious ocular disease which is difficult to diagnose correctly and cure. Development of an effective and safe therapeutic drug for AK is needed. Our preliminary screening of more than 200 extracts from wild plants collected in Korea suggested the potential amoebicidal activity of Phragmites australis (Cav.) Trin. ex Steud. extract (PAE) against Acanthamoeba species. Here, we aimed to analyze the amoebicidal activity of PAE on Acanthamoeba and its underlying amoebicidal mechanism. PAE induced amoebicidal activity against both A. castellanii and A. polyphaga trophozoites, while it showed low cytotoxicity in human corneal epithelial cells (HCE-2) and human retinal pigment epithelial cells (ARPE-19). Transmission electron microscopy analysis showed subcellular morphological changes, such as increased granules, abnormal mitochondria, and atypical cyst wall formation, in the PAE-treated A. castellanii. Fluorometric apoptosis assay and TUNEL assay revealed apoptosis-like programmed cell death (PCD) in the PAE-treated A. castellanii. The PAE treatment increased reactive oxygen species production and reduced mitochondrial membrane potential in the amoeba. The enhanced expression of autophagy-associated genes was also detected. These results suggested that PAE exerted a promising amoebicidal effect on A. castellanii trophozoites via the PCD pathway. PAE could be a potential candidate for developing a therapeutic drug for AK.

Multiple recombinant antigens of Clonorchis sinensis for serodiagnosis of human clonorchiasis.

Antigenic proteins from Clonorchis sinensis have been previously purified and evaluated for their antigenicity to enable the serodiagnosis of clonorchiasis. Though they were of high specificity, molecularly defined proteins were reported to be less sensitive as single antigens than crude antigen. To resolve this issue, 11 clones were selected by immunoscreening an adult C. sinensis cDNA library using infected human sera. Mixed antigens were prepared using recombinant proteins of positive clones and investigated for antigenicity by immunoblotting against C. sinensis- and helminth-infected patient sera. A mixed antigen of recombinant 28 and 26 kDa glutathion S-transferases (Cs28GST and Cs26GST) produced 76% sensitivity and 95% specificity. Furthermore, a triple mix of recombinant Cs26GST and Cs28GST with vitelline precursor protein pushed up the sensitivity to 87% and maintained specificity at 95%. It is proposed that multiple antigen mixes should be further studied to develop rapid serodiagnostic test kits for the serodiagnosis of human clonorchiasis.

Expression of exogenous human hepatic nuclear factor-1α by a lentiviral vector and its interactions with Plasmodium falciparum subtilisin-like protease 2.

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors as well as by individual host responses to these determinants. In both humans and mice, liver injury follows parasite entry, persisting to the erythrocytic stage in the case of infection with the fatal strain of Plasmodium falciparum. Hepatic nuclear factor (HNF)-1α is a master regulator of not only the liver damage and adaptive responses but also diverse metabolic functions. In this study, we analyzed the expression of host HNF-1α in relation to malaria infection and evaluated its interaction with the 5'-untranslated region of subtilisin-like protease 2 (subtilase, Sub2). Recombinant human HNF-1α expressed by a lentiviral vector (LV HNF-1α) was introduced into mice. Interestingly, differences in the activity of the 5'-untranslated region of the Pf-Sub2 promoter were detected in 293T cells, and LV HNF-1α was observed to influence promoter activity, suggesting that host HNF-1α interacts with the Sub2 gene.

Confocal microscopic findings of cysteine protease calpain in Plasmodium falciparum.

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.

Anthelmintic activity of KSI-4088 against Caenorhabditis elegans.

Anthelmintic resistance is a serious global problem because of the worldwide spread of resistant nematodes in animals and humans. This has triggered increasing investment in research for new anthelmintics. Over the past decade, Caenorhabditis elegans has become a popular model organism for parasitic nematode research, and many examples have been published to illustrate its use. In this study, we investigated the effect of KSI-4088 on the egg hatching, larval development, and migration of the nematode worm C. elegans compared with ivermectin and levamisole (well-known anthelmintic drugs). KSI-4088 demonstrated anthelmintic activity on all assays of C. elegans. The anthelmintic activity of KSI-4088 on egg hatching and larval development showed especially strong activity, but assays showed that ivermectin and levamisole had no effects on C. elegans. In addition, KSI-4088 was capable of producing a change in the timing of the development of the worms at the L1-L3 and L4 stage. Also, we demonstrate that C. elegans L3-4 are more sensitive than adults to KSI-4088 in assay of migration. Our results indicate that KSI-4088 is an active anthelmintic compound that should be further investigated with the aim of developing a potent drug against nematodes.

Transcriptional activity of Plasmodium subtilisin-like protease 2 (Pf-Sub2) 5'untranslated regions and its interaction with hepatocyte growth factor.

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.