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Abstract Background Plasmodium falciparum merozoite surface proteins 1 (PfMSP1) and 2 (PfMSP2) are potential candidates for malaria vaccine development. However, the genetic diversity of these genes in the global P. falciparum population presents a significant challenge in developing an effective vaccine. Hence, understanding the genetic diversity and evolutionary trends in the global P. falciparum population is crucial. Methods This study analyzed the genetic variations and evolutionary changes of pfmsp1 and pfmsp2 in P. falciparum isolates from the Central Highland and South-Central regions of Vietnam. DNASTAR and MEGA7 programs were utilized for analyses. The polymorphic nature of global pfmsp1 and pfmsp2 was also investigated. Results A total of 337 sequences of pfmsp1 and 289 sequences of pfmsp2 were obtained. The pfmsp1 and pfmsp2 from Vietnam revealed a higher degree of genetic homogeneity compared to those from other malaria-endemic countries. Remarkably, the allele diversity patterns of Vietnam pfmsp1 and pfmsp2 differed significantly from those of neighboring countries in the Greater Mekong Subregion. Declines in allele diversity and polymorphic patterns of Vietnam pfmsp1 and pfmsp2 were observed. Conclusions The Vietnam P. falciparum population might be genetically isolated from the parasite populations in other neighboring GMS countries, likely due to geographical barriers and distinct evolutionary pressures. Furthermore, bottleneck effects or selective sweeps may have contributed to the genetic homogeneity of Vietnam pfmsp1 and pfmsp2.

A significant outbreak of quartan malaria caused by Plasmodium malariae has occurred in Khanh Vinh District, Khanh Hoa Province, Vietnam in 2023 and the outbreak persists. In this report, we present the epidemiological and clinical characteristics of this extensive outbreak of quartan malaria in Vietnam.

Abstract Plasmodium falciparum erythrocyte binding antigen 175 (PfEBA-175) plays essential role in erythrocyte invasion by the parasite and is a leading vaccine candidate. However, its genetic diversity in global isolates is a concern in developing an universal vaccine incorporating this protein. This study aimed to investigate genetic polymorphisms and natural selection of pfeba-175 region II (RII) in Myanmar and Vietnam P. falciparum isolates. Vietnam pfeba-175 RII displayed a low genetic polymorphism, while Myanmar pfeba-175 RII showed high levels of genetic diversity across the region. Point mutations, deletion, and recombinations were main factors contributing to genetic diversities in P. falciparum populations. Global pfeba-175 RII revealed similar, but not identical, genetic polymorphisms and natural selection profiles. Despite profiles of amino acid substitutions differed among populations, five major amino acid changes (K279E, E403K, K481I, Q584K, and R664) were commonly detected in global pfeba-175 RII populations. Haplotype network and genetic differentiation analyses of global pfeba-175 RII populations demonstrated no geographical relationships. Non-neglectable level of genetic diversity was observed in global pfeba-175 RII populations, emphasizing the need to consider this when designing an effective vaccine based on this protein. This study underscores the importance of the continuous monitoring of genetic diversity of pfeba-175 RII in the global P. falciparum populations.

Acanthamoeba keratitis (AK) is a sight-threatening and difficult-to-treat ocular infection. The significant side effects of current AK treatments highlight the urgent need to develop a safe and effective AK medication. In this study, the amoebicidal activity of Iris setosa Pall. ex Link extract (ISE) against Acanthamoeba was examined and its specific amoebicidal mechanism was explored. ISE induced significant morphological changes in Acanthamoeba trophozoites and exhibited amoebicidal activity against A. castellanii and A. polyphaga. ISE was further fractionated into five subfractions by sequential extraction with n-hexane, chloroform, ethyl acetate, n-butanol, and water, and their amoebicidal activities and underlying amoebicidal mechanisms were investigated. The n-butanol subfraction of ISE (ISE-BuOH) displayed selective amoebicidal activity against the Acanthamoeba species with minimal cytotoxicity in human corneal cells (HCE-2). ISE-BuOH triggered apoptosis-like programmed cell death (PCD) in amoebae, characterized by DNA fragmentation, increased ROS production, and caspase-3 activity elevation. ISE-BuOH also demonstrated a partial cysticidal effect against the amoeba species. ISE-BuOH could be a promising candidate in the development of therapeutic drugs for AK.

Abstract Background Plasmodium vivax apical membrane antigen-1 (pvama-1) is an important vaccine candidate against Malaria. The genetic composition assessment of pvama-1 from wide-range geography is vital to plan the antigen based vaccine designing against Malaria. Methods The blood samples were collected from 84 P. vivax positive malaria patients from different districts of Khyber Pakhtunkhwa (KP) province of Pakistan. The highly polymorphic and immunogenic domain-I (DI) region of pvama-1 was PCR amplified and DNA sequenced. The QC based sequences raw data filtration was done using DNASTAR package. The downstream population genetic analyses were performed using MEGA4, DnaSP, Arlequin v3.5 and Network.5 resources. Results The analyses unveiled total 57 haplotypes of pvama-1 (DI) in KP samples with majorly prevalent H-14 and H-5 haplotypes. Pairwise comparative population genetics analyses identified limited to moderate genetic distinctions among the samples collected from different districts of KP, Pakistan. In context of worldwide available data, the KP samples depicted major genetic differentiation against the Korean samples with Fst = 0.40915 (P-value = 0.0001), while least distinction was observed against Indian and Iranian samples. The statistically significant negative values of Fu and Li’s D* and F* tests indicate the evidence of population expansion and directional positive selection signature. The slow LD decay across the nucleotide distance in KP isolates indicates low nucleotide diversity. In context of reference pvama-1 sequence, the KP samples were identified to have 09 novel non-synonymous single nucleotide polymorphisms (nsSNPs), including several trimorphic and tetramorphic substitutions. Few of these nsSNPs are mapped within the B-cell predicted epitopic motifs of the pvama-1, and possibly modulate the immune response mechanism. Conclusion Low genetic differentiation was observed across the pvama-1 DI among the P. vivax isolates acquired from widespread regions of KP province of Pakistan. The information may implicate in future vaccine designing strategies based on antigenic features of pvama-1.

Abstract Background Coccidiosis is a poultry disease that occurs worldwide and is caused by Eimeria species. The infection is associated with reduced feed efficiency, body weight gain, and egg production. This study aimed to investigate the current status of coccidiosis and anticoccidial resistance to anticoccidial drugs used as part of control strategies for this disease in Korean chicken farms. Results An overall prevalence of 75% (291/388) was found. Positive farms contained several Eimeria species (mean = 4.2). Of the positive samples, E. acervulina (98.6%), E. maxima (84.8%), and E. tenella (82.8%) were the most prevalent species. Compared with cage-fed chickens, broilers and native chickens reared in free-range management were more at risk of acquiring an Eimeria infection. Sensitivities to six anticoccidial drugs (clopidol, diclazuril, maduramycin, monensin, salinomycin, and toltrazuril) were tested using nine field samples. Compared with untreated healthy control chickens, the body weight gains of infected chickens and treated/infected chickens were significantly reduced in all groups. Fecal oocyst shedding was significantly reduced in four clopidol-treated/infected groups, three diclazuril-treated/infected groups, two toltrazuril-treated/infected groups, one monensin-treated/infected group, and one salinomycin-treated/infected group, compared with the respective untreated/infected control groups. Intestinal lesion scores were also reduced in three clopidol-treated/infected groups, one monensin-treated/infected group, and one toltrazuril-treated/infected group. However, an overall assessment using the anticoccidial index, percent optimum anticoccidial activity, relative oocyst production, and reduced lesion score index found that all field samples had strong resistance to all tested anticoccidial drugs. Conclusion The results of this large-scale epidemiological investigation and anticoccidial sensitivity testing showed a high prevalence of coccidiosis and the presence of severe drug resistant Eimeria species in the field. These findings will be useful for optimizing the control of coccidiosis in the poultry industry.

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Abstract Objective To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION’s operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. Results One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

Abstract Background Rapid and accurate diagnosis of central nervous system (CNS) infections is important, and laboratory tests help diagnose CNS infections. Even when the patient has symptoms, laboratory tests often do not reveal any specific findings. The potential of vitamin D-binding protein (VDBP) to be used as a biomarker for viral and bacterial CNS infections was studied. Methods A total of 302 subjects with suspected CNS infection who underwent lumbar puncture were included. Clinical and laboratory data were collected retrospectively. VDBP levels were measured in the cerebrospinal fluid (CSF) samples. Genotyping for the GC gene encoding VDBP was also performed. VDBP levels were analyzed and compared by CNS infection, pathogen, CSF opening pressure, and GC genotype. Results A CNS infection group (n = 90) and a non-CNS infection group (n = 212) were studied. In terms of its receiver operating characteristic, CSF VDBP showed an area under the curve of 0.726 for the diagnosis of CNS infection. CSF VDBP levels were significantly different between the CNS infection and non-infection groups. The CNS infection group with enterovirus showed a statistically lower distribution of CSF VDBP levels than the other virus groups. The group with CSF opening pressure > 25 cmH2O showed higher CSF VDBP levels than the other groups. There was no significant difference in GC gene allele distribution between the CNS infection and non-infection groups. Conclusions CSF VDBP levels were increased in patients with CNS infection. The CSF VDBP showed potential as a new biomarker for viral and bacterial CNS infections.

Abstract Background Malaria rapid diagnostic tests (RDTs) are precious tools to diagnose malaria. Most RDTs used currently are based on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in a patient’s blood. However, concern has been raised in recent years that deletion of pfhrp2 in the parasite could affect the accuracy of PfHRP2-based RDTs. In addition, genetic variation in pfhrp2 might influence the accuracy and sensitivity of RDTs. In this study, the genetic variation in pfhrp2 and pfhrp3 in Myanmar P. falciparum isolates was analysed. Methods Blood samples were collected from malaria patients who were infected with P. falciparum in Mandalay, Naung Cho, Tha Beik Kyin, and Pyin Oo Lwin, Upper Myanmar between 2013 and 2015. The pfhrp2 and pfhrp3 were amplified by nested polymerase chain reaction (PCR), cloned and sequenced. Genetic variation in Myanmar pfhrp2 and pfhrp3 was analysed using the DNASTAR program. Comparative analysis of Myanmar and global pfhrp2 and pfhrp3 isolates was also performed. Results One-hundred and two pfhrp2 and 89 pfhrp3 were amplified from 105 blood samples, of which 84 pfhrp2 and 56 pfhrp3 sequences were obtained successfully. Myanmar pfhrp2 and pfhrp3 showed high levels of genetic variation with different arrangements of distinct repeat types, which further classified Myanmar pfhrp2 and pfhrp3 into 76 and 47 haplotypes, respectively. Novel amino acid changes were also found in Myanmar pfhrp2 and pfhrp3, but their frequencies were very low. Similar structural organization was shared by Myanmar and global pfhrp2 and pfhrp3, and differences in frequencies of repeat types and lengths were also observed between and among global isolates. Conclusion Length polymorphisms and amino acid substitutions generated extensive genetic variation in Myanmar pfhrp2 and pfhrp3. Comparative analysis revealed that global pfhrp2 and pfhrp3 share similar structural features, as well as extensive length polymorphisms and distinct organizations of repeat types. These results provide a better understanding of the genetic structure of pfhrp2 and pfhrp3 in global P. falciparum populations and suggest useful information to develop RDTs with improved quality.

Abstract Background Circumsporozoite surface protein (CSP) of malaria parasites has been recognized as one of the leading vaccine candidates. Clinical trials of vaccines for vivax malaria incorporating Plasmodium vivax CSP (PvCSP) have demonstrated their effectiveness in preventing malaria, at least in part. However, genetic diversity of pvcsp in the natural population remains a major concern. Methods A total of 171 blood samples collected from patients infected with Plasmodium vivax in Myanmar were analysed in this study. The pvcsp was amplified by polymerase chain reaction, followed by cloning and sequencing. Polymorphic characteristics and natural selection of pvcsp population in Myanmar were analysed using DNASTAR, MEGA6 and DnaSP programs. The polymorphic pattern and natural selection of publicly accessible global pvcsp sequences were also comparatively analysed. Results Myanmar pvcsp sequences were divided into two subtypes VK210 and VK247 comprising 143 and 28 sequences, respectively. The VK210 subtypes showed higher levels of genetic diversity and polymorphism than the VK247 subtypes. The N-terminal non-repeat region of pvcsp displayed limited genetic variations in the global population. Different patterns of octapeptide insertion (ANKKAEDA in VK210 and ANKKAGDA in VK247) and tetrapeptide repeat motif (GGNA) were identified in the C-terminal region of global pvcsp population. Meanwhile, the central repeat region (CRR) of Myanmar and global pvcsp, both in VK210 and VK247 variants, was highly polymorphic. The high level of genetic diversity in the CRR has been attributed to the different numbers, types and combinations of peptide repeat motifs (PRMs). Interestingly, 27 and 5 novel PRMs were found in Myanmar VK210 and VK247 variants, respectively. Conclusion Comparative analysis of the global pvcsp population suggests a complex genetic profile of pvcsp in the global population. These results widen understanding of the genetic make-up of pvcsp in the global P. vivax population and provide valuable information for the development of a vaccine based on PvCSP.

Abstract Background Plasmodium falciparum merozoite surface protein-3 (PfMSP-3) is a target of naturally acquired immunity against P. falciparum infection and is a promising vaccine candidate because of its critical role in the erythrocyte invasion of the parasite. Understanding the genetic diversity of pfmsp-3 is important for recognizing genetic nature and evolutionary aspect of the gene in the natural P. falciparum population and for designing an effective vaccine based on the antigen. Methods Blood samples collected from P. falciparum-infected patients in Naung Cho and Pyin Oo Lwin, Myanmar, in 2015 were used in this study. The pfmsp-3 was amplified by polymerase chain reaction, cloned, and sequenced. Genetic polymorphism and natural selection of Myanmar pfmsp-3 were analysed using the programs DNASTAR, MEGA6, and DnaSP 5.10.00. Genetic diversity and natural selection of the global pfmsp-3 were also comparatively analysed. Results Myanmar pfmsp-3 displayed 2 different alleles, 3D7 and K1. The 3D7 allelic type was predominant in the population, but genetic polymorphism was less diverse than for the K1 allelic type. Polymorphic characters in both allelic types were caused by amino acid substitutions, insertions, and deletions. Amino acid substitutions were mainly occurred at the alanine heptad repeat domains, whereas most insertions and deletions were found at the glutamate rich domain. Overall patterns of amino acid polymorphisms detected in Myanmar pfmsp-3 were similar in the global pfmsp-3 population, but novel amino acid changes were observed in Myanmar pfmsp-3 with low frequencies. Complicated patterns of natural selection and recombination events were predicted in the global pfmsp-3, which may act as major driving forces to maintain and generate genetic diversity of the global pfmsp-3 population. Conclusion Global pfmsp-3 revealed genetic polymorphisms, suggesting that the functional and structural consequences of the polymorphisms should be considered in designing a vaccine based on PfMSP-3. Further examination of genetic diversity of pfmsp-3 in the global P. falciparum population is necessary to gain in-depth insight for the population structure and evolutionary aspect of global pfmsp-3.

Abstract Background Plasmodium lactate dehydrogenase (pLDH) is a major target in diagnosing the erythrocytic stage of malaria parasites because it is highly expressed during blood-stage parasites and is distinguished from human LDH. Rapid diagnostic tests (RDTs) for malaria use pLDH as a target antigen; however, genetic variations in pLDH within the natural population threaten the efficacy of pLDH-based RDTs. Methods Genetic polymorphisms of Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) in Myanmar isolates were analysed by nucleotide sequencing analysis. Genetic polymorphisms and the natural selection of PvLDH and PfLDH were analysed using DNASTAR, MEGA6, and DnaSP ver. 5.10.00 programs. The genetic diversity and natural selection of global PvLDH and PfLDH were also analysed. The haplotype network of global PvLDH and PfLDH was constructed using NETWORK ver. 5.0.0.3. Three-dimensional structures of PvLDH and PfLDH were built with YASARA Structure ver. 18.4.24 and the impact of mutations on structural change and stability was evaluated with SDM ver. 2, CUPSAT and MAESTROweb. Results Forty-nine PvLDH and 52 PfLDH sequences were obtained from Myanmar P. vivax and P. falciparum isolates. Non-synonymous nucleotide substitutions resulting in amino acid changes were identified in both Myanmar PvLDH and PfLDH. Amino acid changes were also identified in the global PvLDH and PfLDH populations, but they did not produce structural alterations in either protein. Low genetic diversity was observed in global PvLDH and PfLDH, which may be maintained by a strong purifying selection. Conclusion This study extends knowledge for genetic diversity and natural selection of global PvLDH and PfLDH. Although amino acid changes were observed in global PvLDH and PfLDH, they did not alter the conformational structures of the proteins. These suggest that PvLDH and PfLDH are genetically well-conserved in global populations, which indicates that they are suitable antigens for diagnostic purpose and attractive targets for drug development.

Abstract Background MF6p/host defense molecules (HDMs) are a broad family of small proteins secreted by helminth parasites. Although the physiological role of MF6p/HDMs in trematode parasites is not fully understood, their potential biological function in maintaining heme homeostasis and modulating host immune response has been proposed. Methods A gene encoding the MF6p/HDM of Clonorchis sinensis (CsMF6p/HDM) was cloned. Recombinant CsMF6p/HDM (rCsMF6p/HDM) was expressed in Escherichia coli. The biochemical and immunological properties of rCsMF6/HDM were analyzed. CsMF6p/HDM induced pro-inflammatory response in RAW 264.7 cells was analyzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The structural feature of CsMF6p/HDM was analyzed by three-dimensional modeling and molecular docking simulations. Results The CsMF6p/HDM shares a high level of amino acid sequence similarity with orthologs from other trematodes and is expressed in diverse developmental stages of the parasite. The rCsMF6p/HDM bound to bacteria-derived lipopolysaccharide (LPS), without effectively neutralizing LPS-induced inflammatory response in RAW 264.7 macrophage cells. Rather, the rCsMF6p/HDM induced pro-inflammatory immune response, which is characterized by the expression of TNF-α and IL-6, in RAW 264.7 cells. The rCsMF6p/HDM-induced pro-inflammatory immune response was regulated by JNK and p38 MAPKs, and was effectively down-regulated via inhibition of NF-κB. The structural analysis of CsMF6p/HDM and the docking simulation with LPS suggested insufficient capture of LPS by CsMF6p/HDM, which suggested that rCsMF6p/HDM could not effectively neutralize LPS-induced inflammatory response in RAW 264.7 cells. Conclusions Although rCsMF6p/HDM binds to LPS, the binding affinity may not be sufficient to maintain a stable complex of rCsMF6p/HDM and LPS. Moreover, the rCsMF6p/HDM-induced pro-inflammatory response is characterized by the release of IL-6 and TNF-α in RAW 264.7 macrophage cells. The pro-inflammatory response induced by rCsMF6p/HDM is mediated via NF-κB-dependent MAPK signaling pathway. These results collectively suggest that CsMF6p/HDM mediates C. sinensis-induced inflammation cascades that eventually lead to hepatobiliary diseases.

Abstract Background Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells. Methods Recombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. Results Fowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1. Conclusions Fowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.

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.

Emergence and spreading of antimalarial drug resistant malaria parasites are great hurdles to combating malaria. Although approaches to investigate antimalarial drug resistance status in Myanmar malaria parasites have been made, more expanded studies are necessary to understand the nationwide aspect of antimalarial drug resistance. In the present study, molecular epidemiological analysis for antimalarial drug resistance genes in Plasmodium falciparum and P. vivax from the Mandalay region of Myanmar was performed. Blood samples were collected from patients infected with P. falciparum and P. vivax in four townships around the Mandalay region, Myanmar in 2015. Partial regions flanking major mutations in 11 antimalarial drug resistance genes, including seven genes (pfdhfr, pfdhps, pfmdr-1, pfcrt, pfk13, pfubp-1, and pfcytb) of P. falciparum and four genes (pvdhfr, pvdhps, pvmdr-1, and pvk12) of P. vivax were amplified, sequenced, and overall mutation patterns in these genes were analyzed. Substantial levels of mutations conferring antimalarial drug resistance were detected in both P. falciparum and P. vivax isolated in Mandalay region of Myanmar. Mutations associated with sulfadoxine-pyrimethamine resistance were found in pfdhfr, pfdhps, pvdhfr, and pvdhps of Myanmar P. falciparum and P. vivax with very high frequencies up to 90%. High or moderate levels of mutations were detected in genes such as pfmdr-1, pfcrt, and pvmdr-1 associated with chloroquine resistance. Meanwhile, low frequency mutations or none were found in pfk13, pfubp-1, pfcytb, and pvk12 of the parasites. Overall molecular profiles for antimalarial drug resistance genes in malaria parasites in the Mandalay region suggest that parasite populations in the region have substantial levels of mutations conferring antimalarial drug resistance. Continuous monitoring of mutations linked with antimalarial drug resistance is necessary to provide useful information for policymakers to plan for proper antimalarial drug regimens to control and eliminate malaria in the country.

The circumsporozoite surface protein of Plasmodium vivax (PvCSP) plays a critical role in parasite biology. It has been extensively studied as a leading vivax-malaria-vaccine candidate. In this study, the genetic polymorphism and natural selection of pvcsp in P. vivax isolates collected from the Central Highlands, Vietnam were analyzed to understand the genetic structure of the parasite circulating in the endemic area and to provide baseline information for effective vaccine development based on the protein. Only two major alleles, VK210 and VK247, were detected in Vietnamese pvcsp, with VK247 being the predominant one. The N-terminal and C-terminal regions of Vietnamese VK210 and VK247 variants showed a low genetic diversity. Amino acid substitutions, insertions of a single amino acid or octapeptide (ANKKAEDA in VK210 and ANKKAGDA in VK247), and tetrapeptide repeat motifs (GGNA) were the main factors generating genetic diversity in the two regions of the Vietnamese VK210 and VK247 variants. Interestingly, these two regions of Vietnamese pvcsp displayed a unique natural selection pressure distinct from global pvcsp, particularly with the neighboring Southeast Asian pvcsp population. Meanwhile, the central repeat region (CRR) in both the VK210 and VK247 variants showed a high degree of polymorphic characters, caused by varying numbers, types, and combinations of peptide repeat motifs (PRMs) in Vietnamese pvcsp. Highly complicated polymorphic patterns of the CRR were also detected in global pvcsp. These results expand our understanding of the genetic structure of Vietnamese pvcsp and the population dynamics of P. vivax in the Central Highlands, Vietnam.

Abstract Background Plasmodium falciparum merozoite surface protein-1 (PfMSP-1) and -2 (PfMSP-2) are major blood-stage vaccine candidate antigens. Understanding the genetic diversity of the genes, pfmsp-1 and pfmsp-2, is important for recognizing the genetic structure of P. falciparum, and the development of an effective vaccine based on the antigens. In this study, the genetic diversities of pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum were analysed. Methods The pfmsp-1 block 2 and pfmsp-2 block 3 regions were amplified by polymerase chain reaction from blood samples collected from Myanmar patients who were infected with P. falciparum in 2013–2015. The amplified gene fragments were cloned into a T&A vector, and sequenced. Sequence analysis of Myanmar pfmsp-1 block 2 and pfmsp-2 block 3 was performed to identify the genetic diversity of the regions. The temporal genetic changes of both pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum population, as well as the polymorphic diversity in the publicly available global pfmsp-1 and pfmsp-2, were also comparatively analysed. Results High levels of genetic diversity of pfmsp-1 and pfmsp-2 were observed in the Myanmar P. falciparum isolates. Twenty-eight different alleles of pfmsp-1 (8 for K1 type, 14 for MAD20 type, and 6 for RO33 type) and 59 distinct alleles of pfmsp-2 (18 for FC27, and 41 for 3D7 type) were identified in the Myanmar P. falciparum population in amino acid level. Comparative analyses of the genetic diversity of the Myanmar pfmsp-1 and pfmsp-2 alleles in the recent (2013–2015) and past (2004–2006) Myanmar P. falciparum populations indicated the dynamic genetic expansion of the pfmsp-1 and pfmsp-2 in recent years, suggesting that a high level of genetic differentiation and recombination of the two genes may be maintained. Population genetic structure analysis of the global pfmsp-1 and pfmsp-2 also suggested that a high level of genetic diversity of the two genes was found in the global P. falciparum population. Conclusion Despite the recent remarkable decline of malaria cases, the Myanmar P. falciparum population still remains of sufficient size to allow the generation and maintenance of genetic diversity. The high level of genetic diversity of pfmsp-1 and pfmsp-2 in the global P. falciparum population emphasizes the necessity for continuous monitoring of the genetic diversity of the genes for better understanding of the genetic make-up and evolutionary aspect of the genes in the global P. falciparum population.

BackgroundVitamin D status is presently assessed by measuring total serum concentration of 25-hydroxyvitamin D [25(OH)D]. However, 25(OH)D concentration alone might not accurately reflect vitamin D status owing to its weak relationship with various clinical indices and inconsistency across races. Recently, 24,25-dihydroxyvitamin D [24,25(OH)2D] and vitamin D metabolite ratio [VMR; ratio of 24,25(OH)2D to 25(OH)D] have emerged as vitamin D biomarkers. The present study aimed to determine the values of 24,25(OH)2D and VMR in healthy Koreans and compare them with other vitamin D biomarkers, including 25(OH)D and bioavailable 25(OH)D.MethodsSerum samples and medical information were collected from 200 individuals (100 females and 100 males) who underwent general health checks without self-reported symptoms. We measured 24,25(OH)2D concentration using liquid chromatography-tandem mass spectrometry, and concentrations of 25(OH)D and vitamin D binding protein using immunoassays. VMR and bioavailable 25(OH)D concentration were calculated using the above data. Serum parathyroid hormone level, and bone mineral density (BMD) data were collected as clinical outcomes, and the effects of the vitamin D markers on them were tested using multiple linear regression models.ResultsThe mean values of 25(OH)D, 24,25(OH)2D, VMR, and bioavailable 25(OH)D were 24.3 ± 8.5 ng/mL, 1.9 ± 1.1 ng/mL, 7.6 ± 2.5, and 3.2 ± 1.2 ng/mL, respectively. The concentration of 25(OH)D closely correlated with 24,25(OH)2D (R = 0.868, P < 0.001) and bioavailable 25(OH)D (R = 0.862, P < 0.001). No significant effects of 24,25(OH)2D, VMR, and bioavailable 25(OH)D were observed on the prediction of PTH and BMD in the multiple linear regression models.ConclusionOur study presents the distribution of 24,25(OH)2D concentration and VMR in Korean population for the first time. Overall, our data reaffirm that 25(OH)D is the primary marker for determining vitamin D status in the general population.

Cysteine proteases of malaria parasites have been recognized as potential targets in antimalarial drug development as they play pivotal roles in the biology of these parasites. However, strict regulation of their activities is also necessary to minimize or prevent deleterious damage to the parasite and the host. Previously, we have characterized falcipain family cysteine proteases of Plasmodium malariae, named as malapains (MPs). MPs are active hemoglobinases. They also may participate in the release of merozoites from mature schizonts by facilitating remodeling of erythrocyte skeleton proteins. In this study, we identified and characterized an endogenous inhibitor of cysteine protease of P. malariae (PmICP). PmICP shared similar structural and biochemical properties with ICPs from other Plasmodium species. Recombinant PmICP showed a broad range of inhibitory activities against diverse cysteine proteases such as falcipain family enzymes (MP-2, MP-4, VX-3, VX-4, and FP-3), papain, and human cathepsins B and L, with stronger inhibitory activities against falcipain family enzymes. The inhibitory activity of PmICP was not affected by pH. PmICP was thermo-labile, resulting in rapid loss of its inhibitory activity at a high temperature. PmICP effectively inhibited hemoglobin hydrolysis by MPs and regulated maturation of MPs, suggesting its role as a functional regulator of MPs.

Aedes aegypti is an important mosquito vector transmitting diverse arboviral diseases in Myanmar. Pyrethroid insecticides have been widely used in Myanmar as the key mosquito control measure, but the efforts are constrained by increasing resistance. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) are related to pyrethroid resistance in Ae. aegypti. We analyzed the patterns and distributions of the kdr mutations in Ae. aegypti in the Mandalay area of Myanmar. The segment 6 regions of domains II and III of vgsc were separately amplified from individual Ae. aegypti genomic DNA via polymerase chain reaction. The amplified gene fragments were sequenced. High proportions of three major kdr mutations, including S989P (54.8%), V1016G (73.6%), and F1534C (69.5%), were detected in the vgsc of Ae. aegypti from all studied areas. Other kdr mutations, T1520I and F1534L, were also found. These kdr mutations represent 11 distinct haplotypes of the vgsc population. The S989P/V1016G/F1534C was the most prevalent, followed by S989P/V1016V and V1016G/F1534C. A quadruple mutation, S989P/V1016G/T1520I/F1534C, was also identified. High frequencies of concurrent kdr mutations were observed in vgsc of Myanmar Ae. aegypti, suggesting a high level of pyrethroid resistance in the population. These findings underscore the need for an effective vector control program in Myanmar.

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Abstract Background Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the most extensively studied malaria vaccine candidates, but the genetic polymorphism of PfCSP within and among the global P. falciparum population raises concerns regarding the efficacy of a PfCSP-based vaccine efficacy. In this study, genetic diversity and natural selection of PfCSP in Myanmar as well as global P. falciparum were comprehensively analysed. Methods Blood samples were collected from 51 P. falciparum infected Myanmar patients. Fifty-one full-length PfCSP genes were amplified from the blood samples through a nested polymerase chain reaction, cloned into a TA cloning vector, and then sequenced. Polymorphic characteristics and natural selection of Myanmar PfCSP were analysed using the DNASTAR, MEGA6, and DnaSP programs. Polymorphic diversity and natural selection in publicly available global PfCSP were also analysed. Results The N-terminal and C-terminal non-repeat regions of Myanmar PfCSP showed limited genetic variations. A comparative analysis of the two regions in global PfCSP displayed similar patterns of low genetic diversity in global population, but substantial geographic differentiation was also observed. The most notable polymorphisms identified in the N-terminal region of global PfCSP were A98G and 19-amino acid length insertion in global population with different frequencies. Major polymorphic characters in the C-terminal region of Myanmar and global PfCSP were found in the Th2R and Th3R regions, where natural selection and recombination occurred. The central repeat region of Myanmar PfCSP was highly polymorphic, with differing numbers of repetitive repeat sequences NANP and NVDP. The numbers of the NANP repeats varied among global PfCSP, with the highest number of repeats seen in Asian and Oceanian PfCSP. Haplotype network analysis of global PfCSP revealed that global PfCSP clustered into 103 different haplotypes with geographically-separated populations. Conclusion Myanmar and global PfCSP displayed genetic diversity. N-terminal and C-terminal non-repeat regions were relatively conserved, but the central repeat region displayed high levels of genetic polymorphism in Myanmar and global PfCSP. The observed geographic pattern of genetic differentiation and the points of evidence for natural selection and recombination suggest that the functional consequences of the polymorphism should be considered for developing a vaccine based on PfCSP.

Abstract Background The C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-142) is the target of an immune response. It has been recognised as one of the promising candidate antigens for a blood-stage malaria vaccine. Genetic structure of PfMSP-142 has been considered to be largely conserved in the P. falciparum population. However, only limited information is currently available. This study aimed to analyse genetic diversity and the effect of natural selection on PfMSP-142 among the Myanmar P. falciparum population and compare them with publicly available PfMSP-142 from global P. falciparum populations. Methods A total of 69 P. falciparum clinical isolates collected from Myanmar malaria patients in Upper Myanmar in 2015 were used. The PfMSP-142 region was amplified by polymerase chain reaction, cloned and sequenced. Genetic structure and natural selection of this region were analysed using MEGA4 and DnaSP programs. Polymorphic nature and natural selection in global PfMSP-142 were also investigated. Results All three allele types (MAD20, K1, and RO33) of PfMSP-142 were identified in Myanmar isolates of P. falciparum. Myanmar PfMSP-142 displayed genetic diversity. Most polymorphisms were scattered in blocks 16 and 17. Polymorphisms observed in Myanmar PfMSP-142 showed a similar pattern to those of global PfMSP-142; however, they were not identical to each other. Genetic diversity of Myanmar PfMSP-142 was relatively lower than that of PfMSP-142 from different geographical regions. Evidence of natural selection and recombination were found. Comparative analysis of genetic polymorphism and natural selection in the global PfMSP-142 population suggested that this region was not tightly conserved in global PfMSP-142 as previously thought and is under the complicated influence of natural selection and recombination. Conclusions Global PfMSP-142 revealed limited, but non-negligible, genetic diversity by allele types and geographical origins. Complicated natural selection and potential recombination might have occurred in global PfMSP-142. Comprehensive monitoring of genetic diversity for global PfMSP-142 would be needed to better understand the polymorphic nature and evolutionary aspect of PfMSP-142 in the global P. falciparum population. More thought would be necessary for designing a vaccine based on PfMSP-142.

Abstract Background Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is one of the most common X-linked recessive hereditary disorders in the world. Primaquine (PQ) has been used for radical cure of P. vivax to prevent relapse. Recently, it is also used to reduce P. falciparum gametocyte carriage to block transmission. However, PQ metabolites oxidize hemoglobin and generate excessive reactive oxygen species which can trigger acute hemolytic anemia in malaria patients with inherited G6PD deficiency. Methods A total of 252 blood samples collected from malaria patients in Myanmar were used in this study. G6PD variant was analysed by a multiplex allele specific PCR kit, DiaPlexC™ G6PD Genotyping Kit [Asian type]. The accuracy of the multiplex allele specific PCR was confirmed by sequencing analysis. Results Prevalence and distribution of G6PD variants in 252 malaria patients in Myanmar were analysed. Six different types of G6PD allelic variants were identified in 50 (7 females and 43 males) malaria patients. The predominant variant was Mahidol (68%, 34/50), of which 91.2% (31/34) and 8.8% (3/34) were males and females, respectively. Other G6PD variants including Kaiping (18%, 9/50), Viangchan (6%, 3/50), Mediterranean (4%, 2/50), Union (2%, 1/50) and Canton (2%, 1/50) were also observed. Conclusions Results of this study suggest that more concern for proper and safe use of PQ as a radical cure of malaria in Myanmar is needed by combining G6PD deficiency test before PQ prescription. Establishment of a follow-up system to monitor potential PQ toxicity in malaria patients who are given PQ is also required.

Abstract Background Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. Methods Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. Results Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. Conclusions Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the strong selective pressure of host immunity on the PfAMA-1 gene. These results have significant implications in understanding the nature of Myanmar PfAMA-1 along with global PfAMA-1. They also provide useful information for the development of effective malaria vaccine based on this antigen.

Cathepsin D (CatD; EC 3.4.23.5) family peptidases of parasitic organisms are regarded as potential drug targets as they play critical roles in the physiology and pathobiology of parasites. Previously, we characterized the biochemical features of cathepsin D isozyme 2 (CatD2) in the carcinogenic liver fluke Clonorchis sinensis (CsCatD2). In this study, we performed all-atomic molecular dynamics simulations by applying different systems for the ligand-free/bound forms under neutral and acidic conditions to investigate the pH-dependent structural alterations and associated functional changes in CsCatD2. CsCatD2 showed several distinctive characteristics as follows: (1) acidic pH caused major conformational transitions from open to closed state in this enzyme; (2) during 30–36-ns simulations, acidic pH contributed significantly to the formation of rigid β-sheets around the catalytic residue Asp219, higher occupancy (0% to 99%) of hydrogen bond than that of Asp33, and enhanced stabilization of the CsCatD2-inhibtor complex; (3) neutral pH-induced displacement of the N-terminal part to hinder the accessibility of the active site and open allosteric site of this enzyme; and (4) the flap dynamics metrics, including distance (d1), TriCα angles (θ1 and θ2), and dihedral angle (ϕ), account for the asymmetrical twisting motion of the active site of this enzyme. These findings provide an in-depth understanding of the pH-dependent structural dynamics of free and bound forms of CsCatD2 and basic information for the rational design of an inhibitor as a drug targeting parasitic CatD.

Despite a significant decline in the incidence of malaria in Myanmar recently, malaria is still an important public health concern in the country. Although Plasmodium falciparum is associated with the highest incidence of malaria in Myanmar, the proportion of P. vivax cases has shown a gradual increase in recent years. The genetic diversity of P. vivax merozoite surface protein-1 block 5-6 (pvmsp-1 ICB 5-6) in the P. vivax population of Myanmar was analyzed to obtain a comprehensive insight into its genetic heterogeneity and evolutionary history. High levels of genetic diversity of pvmsp-1 ICB 5-6 were identified in the P. vivax isolates collected from Myanmar between 2013 and 2015. Thirty-nine distinct haplotypes of pvmsp-1 ICB 5-6 (13 for Sal I type, 20 for recombinant type, and 6 for Belem type) were found at the amino acid level. Comparative analyses of the genetic diversity of pvmsp-1 ICB 5-6 sequences in the recent (2013–2015) and the past (2004) P. vivax populations in Myanmar revealed genetic expansion of the pvmsp-1 ICB 5-6 in recent years, albeit with a declined incidence. The recent increase in the genetic heterogeneity of Myanmar pvmsp-1 ICB 5-6 is attributed to a combination of factors, including accumulated mutations and recombination. These results suggest that the size of the P. vivax population in Myanmar is sufficient to enable the generation and maintenance of genetic diversity, warranting continuous molecular surveillance of genetic variation in Myanmar P. vivax.

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (‒)-epicatechin, (‒)-epigallocatechin, rosmarinic acid, and (‒)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS. [ABSTRACT FROM AUTHOR]

Abstract Background The bacteria Orientia tsutsugamushi is the causative agent of scrub typhus, mite-borne disease, which causes an acute febrile illness in patients. An epidemiologic study was conducted to understand the characteristics of scrub typhus in South Korea. Findings Reporting of tsutsugamushi disease is mandatory in South Korea since 1994. To investigate the prevalence of tsutsugamushi disease from 2001 to 2013, medical records from the Korea Center for Disease Control and Prevention were reviewed. In total, 70,914 cases were reported during 2001–2013. Of these, 37.16% (26,349) were male and 62.84% (44,565) were female. The highest number of cases was in the 60–69-year-old age group (19,484; 27.48%), and 72.22% (51,212) were in the 50–79-year-old age group. There were 65,100 cases (91.80%) reported during October (24,964; 35.20%) and November (40,136; 56.60%). An almost four-fold increase in the number of patients was observed in 2013 (10,485 cases) compared to 2001 (2,637 cases). The highest number of patients was reported in the Jeonbuk (9,425; 13.29%) and lowest in the Jeju (362; 0.51%). Conclusions A rapid increase in the incidence of patients with tsutsugamushi disease was observed in most areas from 2001 to 2013, with the majority of cases reported in the western and southern coast.

Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.

South Korea is one of many countries with endemic Plasmodium vivax malaria. Here we report the evaluation of four rapid diagnostic tests (RDTs) for diagnosis of this disease. A total of 253 subjects were enrolled in the study. The sensitivities, specificities and agreement frequencies were estimated by comparing the four RDTs against the standard of nested-PCR and microscopic examination. The CareStart(TM) and SD Bioline had higher test sensitivities (99.4 and 98.8%, respectively) compared with the NanoSign and Asan Easy tests (93.0 and 94.7%, respectively). The CareStart(TM) and SD Bioline tests could detect P. vivax in samples with parasite densities

Malaria continues to be one of the most crucial infectious burdens in endemic areas worldwide, as well as for travelers visiting malaria transmission regions. It has been reported that 8-aminoquinolines are effective against the Plasmodium species, particularly primaquine, for anti-hypnozoite therapy in P. vivax malaria. However, primaquine causes acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Therefore, G6PD deficiency testing should precede hypnozoite elimination with 8-aminoquinoline. Several point-of-care devices have been developed to detect G6PD deficiency. The aim of the present study was to evaluate the performance of a novel, quantitative G6PD diagnostics based on a metagenomic blue fluorescent protein (mBFP). We comparatively evaluated the sensitivity and specificity of the G6PD diagnostic modality with standard methods using 120 human whole blood samples. The G6PD deficiency was spectrophotometrically confirmed. The performance of the G6PD quantitative test kit was compared with that of a licensed control medical device, the G6PD strip. The G6PD quantitative test kit had a sensitivity of 95% (95% confidence interval (CI): 89.3-100%) and a specificity of 100% (95% CI: 94.3-100%). This study shows that the novel diagnostic G6PD quantitative test kit could be a cost-effective and time-efficient, and universally mandated screening tool for G6PD deficiency. [ABSTRACT FROM AUTHOR]

Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking 731Leu-780Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack. [ABSTRACT FROM AUTHOR]

Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.

An understanding of the knowledges, attitudes and perceptions of different populations is key for public health policy makers. Here, a survey was performed on knowledge, attitudes, and perceptions about malaria diagnosis, prevention, control, and treatment. The 407 survey participants included both uninfected inhabitants and patients from 2 cities (Gimpo- and Paju-si) of Northern Gyeonggi-do, known as high-risk areas for vivax malaria. We used community-based study design and non-probability sampling method using the primary data. Association between variables were tested using χ²-tests. In general, the information on malaria reported by the participants in this study was unsystematic and included inaccurate details. The knowledge of malaria symptoms, identified as headache, chills and fever, was high, but the surveyed community lacks knowledge of the specific medications used for malaria treatment, with a large number of respondents having no knowledge of any form of medication. Survey questions with high correct answer rates included questions about easy treatment of malaria in Korea, the high daytime activity of malaria-borne mosquitoes, and the infection risk posed by outdoor activities. However, a large portion of the respondents was unable to provide simple medical and biological information about the disease. This study aimed to comprehensively evaluate the knowledge, attitude, and practical behavior of the surveyed community with respect to malaria and the implications reported here could be applicable to other malaria endemic areas in Korea. [ABSTRACT FROM AUTHOR]

The prevalence and intensity of Opisthorchis viverrini metacercariae (OvMc) were investigated in fish from 3 southern administrative regions along the Mekong River in Cambodia, i.e., Phnom Penh, Takeo, and Kandal Provinces from 2017 to 2020. A total of 295 freshwater fish (24 species) were transported to our laboratory with ice and examined using the artificial digestion method. In Phnom Penh, among 4 fish species positive for OvMc, 9 (23.7%) of 38 specimens examined were infected, and their intensity of infection averaged 4.3 metacercariae per infected fish. In Takeo Province, among 10 fish species positive for OvMc, 24 (38.1%) out of 63 fish examined were infected, and their intensity of infection was av. 14.4 metacercariae per infected fish. In particular, all of 3 Osteochilus schlegelii fish examined were infected, and their infection intensity was high, 34.7 metacercariae per fish. In Kandal Province, among 6 fish species positive for OvMc, 46 (90.2%) out of 51 specimens examined were infected, and their infection intensity was 24.0 metacercaraie per infected fish. All fish of Systomus orphoides (n=17), Barbonymus altus (n=14), and Rasbora aurotaenia (n=2) were infected, and their intensity of infection averaged 37.7, 21.6, and 18.5 metacercariae per fish, respectively. Metacercariae of Haplochis yokogawai, Haplorchis taichui, and Centrocestus formosanus were detected in fish from Takeo and Kandal Provinces. From these results, it has been confirmed that a variety of fish species from Phnom Penh, Takeo, and Kandal Provinces are commonly infected with OvMc, and preventive measures to avoid human O. viverrini infection should be performed in Cambodia. [ABSTRACT FROM AUTHOR]

The endemicity of zoonotic trematode metacercariae (ZTM) was investigated with total 871 freshwater fishes (19 species) from Deokcheon-gang (a branch stream of Gyeongho-gang) in Sancheong-gun, Gyeongsangnam-do, Korea for 3 years (2018-2020). All fishes were examined with the artificial digestion method. The metacercariae of Clonorchis sinensis (CsMc) were detected in 233 (36.3%) out of 642 fish in 11 positive fish species (PFS), and their infection intensity was 27 per fish infected (PFI). Especially, in index fish, Puntungia herzi, of CsMc infection, prevalence was 64.2% and infection intensity was 37 PFI. Metagonimus spp. metacercariae (MsMc) were found in 760 (87.5%) out of 869 fish in 18 PFS and their infection intensity was 228 PFI. In sweet smelt, Plecoglossus altivelis, the prevalence of MsMc was 97.6% and their infection intensity was 3,570 PFI. Centrocestus armatus metacercariae were detected in 209 (29.4%) out of 710 fish in 8 PFS and their infection intensity was 1,361 PFI. Echinostoma spp. metacercariae were found in 293 (42.6%) out of 688 fish in 15 PFS and their infection intensity was 5 PFI. Metacercariae of Clinostomum complanatum and Metorchis orientalis were also detected in 2.7% and 21.2% fish in 4 PFS and their infection intensities were 3.1 and 3.4 PFI respectively. By the present study, it was confirmed that some species of ZTM including CsMc and MsMc are more or less prevalent in fishes from Deokcheon-gang in Sancheong-gun, Gyeongsangnam-do, Korea. [ABSTRACT FROM AUTHOR]

The incidence of vivax malaria in Korea was reduced to a low plateau. For successful elimination of vivax malaria, socio-behavioral changes in the communities are essential. This study aimed to figure out awareness of the inhabitants on the vivax malaria endemicity. The 407 participants including vivax malaria patients and uninfected inhabitants in Gimpo- and Paju-si, Gyeonggi-do, known as high-risk areas in Korea. We used a community-based study design and non-probability sampling method using primary data. Except for the perception about the public health facilities' capability to cope with anti-malaria programs, the 2 groups of participants shared the same level of awareness about public promotional and educational measures and opinions for malaria elimination from the community. Thus, our future goals for malaria prevention and elimination are to develop more active and well-organized community-based education and evaluation programs collaborating with the community healthcare authorities and local governments. [ABSTRACT FROM AUTHOR]

The infection status of zoonotic trematode metacercariae (ZTM) was investigated in total 568 freshwater fishes (19 species) from the irrigation canal of Togyo-jeosuji (Reservoir) in Cheorwon-gun, Gangwon-do, the Republic of Korea for 3 years (2018-2020). All fishes were examined using the artificial digestion method. The metacercariae of Clonorchis sinensis (CsMc) were detected in 180 (43.8%) out of 411 fish of positive species, and their infection intensity was 38 per fish infected (PFI). Especially, in 2 fish species, i.e., Pseudorasbora parva and Puntungia herzi, the prevalence was 82.1% and 31.3%, and the infection intensity with CsMc was 88 and 290 PFI, respectively. Metagonimus spp. metacercariae (MsMc) were found in 403 (74.1%) out of 544 fish of positive species, and their infection intensity was 62 PFI. In the pale chub, Zacco platypus, the prevalence of MsMc was 98.6%, and their infection intensity was 144 PFI. Centrocestus armatus metacercariae were detected in 171 (38.9%) out of 440 fish of positive species, and their infection intensity was 1,844 PFI. Echinostoma spp. metacercariae were found in 94 (19.6%) out of 479 fish of positive species, and their infection intensity was 3 PFI. Metorchis orientalis metacercariae were detected in 43 (29.3%) out of 147 fish of positive species, and their infection intensity was 4 PFI. By the present study, it has been confirmed that some species of ZTM, including CsMc and MsMc, are prevalent in fishes from the irrigation canal of Togyo-jeosuji in Cheorwon-gun, Gangwon-do, Korea. [ABSTRACT FROM AUTHOR]

The circumsporozoite protein (CSP) of Plasmodium spp. is a diagnostic antigen and useful biomarker for monitoring short-term/seasonal changes to malaria transmission. Using P. vivax CSP antibody ELISA, epidemiological characteristics were analyzed in the residents of Ganghwa, Cheorwon, Paju, and Goseong from 2017 to 2018. In Ganghwa and Cheorwon, 1.6% and 1.2% of residents, respectively, were PvCSP-antibody-positive in 2018, which indicates a decrease of 0.4% in the positive rate compared to 2017. The annual parasite incidence (API) in Ganghwa and Cheorwon was 24.9 and 10.5 in 2017 and 20.3 and 10.7 in 2018, respectively. Although the changes were not significant, the API in Ganghwa decreased slightly by 4.5 in 2018 compared to the previous year. In Paju and Goseong, 3.9% and 2.0% of residents were positive for the PvCSP antibody. The API in Paju was 13.1 in 2017 and 16.0 in 2018, although no malaria patients were reported for the 2 years. Therefore, the results suggest that PvCSP is a useful antigen for confirming initial malaria infection. Additionally, considering that the antibody is relatively transient, it can be employed for sero-epidemiological studies to determine the extent of malaria transmission in the current year. [ABSTRACT FROM AUTHOR]

We surveyed on the infection status of zoonotic trematode metacercariae (ZTM) in freshwater fishes from Soyang-cheon (a branch stream of Mangyeong-gang) in Wanju-gun, Jeollabuk-do, the Republic of Korea. A total of 927 fishes were individually examined with the artificial digestion method during 2013-2015 (462 fish in 15 spp.) and 2018-2019 (465 fish in 25 spp.). Clonorchis sinensis metacercariae were detected in 207 (31.4%) out of 659 fishes in 14 positive fish species (PFS), and their mean intensity was 114 per fish infected (PFI). Metagonimus spp. metacercariae were found in 302 (37.4%) out of 808 fishes in 21 PFS, and their mean intensity was 12 PFI. Centrocestus armatus metacercariae were detected in 222 (59.0%) out of 376 fishes in 12 PFS, and their mean intensity was 383 PFI. Echinostoma spp. metacercariae were found in 139 (22.1%) out of 628 fishes in 10 PFS, and their mean intensity was 7 PFI. Clinostomum complanatum metacercariae were detected in 14 (6.5%) out of 214 fishes in 4 PFS, and their mean intensity was 2.4 PFI. Metorchis orientalis metacercariae were detected in 36 (13.5%) out of 267 fishes in 5 PFS, and their mean intensity was 4.3 PFI. Conclusively, the prevalence and infection intensity of ZTM is generally not so high in fishes from Soyang-cheon. However, those of C. sinensis metacercariae are more or less higher in 2 fish species, Pungtungia herzi and Sarcocheilichthys variegatus wakiyae. [ABSTRACT FROM AUTHOR]

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yongjeon-cheon (a branch of Nakdong-gang) located in Cheongsong-gun, Gyeongsangbuk-do, the Republic of Korea (Korea). A total of 750 fishes in 19 species were examined by the artificial digestion method for 2 years (2019 and 2020). CsMc were detected in 378 (51.4%) out of 735 fishes in 14 species (73.7%), and the infection intensity was 666 per fish infected. In 2019, CsMc were found in 172 (68.0%) out of 253 fishes in 10 species, and the infection intensity was 565 per fish infected. In 2020, CsMc were detected in 206 (62.2%) out of 331 fishes in 10 species, and the infection intensity was 751 per fish infected. The other zoonotic trematode, ie. Metagonimus spp., Centrocestus armatus, Echinostoma spp. and Clinostomum complanatum, metacercariae were also detected in fishes from the survey streams, but their endemicities were relatively low. Conclusively, it was first confirmed that CsMc are highly endemic in fishes from Yongjeon-cheon in Cheongsong-gun, Gyeongsangbuk-do, Korea. [ABSTRACT FROM AUTHOR]

The infection status of zoonotic trematode metacercariae (ZTM) was surveyed in freshwater fishes from the water systems of Geum-gang (River) in the Republic of Korea (Korea). A total of 1,161 freshwater fishes from 6 local sites of Geum-gang were examined with the artificial digestion method for 4 years (2012-2015). Clonorchis sinensis metacercariae were detected in 122 (37.2%) out of 328 fishes in the positive fish species from 4 surveyed areas, and their mean intensity was 43 per fish infected. Metagonimus spp. metacercariae were found in 432 (51.7%) out of 835 fishes in the positive fish species from all 6 surveyed areas, and their mean intensity was 30 per fish infected. Centrocestus armatus metacercariae were detected in 285 (75.0%) out of 380 fishes in the positive fish species from 6 surveyed areas, and their mean intensity was 2,100 per fish infected. Echinostoma spp. metacercariae were found in 56 (19.7%) out of 284 fishes in the positive fish species from 5 surveyed areas, and their mean intensity was 10 per fish infected. Clinostomum complanatum metacercariae were detected in 98 (57.3%) out of 171 fishes in the positive fish species from only 2 surveyed areas, and their mean intensity was 11 per fish infected. Conclusively, the endemicity of ZTM is not so high in fishes from water systems of Geum-gang in Korea although it is more or less different by fish species, surveyed areas and ZTM species. [ABSTRACT FROM AUTHOR]

A survey was performed to investigate the infection status of zoonotic helminth larvae in fish from a local market of North Dagon District in Yangon City, Myanmar. A total of 486 fish in 13 species were collected 8 times from December 2015 to December 2019. All fish were transported under ice to a laboratory in Korea and examined for helminth larvae using artificial digestion method. Larval gnathostomes and metacercariae of more than 8 zoonotic trematode species, i.e., Opisthorchis viverrini, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp., were detected. Larval gnathostomes were found in 58 (16.0%) out of 362 fish of 6 species, with mean intensity of 2.8 per fish infected. Metacercariae of O. viverrini were detected in 10 (2.9%) out of 349 fish of 5 species, with mean intensity of 16.9 per fish infected. Metacercarial prevalences of 4 intestinal flukes, H. taichui, H. pumilio, H. yokogawai, and Centrocestus spp., were 16.8%, 26.0%, 12.5%, and 15.0% in the positive fish species, respectively, and mean metacercarial intensity was 63.3, 26.8, 86.2, and 8.7 per fish infected. Metacercariae of S. falcatus and P. cambodiensis were detected only from the mullet, Chelon macrolepis. Metacercariae of Procerovum sp. were found in Channa striata and Anabas testudineus. Collectively, it was confirmed that the fish were infected with gnathostome larvae and metacercariae of O. viverrini and intestinal flukes in Yangon City, Myanmar. [ABSTRACT FROM AUTHOR]

Present study was performed to know the infection status of Gnathostoma sp. larvae in swamp eels from Cambodia. We purchased total 30 Asian swamp eels, Monopterus albus, from local markets in Pursat and Takeo Provinces and Phnom Penh on May and November 2017 and May 2018. All collected eels were transferred to our laboratory with ice and each of them was examined by artificial digestion method. A total of 15 larval gnathostomes (1-5 larvae) were detected from 55.6% (5/9) swamp eels in Pursat Province. No larval gnathostomes were found in 21 swamp eels in Takeo Province and Phnom Penh. The advanced third-stage larvae (AdL3) detected were 2.575-3.825 (3.250) mm in length and 0.375-0.425 (0.386) mm in width. They had the characteristic head bulb (av. 0.104-0.218 mm) with 4 rows of hooklets, long muscular esophagus (1.048 mm), and 2 pairs of cervical sacs (0.615 mm). The number of hooklets in 4 rows on the head bulb was 41, 44, 47, and 50. In scanning electron microscopy, characteristic features were 4 rows of hooklets on the head bulb, cervical papillae, tegumental spines regularly arranged in transverse striations, and anus. The larval gnathostomes were identified as AdL3 of Gnathostoma spinigerum based on the morphological characters. By the present study, it has been confirmed that G. spinigerum larvae are infected in Asian swamp eels, M. albus, in Pursat Province, Cambodia. [ABSTRACT FROM AUTHOR]

Morphological and molecular characterization of clinostomid metacercariae (CMc) was performed with the specimens collected in fish from Korea and Myanmar. Total 6 batches of clinostomid specimens by the fish species and geographical localities, 5 Korean and 1 Myanmar isolates, were analyzed with morphological (light microscopy and SEM) and molecular methods (the cytochrome c oxidase 1 gene and internal transcribed spacer 1/5.8S rRNA sequence). There were some morphological variations among CMc specimens from Korea. However, some morphometrics, i.e., the size of worm body and each organ, ratio of body length to body width, and morphology of cecal lumens, were considerably different between the specimens from Korea and Myanmar. The surface ultrastructures were somewhat different between the specimens from Korea and Myanmar. The CO1 sequences of 5 Korean specimens ranging 728-736 bp showed 99.6- 100% identity with Clinostomum complanatum (GenBank no. KM923964). They also showed 99.9-100% identity with C. complanatum (FJ609420) in the ITS1 sequences ranging 692-698 bp. Meanwhile, the ITS1 sequences of Myanmar specimen showed 99.9% identity with Euclinostomum heterostomum (KY312847). Five sequences from Korean specimens clustered with the C. complanatum genes, but not clustered with Myanmar specimens. Conclusively, it was confirmed that CMc from Korea were morphologically and molecularly identical with C. complanatum and those from Myanmar were E. heterostomum. [ABSTRACT FROM AUTHOR]

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to insecticides. Although insecticide resistance has been suspected to be widespread in the natural population of Aedes aegypti in Myanmar, only limited information is currently available. The overall prevalence and distribution of kdr mutations was analyzed in Ae. aegypti from Mandalay areas, Myanmar. Sequence analysis of the VGSC in Ae. aegypti from Myanmar revealed amino acid mutations at 13 and 11 positions in domains II and III of VGSC, respectively. High frequencies of S989P (68.6%), V1016G (73.5%), and F1534C (40.1%) were found in domains II and III. T1520I was also found, but the frequency was low (8.1%). The frequency of S989P/V1016G was high (55.0%), and the frequencies of V1016G/F1534C and S989P/V1016G/F1534C were also high at 30.1% and 23.5%, respectively. Novel mutations in domain II (L963Q, M976I, V977A, M994T, L995F, V996M/A, D998N, V999A, N1013D, and F1020S) and domain III (K1514R, Y1523H, V1529A, F1534L, F1537S, V1546A, F1551S, G1581D, and K1584R) were also identified. These results collectively suggest that high frequencies of kdr mutations were identified in Myanmar Ae. aegypti, indicating a high level of insecticide resistance. [ABSTRACT FROM AUTHOR]