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Surveillance is an important component of influenza control. This report describes the establishment and first results of the Korean Influenza Surveillance Scheme (KISS), an integrated clinical and laboratory surveillance network involving 622 public health centres (PHCs) and private clinics. Sentinel physicians reported cases of influenza-like illness (ILI) weekly and forwarded specimens for virus isolation and characterization. Influenza activity during the opening 2000-2001 season was milder and delayed compared with previous years. The ILI consultation rate corresponded well with the number of influenza virus isolates, both peaking in week 10 of 2001. Influenza A(H3N2) was the dominant isolate. The peak ILI consultation rate was higher in private clinics than in PHCs (5.04 vs 1.79 cases/1000 visits). An evaluation questionnaire generated potential enhancements to the scheme. KISS appears to represent the pattern of influenza activity accurately and will have a valuable role in monitoring and preventing epidemics in Korea. [ABSTRACT FROM AUTHOR]

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., (© 2024. The Author(s).)

Plasmodium vivax Duffy binding protein (PvDBP) is crucial for erythrocyte invasion, interacting with the Duffy Antigen Receptor for Chemokines (DARC) on the erythrocyte surface. The amino-terminal cysteine-rich region II of PvDBP (PvDBPII) is a promising blood stage vaccine candidate, yet the genetic polymorphisms of this protein in global P. vivax isolates complicate the design of effective vaccines against vivax malaria. This study analyzed the genetic polymorphism of PvDBPII in Pakistan P. vivax isolates. A total of 29 single nucleotide polymorphisms (SNPs), including 22 nonsynonymous SNPs, were identified in 118 Pakistan PvDBPII. Most amino acid substitutions occurred in subdomains II and III, with six commonly observed in the global PvDBPII population. The amino acid change patterns in Pakistan PvDBPII generally mirrored those in global PvDBPII, although the frequencies of amino acid changes varied by country. Nucleotide diversity in Pakistan PvDBPII was comparable to that found in global PvDBPII. Evidence of natural selection and recombination in Pakistan PvDBPII aligned with observations in global PvDBPII. Analysis of the haplotype network of global PvDBPII revealed a complexed network of 167 haplotypes, but no geographical clustering was observed. The findings are crucial for understanding the genetic characteristics of Pakistan PvDBPII. A comprehensive analysis of nucleotide diversity and evolutionary trends in the global PvDBPII population offers valuable insights for the development of vivax malaria vaccines based on this antigen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial or non-financial interests which may be considered as potential conflicts of interest:, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Myanmar aims to eliminate malaria by 2030. However, recent increase of malaria incidence is a great challenge to archive that goal. Increasing prevalence of Plasmodium vivax also hinders this endeavor. Monitoring genetic structure of the parasite is necessary to understand genetic nature and evolutionary aspect of P. vivax population in Myanmar. Partial fragment flanking blocks I and II of merozoite surface protein-3 alpha of P. vivax (pvmsp-3α) was amplified from P. vivax isolates collected in Pyin Oo Lwin, Mandalay Region, Myanmar in 2013-2015. Sequence analysis of pvmsp-3α was performed to determine genetic diversity and natural selection of this gene. Spatio-temporal genetic changes of pvmsp-3α in Myanmar P. vivax population were also investigated via comparative analysis of gene sequences obtained in this study and previously reported Myanmar pvmsp-3α sequences. Genetic diversity of Myanmar pvmsp-3α was detected in P. vivax isolates analyzed. Size polymorphisms in block I and amino acid changes and recombination events in block II were main factors contributing to the genetic diversity of pvmsp-3α. Comparative spatio-temporal analysis with previously reported Myanmar pvmsp-3α populations revealed the presence of genetic differences by population with moderate genetic differentiation between populations. Similar pattern of natural selection was also detected in Myanmar pvmsp-3α populations. These suggested that enough size of the P. vivax population sufficient to generate or maintain the genetic diversity remains in the population. Thus, continuous molecular surveillance of genetic structure of Myanmar P. vivax is necessary., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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.

Plasmodium vivax variant interspersed repeats (vir) refer to the key protein used for escaping the host immune system. Knowledge in the genetic variation of vir genes can be used for the development of vaccines or diagnostic methods. Therefore, we evaluated the genetic diversity of the vir genes of P. vivax populations of several Asian countries, including Pakistan, which is a malaria-endemic country experiencing a significant rise in malaria cases in recent years. We analyzed the genetic diversity and population structure of 4 vir genes (vir 4, vir 12, vir 21, and vir 27) in the Pakistan P. vivax population and compared these features to those of the corresponding vir genes in other Asian countries. In Pakistan, vir 4 (S=198, H=9, Hd=0.889, Tajima's D value=1.12321) was the most genetically heterogenous, while the features of vir 21 (S=8, H=7, Hd=0.664, Tajima's D value =-0.63763) and vir 27 (S =25, H =11, Hd =0.682, Tajima's D value=-2.10836) were relatively conserved. Additionally, vir 4 was the most genetically diverse among Asian P. vivax populations, although within population diversity was low. Meanwhile, vir 21 and vir 27 among all Asian populations were closely related genetically. Our findings on the genetic diversity of vir genes and its relationships between populations in diverse geographical locations contribute toward a better understanding of the genetic characteristics of vir. The high level of genetic diversity of vir 4 suggests that this gene can be a useful genetic marker for understanding the P. vivax population structure. Longitudinal genetic diversity studies of vir genes in P. vivax isolates obtained from more diverse geographical areas are needed to better understand the function of vir genes and their use for the development of malaria control measures, such as vaccines.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is one of the most common X-linked hereditary disorders worldwide. G6PD deficiency provides resistance against severe malaria, but paradoxically, G6PD deficiency is also a stumbling block in fighting against malaria. Primaquine (PQ), a drug for the radical cure of Plasmodium vivax, can cause lethal acute hemolytic anemia in malaria patients with inherited G6PD deficiency. In this study, we analyzed the phenotypic and genotypic G6PD deficiency status in 1721 individuals (963 males and 758 females) residing in three malaria-endemic areas within the Gia Lai province, Vietnam. The G6PD activity in individuals ranged from 3.04 to 47.82 U/g Hb, with the adjusted male median (AMM) of 7.89 U/g Hb. Based on the G6PD activity assay results, no phenotypic G6PD deficiency was detected. However, the multiplex polymerase chain reaction to detect G6PD variations in the gene level revealed that 26 individuals (7 males, 19 females) had Viangchan mutations (871 G > A). Sequencing analyses suggested that all the males were hemizygous Viangchan, whereas one was homozygous, and 18 were heterozygous Viangchan in females. These results suggested a relatively low prevalence of G6PD deficiency mutation rate (1.51%) in the minor ethnic populations residing in the Gia Lai province, Vietnam. However, considering these areas are high-risk malaria endemic, concern for proper and safe use of PQ as a radical cure of malaria is needed by combining a G6PD deficiency test before PQ prescription., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 μg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Background: Acanthamoeba is an opportunistic pathogen that can cause human infections such as granulomatous amebic encephalitis and acanthamoeba keratitis. However, no specific drug to treat the diseases has been developed. Therefore, the discovery or development of novel drugs for treating Acanthamoeba infections is urgently needed. The anti-protozoan activity of (‒)-epicatechin (EC) has been reported, suggesting it is an attractive anti-protozoal drug candidate. In this study, the amoebicidal activity of EC against A. castellanii was assessed and its mechanism of action was unveiled., Methods: The amoebicidal activity of EC against A. castellanii trophozoites and the cytotoxicity of EC in HCE-2 and C6 cells were determined with cell viability assay. The underlying amoebicidal mechanism of EC against A. castellanii was analyzed by the apoptosis/necrosis assay, TUNEL assay, mitochondrial dysfunction assay, caspase-3 assay, and quantitative reverse transcription polymerase chain reaction. The cysticidal activity of EC was also investigated., Results: EC revealed amoebicidal activity against A. castellanii trophozoites with an IC 50 of 37.01 ± 3.96 µM, but was not cytotoxic to HCE-2 or C6 cells. EC induced apoptotic events such as increases in DNA fragmentation and intracellular reactive oxygen species production in A. castellanii. EC also caused mitochondrial dysfunction in the amoebae, as evidenced by the loss of mitochondrial membrane potential and reductions in ATP production. Caspase-3 activity, autophagosome formation, and the expression levels of autophagy-related genes were also increased in EC-treated amoebae. EC led to the partial death of cysts and the inhibition of excystation., Conclusion: EC revealed promising amoebicidal activity against A. castellanii trophozoites via programmed cell death events. EC could be a candidate drug or supplemental compound for treating Acanthamoeba infections., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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., (© 2023. The Author(s).)

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.

Background: Naegleria fowleri is a brain-eating amoeba causing a fatal brain infection called primary amoebic meningoencephalitis (PAM). Despite its high mortality over 95%, effective therapeutic drug for PAM has not been developed yet. Therefore, development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated anti-amoebic effect of kaempferol (KPF) against N. fowleri and its underlying anti-amoebic molecular mechanisms., Methods: Anti-amoebic activity of KPF against N. fowleri trophozoites, as well as cytotoxicity of KPF in C6 glial cells and CHO-K1 cells were investigated. The programmed cell death mechanisms in KPF-treated N. fowleri were also analyzed by apoptosis-necrosis assay, mitochondrial dysfunction assay, TUNEL assay, RT-qPCR, and CYTO-ID assay., Results: KPF showed anti-amoebic activity against N. fowleri trophozoites with an IC 50 of 29.28 ± 0.63 μM. However, it showed no significant cytotoxicity to mammalian cells. KPF induced significant morphological alterations of the amoebae, resulting in death. Signals associated with apoptosis were detected in the amoebae upon treatment with KPF. KPF induced an increase of intracellular reactive oxygen species level, loss of mitochondrial membrane potential, increases of expression levels of genes associated with mitochondria dysfunction, and reduction of ATP levels in the amoebae. Autophagic vacuole accumulations with increased expression levels of autophagy-related genes were also detected in KPF-treated amoebae., Conclusion: KPF induces programmed cell death in N. fowleri trophozoites via apoptosis-like pathway and autophagy pathway. KPF could be used as a candidate of anti-amoebic drug or supplement compound in the process of developing or optimizing therapeutic drug for PAM., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Extracellular vesicles (EVs) of protozoan parasites have diverse biological functions that are essential for parasite survival and host-parasite interactions. In this study, we characterized the functional properties of EVs from Naegleria fowleri , a pathogenic amoeba that causes a fatal brain infection called primary amoebic meningoencephalitis (PAM). N. fowleri EVs (NfEVs) have been shown to be internalized by host cells such as C6 glial cells and BV-2 microglial cells without causing direct cell death, indicating their potential roles in modulating host cell functions. NfEVs induced increased expression of proinflammatory cytokines and chemokines such as TNF-α, IL-1α, IL-1β, IL-6, IL-17, IFN-γ, MIP-1α, and MIP-2 in BV-2 microglial cells; these increases were initiated via MyD88-dependent TLR-2/TLR-4. The production levels of proinflammatory cytokines and chemokines in NfEVs-stimulated BV-2 microglial cells were effectively downregulated by inhibitors of MAPK, NF-κB, or JAK-STAT. Phosphorylation levels of JNK, p38, ERK, p65, JAK-1, and STAT3 were increased in NfEVs-stimulated BV-2 microglial cells but were effectively suppressed by each corresponding inhibitor. These results suggest that NfEVs could induce proinflammatory immune responses in BV-2 microglial cells via the NF-κB-dependent MAPK and JAK-STAT signaling pathways. Taken together, these findings suggest that NfEVs are pathogenic factors involved in the contact-independent pathogenic mechanisms of N. fowleri by inducing proinflammatory immune responses in BV-2 microglial cells, further contributing to deleterious inflammation in infected foci by activating subsequent inflammation cascades in other brain cells.

Rationale: Clinostomum complanatum is a laryngeal fluke whose hosts include birds and mammals.[1] In humans, infection occurs accidentally during the consumption of raw freshwater fish.[1,2]., Patient Concerns: A 59-year-old female presented to our hospital with throat pain and globus sensation. The patient had been prescribed Proton Pump Inibitor for 3 weeks at another hospital. The patient continued the medication, but the discomfort persisted, and she was admitted to our hospital for further examination. The patient had eaten raw fish 24 days before, and the symptoms occurred after eating the raw fish. Endoscopy under sedation showed a fluke, with an approximate length of 8.0 mm and width of 3.2 mm, on the interaryepiglottic fold, with active motility on the mucosa., Diagnosis Interventions: It was extracted from the larynx using biopsy forceps and identified as C complanatum., Outcomes: After the fluke was removed, symptoms improved, and the patient was discharged. The globus symptoms completely resolved at the last follow-up visit., Lessons: To the best of our knowledge, this is an endoscopically diagnosed and treated case of human infection by C complanatum in Korea after the longest period of infection. This suggests that C complanatum can survive for up to 3 weeks or more in the gastrointestinal tract. Endoscopy is a useful tool for the diagnosis and treatment of patients with atypical extraesophageal symptoms who do not respond to Proton Pump Inibitors., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Competing Interests: Conflict of Interest Non-declared.

Background The nucleocapsid protein (N protein) of SARS-CoV-2 is undeniably a potent target for the development of diagnostic tools due to its abundant expression and lower immune evasion pressure compared to spike (S) protein. Methods Blood samples of active COVID-19 infections (n=71) and post-COVID-19 (n=11) were collected from a tertiary care hospital in India; pre-COVID-19 (n=12) sera samples served as controls. Real-time reverse transcriptase-PCR (rRT-PCR) confirmed pooled sera samples (n=5) were used with PEPperCHIP® SARS-CoV-2 Proteome Microarray (PEPperPRINT GmbH, Germany) to screen immunodominant epitopes of SARS-CoV-2. Highly immunodominant epitopes were then commercially synthesized and further validated for their immunoreactivity by dot-blot and ELISA. Results The lowest detectable concentration (LDC) of the N1 peptide in the dot-blot assay was 12.5 µg demonstrating it to be fairly immunoreactive compared to control sera. IgG titers against the contiguous peptide (N2: 156AIVLQLPQGTTLPKGFYAEGS176) was found to be significantly higher (p=0.018) in post-COVID-19 compared to pre-COVID-19 control sera. These results suggested that N2-specific IgG titers buildup over time as expected in post-COVID-19 sera samples, while a non-significant immunoreactivity of the N2 peptide was also observed in active-COVID-19 sera samples. However, there were no significant differences in the total IgG titers between active COVID-19 infections, post-COVID-19 and pre-COVID-19 controls. Conclusion The N2-specific IgG titers in post-COVID-19 samples demonstrated the potential of N protein as an exposure biomarker, particularly in sero-surveillance studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Vashisht et al.)

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var multigene family, is a highly polymorphic antigen that plays a crucial role in the pathology of malaria. The contribution of the genetic diversity of var toward the immune escape of P. falciparum has not yet been fully elucidated. This study aimed to characterize the diversity of var repertoires by screening P. falciparum Duffy-binding-like α domain (PfDBLα) among field isolates from central Myanmar. Genetic analysis revealed that the D-H segments of var in Myanmar populations have an extensive polymorphic repertoire, with high numbers of unique sequence types in each individual. However, var genes from the global population, including Myanmar, shared close genetic lineages regardless of their geographic origins, indicating that they have not undergone rapid evolutionary changes.

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.

Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian, and Latin American regions. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often accomplished by the administration of mass-scale Rapid diagnostic tests (RDTs). The inherent limitations of RDTs- insensitivity in scenarios of low transmission settings and deletion of one of the target proteins- Histidine rich protein 2/3 (HRP-2/3) are evident from multiple reports, thus necessitating the need to explore novel diagnostic tools/targets. The present study used peptide microarray to screen potential epitopes from 13 antigenic proteins (CSP, EXP1, LSA1, TRAP, AARP, AMA1, GLURP, MSP1, MSP2, MSP3, MSP4, P48/45, HAP2) of P. falciparum. Three cyclic constrained immunoreactive peptides- C6 (EXP1), A8 (MSP2), B7 (GLURP) were identified from 5458 cyclic constrained peptides (in duplicate) against P. falciparum-infected sera. Peptides (C6, A8, B7- cyclic constrained) and (G11, DSQ, NQN- corresponding linear peptides) were fairly immunoreactive towards P. falciparum-infected sera in dot-blot assay. Using direct ELISA, cyclic constrained peptides (C6 and B7) were found to be specific to P. falciparum-infected sera. A substantial number of samples were tested and the peptides successfully differentiated the P. falciparum positive and negative samples with high confidence. In conclusion, the study identified 3 cyclic constrained immunoreactive peptides (C6, B7, and A8) from P. falciparum secretory/surface proteins and further validated for diagnostic potential of 2 peptides (C6 and B7) with field-collected P. falciparum-infected sera samples., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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.

Clonorchis sinensis is a carcinogenic liver fluke that causes clonorchiasis in humans. Clonorchiasis is prevalent in East Asian countries, and approximately 15–20 million individuals are estimated to be infected with this fluke globally. This review highlights the current status of C. sinensis and clonorchiasis in Korea from the epidemiological perspective involving the analysis of humans and intermediate hosts. Despite the recent decline in C. sinensis infection rate in Korea, C. sinensis infections remain endemic in 5 major river basins (Han-gang, Geum-gang, Seomjin-gang, Yeongsan-gang and Nakdong-gang; gang means river) with a high incidence of cholangiocarcinoma. A noticeable pattern involves increasing mild infections among patients diagnosed positive for C. sinensis eggs. The infection rate of C. sinensis metacercariae in the second intermediate host, freshwater fish, is also maintained at a substantial level. Thus, the One Health approach integrating different sectors and disciplines is recommended to accelerate and sustain control of C. sinensis , thereby leading to successful eradication. Health promotion via information dissemination and health education should be extended to prevent the consumption of raw freshwater fish by residents living in high-risk areas.

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.

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 flanking731Leu-780 Leu 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.

Naegleria fowleri is a ubiquitous protozoa parasite that can cause primary amoebic meningoencephalitis (PAM), a fatal brain infection in humans. Cathepsin Bs of N. fowleri (NfCBs) are multifamily enzymes. Although their pathogenic mechanism in PAM is not clearly understood yet, NfCBs have been proposed as pathogenic factors involved in the pathogenicity of amoeba. In this study, the immune response of BV-2 microglial cells induced by NfCB was analyzed. Recombinant NfCB (rNfCB) evoked enhanced expressions of TLR-2, TLR-4, and MyD88 in BV-2 microglial cells. This enzyme also induced an elevated production of several pro-inflammatory cytokines such as TNF-α, IL-1α, IL-1β, and IL-6 and iNOS in cells. The inhibition of mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK, effectively reduced the production of these pro-inflammatory cytokines. The rNfCB-induced production of pro-inflammatory cytokines in BV-2 microglial cells was suppressed by inhibiting NF-kB and AP-1. Phosphorylation and nuclear translocation of p65 in cells were also enhanced by rNfCB. These results suggest that NfCB can induce a pro-inflammatory immune response in BV-2 microglial cells via the NF-κB- and AP-1-dependent MAPK signaling pathways. Such a NfCB-induced pro-inflammatory immune response in BV-2 microglial cells might contribute to the pathogenesis of PAM caused by amoeba, by exacerbating deleterious immune responses and tissue damages in N. fowleri -infected foci of the brain.

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., (© 2022. The Author(s).)

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.

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., (© 2022. The Author(s).)

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.

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.

Apical membrane antigen-1 of Plasmodium falciparum (PfAMA-1) is a leading malaria vaccine candidate antigen. However, the genetic diversity of pfama-1 and associated antigenic variation in global P. falciparum field isolates are major hurdles to the design of an efficacious vaccine formulated with this antigen. Here, we analyzed the genetic structure and the natural selection of pfama-1 in the P. falciparum population of Vietnam. A total of 37 distinct haplotypes were found in 131 P. falciparum Vietnamese isolates. Most amino acid changes detected in Vietnamese pfama-1 were localized in the ectodomain, domains I, II, and III. Overall patterns of major amino acid changes in Vietnamese pfama-1 were similar to those of global pfama-1 , but the frequencies of the amino acid changes slightly differed by country. Novel amino acid changes were also identified in Vietnamese pfama-1 . Vietnamese pfama-1 revealed relatively lower genetic diversity than currently analyzed pfama-1 in other geographical regions, and suggested a distinct genetic differentiation pattern. Evidence for natural selection was detected in Vietnamese pfama-1 , but it showed purifying selection unlike the global pfama-1 analyzed so far. Recombination events were also found in Vietnamese pfama-1 . Major amino acid changes that were commonly identified in global pfama-1 were mainly localized to predicted B-cell epitopes, RBC-binding sites, and IUR regions. These results provide important information for understanding the genetic nature of the Vietnamese pfama-1 population, and have significant implications for the design of a vaccine based on PfAMA-1.

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.

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.

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.

Avian coccidiosis is a disease caused by members of the genus Eimeria . Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

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 Asp 219 , higher occupancy (0% to 99%) of hydrogen bond than that of Asp 33 , 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 ( d 1 ), TriCα angles ( θ 1 and θ 2 ), 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.

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.

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.

Vietnam achieved outstanding success against malaria in the last few decades. The mortality and morbidity of malaria in Vietnam have decreased remarkably in recent years, but malaria is still a major public health concern in the country, particularly in the Central Highlands region. In this study, molecular analyses of malaria parasites in the Central Highlands were performed to understand the population structure and genetic diversity of the parasites circulating in the region. Plasmodium falciparum (68.7%) and P. vivax (27.4%) along with mixed infections with P. falciparum/P. vivax (3.9%) were detected in 230 blood samples from patients with malaria. Allele-specific nested-polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of pfmsp-1, pfama-1, pvcsp, and pvmsp-1 revealed complex genetic makeup in P. falciparum and P. vivax populations of Vietnam. Substantial multiplicity of infection (MOI) was also identified, suggesting significant genetic diversity and polymorphism of P. falciparum and P. vivax populations in the Central Highlands of Vietnam. These results provide fundamental insight into the current patterns of dispersion and genetic nature of malaria parasites as well as for the development of malaria elimination strategies in the endemic region., (Copyright © 2021. Published by Elsevier B.V.)

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 .

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.

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.

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 cmH 2 O 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.

Background: Vitamin 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., Methods: Serum 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., Results: The 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., Conclusion: Our 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., Competing Interests: This study was funded by Roche Diagnostics Korea. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

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.

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.

The Plasmodium vivax variant proteins encoded by vir genes are highly polymorphic antigens and are considered as one of key proteins of P. vivax for host immune evasion via antigenic variations. Because genetic diversity of these antigens is a critical hurdle in the development of an effective vaccine, understanding the genetic nature of the vir genes in natural population is important. In this study, we selected four vir genes (vir 4, vir 12, vir 21, and vir 27) previously used for genetic analysis in several studies and evaluated the genetic polymorphisms and phylogenetic relationship of these 4 vir genes in Myanmar P. vivax population. Taken all genetic diversity values, the vir 12 (S = 168, H = 17, Hd = 0.854, Tajima's D value = 2.91524) was the most genetically diverse gene and the vir 4 (S = 9, H = 4, Hd = 0.744, Tajima's D value = -0.49151) was the most conserved gene. All phylogenetic trees showed two clades, and vir 4 and 12 haplotypes from Myanmar were clustered in a distinct clade with those from India and Republic of Korea. These results confirmed the pattern of high genetic polymorphism of vir genes and provided information on vir gene for further functional research and studies focused toward the practical use of vir genes., (Copyright © 2020 Elsevier B.V. All rights reserved.)