(‒)-Epicatechin reveals amoebicidal activity against Acanthamoeba castellanii by activating the programmed cell death pathway.

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 IC50 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.

Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Gia Lai Province, Vietnam.

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.

Antiamoebic activities of flavonoids against pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba species.

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.

Genetic polymorphism and natural selection of the erythrocyte binding antigen 175 region II in Plasmodium falciparum populations from Myanmar and Vietnam.

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.

Naegleria fowleri Extracellular Vesicles Induce Proinflammatory Immune Responses in BV-2 Microglial Cells.

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-1ß, 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.

Kaempferol induces programmed cell death in Naegleria fowleri.

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 IC50 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.

Case report of laryngeal infection by Clinostomum complanatum 24 days after ingestion of raw fish.

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.

Genetic diversity of Plasmodium falciparum erythrocyte membrane protein 1 in field isolates from central Myanmar.

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.

Exploring the Immunodominant Epitopes of SARS-CoV-2 Nucleocapsid Protein as Exposure Biomarker.

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.

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

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

Molecular Profiles of Multiple Antimalarial Drug Resistance Markers in Plasmodium falciparum and Plasmodium vivax in the Mandalay Region, Myanmar.

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.

Genetic Diversity of Circumsporozoite Surface Protein of Plasmodium vivax from the Central Highlands, Vietnam.

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.

Population genetic analyses inferred a limited genetic diversity across the pvama-1 DI domain among Plasmodium vivax isolates from Khyber Pakhtunkhwa regions of Pakistan.

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.

Mapping of the Complement C9 Binding Region on Clonorchis sinensis Paramyosin.

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.

An Evaluation of a New Quantitative Point-of Care Diagnostic to Measure Glucose-6-phosphate Dehydrogenase Activity.

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.

Naegleria fowleri Cathepsin B Induces a Pro-Inflammatory Immune Response in BV-2 Microglial Cells via NF-κB and AP-1 Dependent-MAPK Signaling Pathway.

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-1ß, 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.

Epidemiological investigation and drug resistance of Eimeria species in Korean chicken farms.

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.

Current status of Clonorchis sinensis and clonorchiasis in Korea: epidemiological perspectives integrating the data from human and intermediate hosts.

Clonorchis sinensis is a carcinogenic liver fluke that causes clonorchiasis in humans. Clonorchiasis is prevalent in East Asian countries, and approximately 15­20 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.

Cyclic constrained immunoreactive peptides from crucial P. falciparum proteins: potential implications in malaria diagnostics.

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.