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.

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.

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.

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.

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.

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.

Clinical utility of cerebrospinal fluid vitamin D-binding protein as a novel biomarker for the diagnosis of viral and bacterial CNS infections.

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

Awareness and Opinions of Inhabitants on Vivax Malaria in Two Endemic Areas, Gyeonggi-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.

Seroprevalence of Plasmodium vivax Circumsporozoite Protein Antibody in High-Risk Malaria Areas in Korea.

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.

Survey of Zoonotic Trematode Metacercariae in Fish from Water Systems of Geum-gang (River) in Republic of Korea.

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.

High Endemicity with Clonorchis sinensis Metacercariae in Fish from Yongjeon-cheon (Stream) in Cheongsong-gun, Gyeongsangbuk-do, Korea.

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.

Endemicity of Zoonotic Trematode Metacercariae in Fish from Deokcheon-gang (River) in Sancheong-gun, Gyeongsangnam-do, Republic of Korea.

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.

Prevalence and Intensity of Opisthorchis viverrini Metacercarial Infection in Fish from Phnom Penh, Takeo, and Kandal Provinces, Cambodia.

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.

Prevalence and Infection Intensity of Zoonotic Trematode Metacercariae in Fish from Soyang-cheon (Stream), in Wanju-gun, Jeollabuk-do, Korea.

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.

Knowledge, Attitudes and Perceptions Regarding Endemic Vivax Malaria in Inhabitants and Patients in Two Cities of Northern Gyeonggi-do, Korea, 2020.

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.

Survey of Zoonotic Trematode Metacercariae in Fish from Irrigation Canal of Togyo-jeosuji (Reservoir) in Cheorwon-gun, Gangwon-do, Republic of Korea.

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.

Genetic variations in histidine-rich protein 2 and histidine-rich protein 3 of Myanmar Plasmodium falciparum isolates.

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

Genetic diversity of Plasmodium vivax and Plasmodium falciparum lactate dehydrogenases in Myanmar isolates.

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

Genetic polymorphism and natural selection of circumsporozoite protein in Myanmar Plasmodium vivax.

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