Corrigendum: The first molecular genotyping of Naegleria fowleri causing primary amebic meningoencephalitis in Thailand with epidemiology and clinical case reviews.

[This corrects the article DOI: 10.3389/fcimb.2022.931546.].

The First Molecular Genotyping of Naegleria fowleri Causing Primary Amebic Meningoencephalitis in Thailand With Epidemiology and Clinical Case Reviews.

Primary amebic meningoencephalitis (PAM) is a rare and fatal central nervous system infection caused by Naegleria fowleri, a free-living amoeba found in the environment. To date, eight pathogenic N. fowleri genotypes have been reported worldwide. We aimed to explore the genotypes of N. fowleri that cause primary amebic meningoencephalitis in Thailand. In 2021, the 17th PAM case was reported, and a retrospective literature search of PAM cases in Thailand from 1982 through April 2021 was performed. Phylogenetic and genotyping analyses of the two mitochondrial (12S rRNA and 16S rRNA) and nuclear (ITS1 and 5.8s rRNA) genes of N. fowleri were performed on four available clinical isolates. Based on the mitochondrial and nuclear genes, N. fowleri genotype T3 was found to cause PAM in three out of four cases. However, disagreement between the genotype based on the mitochondrial and nuclear genes was found in one of the PAM cases, in which the 12S rRNA locus suggested the causative genotype as T1, while the ITS1 implied genotype T4. The discrepancy between the mitochondrial and nuclear genome was previously observed, which suggests the possible horizontal gene transfer among N. fowleri species. Based on the ITS1 gene, two N. fowleri genotypes, T3 and T4, were found to be the genotypes causing PAM in this study. In addition, N. fowleri genotype T2 was previously reported in a traveler who was infected in Thailand. Thus, at least three genotypes (T2, T3, and T4) of N. fowleri are found to be associated with PAM in Thailand.

Extracellular Vesicles from Naegleria fowleri Induce IL-8 Response in THP-1 Macrophage.

Extracellular vesicles (EVs) released from pathogenic protozoans play crucial roles in host-parasite communication and disease pathogenesis. Naegleria fowleri is a free-living protozoan causing primary amoebic meningoencephalitis, a fatal disease in the central nervous system. This study aims to explore the roles of N. fowleri-derived EVs (Nf-EVs) in host-pathogen interactions using the THP-1 cell line as a model. The Nf-EVs were isolated from the N. fowleri trophozoite culture supernatant using sequential centrifugation and characterized by nanoparticle tracking analysis and transmission electron microscopy. The functional roles of Nf-EVs in the apoptosis and immune response induction of THP-1 monocytes and macrophages were examined by flow cytometry, quantitative PCR, and ELISA. Results showed that Nf-EVs displayed vesicles with bilayer membrane structure approximately 130-170 nm in diameter. The Nf-EVs can be internalized by macrophages and induce macrophage responses by induction of the expression of costimulatory molecules CD80, CD86, HLA-DR, and CD169 and the production of cytokine IL-8. However, Nf-EVs did not affect the apoptosis of macrophages. These findings illustrate the potential role of Nf-EVs in mediating the host immune cell activation and disease pathogenesis.

ANTI-AMEBIC ACTIVITY OF DIOSGENIN ON NAEGLERIA FOWLERI TROPHOZOITES.

The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 µg/ml; 241.2 µM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.

Effect of synthetic antimicrobial peptides on Naegleria fowleri trophozoites.

We evaluated the effect of tritrpticin, lactoferrin, killer decapeptide and scrambled peptide in vitro against Naegleria fowleri trophozoites compared with amphotericin B. Tritrpticin (100 microg/ml) caused apoptosis of N. fowleri trophozoites (2x10(5) cells/ml), while lactoferrin, killer decapeptide and scrambled peptide did not. On Gormori trichrome staining, tritrpticin affected the elasticity of the surface membrane and reduced the size of the nuclei of N. fowleri trophozoites. The ultrastructure surface membrane and food cup formation of the trophozoites were 100% inhibited. These results are consistent with inhibition of the nfa1, Mp2CL5 of the treated trophozoite, which plays a role in food cup formation. Tritrpticin 100 microg/ml was not toxic against SK-N-MC cells. Our findings suggest tritrpticin has activity against the surface membrane and nfa1 and Mp2CL5 of N. fowleri trophozoites and could be developed as a potential therapeutic agent.

Multiplex PCR for identifying common dust mites species (Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis).

INTRODUCTION: Dust mites are known to be an important source of inhalant allergens causing allergic rhinitis and asthma worldwide. The sizes of dust mite populations in patients' houses are useful to monitor the risk of allergen exposure. However, mite identification using the conventional microscopic technique requires specific expertise and is time consuming; therefore a molecular technique has been developed in order to solve these drawbacks. OBJECTIVE: To develop a multiplex PCR assay for identifying the three common dust mite species in Thailand, namely Dermatophagoides pteronyssinus (Dp), D. farinae (Df) and Blomia tropicalis (Bt), and to evaluate the efficacy of the technique. METHODS: Pairs of primers were designed and tested in either singleplex PCR or multiplex PCR. The multiplex PCR technique was also optimized in order to obtain specific products. The reaction mixture contained 5 pmole of individual primers, 10 mM dNTP, 5 units Taq DNA polymerase and genomic DNA (gDNA). The reaction was run for 25 cycles at 94 degrees C for 20 seconds, 58 degrees C for 20 seconds and 72 degrees C for 30 seconds. The PCR products were analyzed by 1.5% agarose gel electrophoresis with GelRed fluorescence dye. The optimized multiplex technique was also tested with 30 house dust samples and dust samples spiked with DNA from other insect and mite species. RESULTS: Three PCR products were obtained with the relevant gDNA templates as expected; 143 bp for DF, 221 bp for DP and 318 bp for BT, respectively. The detection limit of the tests was found to be as low 1 ng of gDNA, whereas mixed gDNA species confirmed the 100% specificity of this assay. The total duration from the preparation of the PCR reaction mixture until the analysis by agarose gel electrophoresis was approximately 2 hours. No amplified product was obtained from mites and insects of other species. CONCLUSION: The multiplex PCR was successfully developed for identifying 3 common dust mite species. This technique can be helpful, not only for non-acarologist personnel for dust mite identification, but also for patients who are allergic to dust mites.

Scanning electron microscopic study of human neuroblastoma cells affected with Naegleria fowleri Thai strains.

In order to understand the pathogenesis of Naegleria fowleri in primary amoebic meningoencephalitis, the human neuroblastoma (SK-N-MC) and African green monkey kidney (Vero) cells were studied in vitro. Amoeba suspension in cell-culture medium was added to the confluent monolayer of SK-N-MC and Vero cells. The cytopathic activity of N. fowleri trophozoites in co-culture system was elucidated by scanning electron microscope at 3, 6, 9, 12, and 24 h. Two strains of N. fowleri displayed well-organized vigorous pseudopods in Nelson's medium at 37 degrees C. In co-culture, the target monolayer cells were damaged by two mechanisms, phagocytosis by vigorous pseudopods and engulfment by sucker-like apparatus. N. fowleri trophozoites produced amoebostomes only in co-culture with SK-N-MC cells. In contrast, we could not find such apparatus in the co-culture with Vero cells. The complete destruction time (100%) at 1:1 amoeba/cells ratio of SK-N-MC cells (1 day) was shorter than the Vero cells (12 days). In conclusion, SK-N-MC cells were confirmed to be a target model for studying neuropathogenesis of primary amoebic meningoencephalitis.

Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells.

The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient's cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7 days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients.

Zymogram patterns of Naegleria spp isolated from natural water sources in Taling Chan district, Bangkok.

A genetic approach was cited for species detection of the ameba genus Naegleria using allozyme electrophoresis to characterize the trophozoite stage of three strains of Naegleria fowleri isolated from patients with primary amebic meningoencephalitis, five thermophilic (45 degrees C) Naegleria spp isolated from natural water sources in the Taling Chan district, and a reference control strain, Naegleria fowleri CDC VO 3081. Isoenzymes of ameba whole-cell extracts were analyzed by vertical polyacrylamide slab gel electrophoresis to determine whether there was any correlation between different strains of the ameba. The results showed that five out of fifteen enzymes; aldehyde oxidase (ALDOX), aldolase (ALD), a-glycerophosphate dehydrogenase (a-GPDH), xanthine dehydrogenase (XDH), and glutamate oxaloacetate transaminase (GOT), were undetectable in the pathogenic strains, while the other enzymes; esterase (EST), fumerase (FUM), glucose-6-phosphate dehydrogenase (G-6-PDH), glucose phosphate isomerase (GPI), isocitate dehydrogenase (IDH), lactate dehydrogenase (LDH), leucine aminopeptidase (LAP), malic enzyme (ME), glucose phosphomutase (GPM), and malate dehydrogenase (MDH), were detected. Naegleria fowleri strains were biochemically the most homogeneous. They showed intraspecific isoenzyme variation that allowed them to be grouped. In contrast, the allozyme patterns (EST 1-7, IDH) of Naegleria spp isolated from the environment showed interspecific isoenzyme variations from the pathogenic Naegleria strain. In conclusion, this study recognized the zymograms of the Naegleria fowleri strains were heterogenically different from the thermophilic 45 degrees C Naegleria spp isolated from the environment.

Effect of antifungal drugs on pathogenic Naegleria spp isolated from natural water sources.

UNLABELLED: Five of 16 strains of pathogenic Naegleria spp isolated from 350 natural water sources in Taling Chan District, Bangkok had similar molecular weights and zymogram patterns to those of Naegleria fowleri CDCVO 3081 and Thai strain. The in vitro effects of antifungal drugs (amphotericin B, ketoconazole, fluconazole and itraconazole) were tested at the following concentrations: amphotericin B 0.01-0.55 microg/ml, ketoconazole 0.01-0.3 microg/ml, fluconazole 0.75-3.5 mg/ml and itraconazole 4-12 mg/ml respectively. Aliquots (15,000 cells/ml) of the amoebae were placed in the cells of the microtiter plate and incubated at 37 degrees C. Amoebae from each treatment sample were exposed to one of the four antifungal drugs. Statistical analysis was done by dependent t-test. The sensitivity of the antifungal drugs (MIC50) was as follows: amphotericin B 0.03-0.035 microg/ml ketoconazole 0.05-0.15 microg/ml fluconazole 1.75 mg/ml and itraconazole 8-9 mg/ml respectively (p < 0.005). CONCLUSION: Amphotericin B and ketoconazole are more active against Naegleria fowleri in vitro. The results of the present study should be used as an in vitro screening test for drugs that have potential amebicidal activity.

In vitro effect of antifungal drugs on pathogenic Naegleria spp.

An ameba of the genus Naegleria causing fatal meningoencephalitis in human subjects was investigated for its sensitivity to antifungal drugs: amphotericin B, ketoconazole, fluconazole and itraconazole. The efficacy of these antifungal drugs for pathogenic Naegleria spp was investigated in three strains isolated from patients who had died of primary amebic meningoencephalitis infection at Siriraj Hospital (1986), Ramathibodi Hospital (1987) and Chachoengsao Hospital (1987). All of the isolates were maintained in axenic culture in the Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand. The sensitivities of the antifungal drugs (MIC50) were: amphotericin B (0.05-0.5 microg/ml), ketoconazole (0.125 microg/ml), fluconazole (0.5-2.0 mg/ml), and itraconazole (10 mg/ml) (p < 0.05). It is important to explain that ketoconazole is slightly more effective than amphotericin B because its action is directed of the permeability of the amebic membrane. The amebae were more resistant ot fluconazole and itraconazole due to the action of the cytochrome P450 multienzyme (in the case of fluconazole) and the direct effect on heme-iron, blocking cytochrome P450-dependent chitin synthesis (in the case of itraconzole). We conclude that amphotericin B and ketoconazole remain the main drugs with proven activity against pathogenic Naegleria spp.