Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse

Kang, Hyung Suk; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho‐Joon

doi:10.1016/j.ejop.2015.07.003

Cited by 14 publications

(12 citation statements)

References 34 publications

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“…N. fowleri , known as “brain-eating amoeba”, causes acute PAM in children and young adults with a history of diving in freshwater and recreational activities in amoebae-contaminated water in the hot summer [2,16]. In these latter days, death due to PAM is rearing its ugly head as a Neglected Tropical Disease (NTD) in countries of Asia, such as India, Pakistan, Taiwan, Thailand, and Vietnam [17].…”

Section: Discussionmentioning

confidence: 99%

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae

et al. 2017

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Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page’s amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

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Section: Discussionmentioning

confidence: 99%

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae

et al. 2017

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“…Conventional PCR was performed to detect the protozoa Entamoeba histolytica , Toxoplasma gondii , Naegleria fowleri , and Acanthamoeba spp. based on previous studies [ 6 - 9 ]. N. fowleri -specific PCR was performed using the following primer set to amplify the amplicon of predicted size 183 bp: Nae3-F (5´-CAAACACCGTTATGACAGGG-3´) and Nae3-R (5´-CTGGTTTCCCTCACCTTACG-3´) [ 6 ].…”

Section: Methodsmentioning

confidence: 99%

Diagnosis of Balamuthia mandrillaris Encephalitis by Thymine–Adenine Cloning Using Universal Eukaryotic Primers

Kim

et al. 2022

Ann Lab Med

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Background: Identifying the causal pathogen of encephalitis remains a clinical challenge. A 50-year-old man without a history of neurological disease was referred to our department for the evaluation of an intracranial lesion observed on brain magnetic resonance imaging (MRI) scans, and the pathology results suggested protozoal infection. We identified the species responsible for encephalitis using thymine-adenine (TA) cloning, suitable for routine clinical practice.Methods: We extracted DNA from a paraffin-embedded brain biopsy sample and performed TA cloning using two universal eukaryotic primers targeting the V4-5 and V9 regions of the 18S rRNA gene. The recombinant plasmids were extracted, and the inserted amplicons were identified by Sanger sequencing and a homology search of sequences in the National Center for Biotechnology Information Basic Local Alignment Search Tool. Results:The infection was confirmed to be caused by the free-living amoeba Balamuthia mandrillaris. Two of 41 colonies recombinant with 18S V4-5 primers and 35 of 63 colonies recombinant with the 18S V9 primer contained B. mandrillaris genes; all other colonies contained human genes. Pathogen-specific PCR ruled out Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp., and Toxoplasma gondii infections.Conclusions: This is the first report of B. mandrillaris-induced encephalitis in Korea based on molecular identification. TA cloning with the 18S rRNA gene is a feasible and affordable diagnostic tool for the detection of infectious agents of unknown etiology.

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“…The amoebae were sub-cultured on fresh NN-agar plates two or three times. Further, N. fowleri, A. castellanii, and A. polyphaga used as positive control amoebae were cultured on Nelson's and PYG medium according to previous reports 29,30 . The morphological identification of cultured amoebae was observed with an inverted microscope (Olympus CKX 31, Japan).…”

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Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea

Kang

Sohn

Seo

et al. 2020

Sci Rep

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the free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies. The free-living amoebae (FLA) Naegleria spp. and Acanthamoeba spp. are mainly distributed in ponds, rivers, and fresh waters worldwide. Their existing stages are trophozoite and cyst and additional biflagellate form in in case of Naegleria. Trophozoites shows moving, feeding, and proliferation activity. However, cysts are formed in poor environments, such as under abrupt temperature changes, drying, and food depletion, and can survive for long periods 1,2. N. fowleri is a pathogenic agent that causes primary amoebic meningoencephalitis (PAM), which is fatal to humans and laboratory animals 2,3. Acanthamoeba spp. and Balamuthia mandrillaris cause chronic granulomatous amebic encephalitis (GAE) 4-6. Further, A. castellanii and A. polyphaga can infect the eye, resulting in acanthamoeba keratitis (AK) 2,7,8. PAM is mainly associated with activities in amoeba-contaminated water (swimming or water leisure activity), use of Neti-pots in rhinitis treatment, and religious ceremonies in some Asian countries 5,9,10. The amoeba enters through the nasal cavity to invade the mucosal membrane. Subsequently, it moves into the olfactory bulb and meninges via the nasal nerve system, leading to development of meningoencephalitis 11-13. Symptoms of PAM include acute headache, anorexia, nausea, vomiting, high fever (38-40 °C), and limb dysfunction symptoms. It also progresses acutely, with a mortality rate of over 95%. Amphotericin B is mainly used as a therapeutic agent, as a combination treatment by mixing with micronazole, rifampin, and doxycycline; however, only limited therapeutic effects have been demonstrated 1,2,14-16. AK usually occurs after wearing contaminated contact lenses, improper ophthalmic surgery, or corneal injury in various cases. With the popularization of contact lenses and careless lens management, the number of AK patients continues to increase 2,7,17,18. PAM due to N. fowleri occurs annually worldwide. In the United States, 143 cases of PAM occurred from 1962 to...

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Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse

Cited by 14 publications

References 34 publications

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae

Diagnosis of Balamuthia mandrillaris Encephalitis by Thymine–Adenine Cloning Using Universal Eukaryotic Primers

Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea

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