Ann Lab Med 2022; 42(2): 196-202
Published online March 1, 2022 https://doi.org/10.3343/alm.2022.42.2.196
Copyright © Korean Society for Laboratory Medicine.
Diagnosis of Balamuthia mandrillaris Encephalitis by Thymine–Adenine Cloning Using Universal Eukaryotic Primers
1Department of Environmental Medical Biology, Institute of Tropical Medicine, Arthropods of Medical Importance Resource Bank, Yonsei University College of Medicine, Seoul, Korea; 2Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea; 3Department of Neurosurgery, Eulji University Hospital, College of Medicine, Eulji University, Daejeon, Korea
Correspondence to: Tai-Soon Yong, M.D., Ph.D.
Department of Environmental Medical Biology, Institute of Tropical Medicine, Arthropods of Medical Importance Resource Bank, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Seong Min Kim, M.D., Ph.D.
Department of Neurosurgery, Eulji University Hospital, College of Medicine, Eulji University, 95 Dunsanseo-ro, Seo-gu, Daejeon 35233, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Keywords: Amoeba, Balamuthia mandrillaris, Encephalitis, TA cloning, 18S rRNA