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

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 depart-ment for the evaluation of an intracranial lesion observed on brain magnetic resonance imaging (MRI) scans, and the pathology results suggested protozoal infection. We identi-fied 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 per-formed TA cloning using two universal eukaryotic primers targeting the V4-5 and V9 re-gions 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, Naegle-ria 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 afford-able diagnostic tool for the detection of infectious agents of unknown etiology.

Automated summary

This study reported the first case of B. mandrillaris-induced encephalitis in Korea. Molecular identification using TA cloning with the 18S rRNA gene confirmed the presence of the pathogen. This method can serve as a feasible and affordable diagnostic tool for detecting infectious agents.