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

Abstract

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: 18S rRNA; Amoeba; Balamuthia mandrillaris; Encephalitis; TA cloning.

Fig. 1

Magnetic resonance image (MRI) and brain biopsy. T2-weighted MRI showing (A) a lesion (~21 mm×18 mm) with an irregular, marginated, heterogeneous dark signal and (B) marginal thin-rim enhancement of gadolinium at the left parietal cortex with surrounding edema (red arrows). (C, D) Hematoxylin and eosin staining of brain biopsy shows numerous amoebic trophozoites (20–25 μm) in the hemorrhagic necrosis background (arrowheads). Magnification: ×200 (C), ×1,000 (D).

Fig. 2

Pathogen-specific PCR targeting (A) Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp., Toxoplasma gondii, and (B) Balamuthia mandrillaris. Red arrows show approximate band sizes of 270, 183, and 463 bp for E. histolytica, N. fowleri, and Acanthamoeba spp., respectively. Toxoplasma gondii DNA (469 bp) was used as the positive control. Balamuthia mandrillaris-specific PCR using the patient DNA was performed with three sets of primers. Lane 1: 100 bp ladder. Lanes 2, 3, and 4: 5′Balspec 16S and 3′Balspec16S, 5′Balspec 16S and Balspec 16Sr 610, and BalaF1451 and BalaR1621 primers, respectively.

Fig. 3

Phylogenetic trees of the sequenced sample based on the (A) V4-5 region and (B) V9 region of the 18S rRNA gene. The total branch lengths of phylogenetic trees for the V4-5 and V9 regions were 0.8465 and 2.2305, respectively, computed using the maximum composite likelihood method and measured in base substitutions per site. Sequences of 18S rRNA genes of reference organisms were obtained from GenBank (National Center for Biotechnology Information).