Demonstration of Balamuthia and Acanthamoeba mitochondrial DNA in sectioned archival brain and other tissues by the polymerase chain reaction

Abstract

Granulomatous amoebic encephalitis (GAE) is a usually fatal disease caused by the free-living amoebae Balamuthia mandrillaris and Acanthamoeba spp. The intent of this study was to determine if the polymerase chain reaction (PCR) could be used retrospectively to detect amoeba mitochondrial 16S ribosomal ribonucleic acid gene deoxyribonucleic acid (DNA) in confirmed archival tissue sections from GAE cases stored in our laboratories for 1 to 34 years. The DNA was extracted from deparaffinized sections, and appropriate primer sets for each of the two amoebae were used for DNA detection. Indirect immunofluorescent staining (IIF) of tissue sections was used as the standard for identification of amoebae against which the PCR results were compared. Sixty slides from a total of 56 cases were processed by PCR for amoeba 16S DNA. In 28 (47%) slides, there was agreement between the IIF and PCR results. In 41 of the slides (52%), no DNA was detected after PCR. In one slide (1%), the PCR and IIF results did not agree. While PCR supported IIF findings in about half of the slides, there are significant limitations in amoeba DNA identifications in formalin-fixed brain tissues. Degradation of amoeba DNA because of formalin fixation was probably a factor in limiting valid results.

Diagnosing Balamuthia mandrillaris encephalitis via next-generation sequencing in a 13-year-old girl

“…None showed cysts in the meninges [35]. Because of low trophozoite and cyst densities, indirect immunofluorescence and PCR assays have been developed to rapidly identify BAE [36,37]. Nevertheless, in most countries, the lack of availability of indirect immunofluorescence and PCR tests for B. mandrillaris limits the opportunity for diagnosis.…”

Balamuthia mandrillaris brain abscess successfully treated with complete surgical excision and prolonged combination antimicrobial therapy

“…14 A multiplex PCR for Acanthamoeba, Balamuthia, and Naegleria based on sequence of mitochondrial 16S rRNA gene has a high specificity and sensitivity (96%-98% and 99%, respectively) from fresh brain tissue, but a lower sensitivity from deformalinized specimens, and a sensitivity of only 25% from CSF. 13,17,18 Antimicrobial recommendations are based only on in vitro evidence of activity against Balamuthia mandrillaris isolates or limited case reports of in vivo use.…”

Acanthamoeba spp. un agente oportunista en infecciones humanas

“…Recientemente se ha desarrollado un PCR para el diagnóstico de Acanthamoeba 100,101,102,103,104 . El diagnóstico puede ser realizado en LCR, lavado bronquio-alveolar, tejido cerebral y otros tejidos.…”