[Morphological and Molecular Identification of Acanthamoeba sp., A New Pathogen for Human Respiratory Tract...

To identify the species of a morphologically Acanthamoeba-like pathogen in sputum from a patient with repeated cough.

Protozoa were isolated from the sputum and cultured for morphological observation of the trophozoites and cysts. DNA was extracted from the cultivated sample, and PCR was performed using primers as follows: 18S universal primers for amoeba family（Ami6F1 and Ami9R） and for amoeba genus（JDP1 and JDP2）, and primers for 18S full-length sequence of S-7 ATCC reference strain of Acanthamoeba griffini （AacGF and AscGR）. The 18S rRNA was sequenced, followed by homology analysis. The maximum likelihood method was used to construct phylogenetic tree.

Microscopic examination showed that the trophozites had spine and irregular-shape pseudopodia bulge. The cysts were encapsulated by double membrane layer with the inner membrane having star-like processes. As expected, PCR amplification resulted in bands of 830, 479 and 1 957 bp, respectively, which were blasted to be 99%, 99% and 100% homologous to those of A. griffini（U07412.1）. Phylogenetic tree indicated that this acanthamobe in the patient’s sample was 91.4%, 99.6%, 94.5% and 91.8% homologous to keratitis-associated A. castellanii, A. polyphage, A. cullbertsoni and A. rhysodes.

The parasite in sputum of the patient with respiratory tract infection is Acanthamoeba griffini.