AUTHOR=Aboutalebian Shima , Ahmadikia Kazem , Fakhim Hamed , Chabavizadeh Javaher , Okhovat Ahmadreza , Nikaeen Mahnaz , Mirhendi Hossein 

TITLE=Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2021.644060

DOI=10.3389/fcimb.2021.644060

ISSN=2235-2988

ABSTRACT=<sec><title>Background</title><p>Considering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients.</p></sec><sec><title>Methods and Materials</title><p>A total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol–chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers. <italic>Pseudomonas</italic> and <italic>Staphylococcus</italic> specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, <italic>Candida</italic> species-specific multiplex primers targeting the most clinically important <italic>Candida</italic> species causing OE (<italic>i.e</italic>., <italic>C. albicans</italic>, <italic>C. parapsilosis</italic>, and <italic>C. auris</italic>), as well as <italic>Aspergillus</italic> related multiplex PCR identifying the most prevalent <italic>Aspergillus</italic> species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.</p></sec><sec><title>Results</title><p>The overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for <italic>Staphylococcus</italic> and <italic>Pseudomonas</italic>, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained <italic>Candida</italic> and <italic>Aspergillus</italic> species, respectively. It should be noted that dual amplicon bands of <italic>Candida</italic> and <italic>Aspergillus</italic>-related multiplex PCR were yielded in 30 specimens.</p></sec><sec><title>Conclusion</title><p>The stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.</p></sec>