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

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

doi:10.3389/fcimb.2021.644060

Abstract

BackgroundConsidering 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.Methods and MaterialsA 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. Pseudomonas and Staphylococcus specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, Candida species-specific multiplex primers targeting the most clinically important Candida species causing OE (i.e., C. albicans, C. parapsilosis, and C. auris), as well as Aspergillus related multiplex PCR identifying the most prevalent Aspergillus species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.ResultsThe 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 Staphylococcus and Pseudomonas, 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 Candida and Aspergillus species, respectively. It should be noted that dual amplicon bands of Candida and Aspergillus-related multiplex PCR were yielded in 30 specimens.ConclusionThe 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.

Highlights

Otitis externa (OE) describes a group of acute or chronic inflammatory disorders of the external auditory canal and auricle following disruption of the protective squamous epithelial layer of the ear canal (Thorne and Wetmore, 2009)
Given the great number of mixed fungal and bacterial ear infections, the current study investigated the diagnostic value of pan-fungal/pan-bacterial multiplex polymerase chain reaction (PCR) in order to differentially detect fungal and bacterial OE in the ear specimens aspirated from patients previously proven to carry fungal and bacterial ear infections
The distinction of the etiological agents affecting the ear canal is a crucial issue to ensure the accurate choice of drug warranting the successful therapy considering the different management and variations of the in vitro antimicrobial susceptibility patterns among different fungal and bacterial genera, emergence of infections caused by C. auris, fewer susceptible species to routinely used antifungal agents, and differential diagnosis (Adegbiji et al, 2017; Abastabar et al, 2019; Aboutalebian et al, 2020)

Summary

Introduction

Otitis externa (OE) describes a group of acute or chronic inflammatory disorders of the external auditory canal and auricle following disruption of the protective squamous epithelial layer of the ear canal (Thorne and Wetmore, 2009). To overcome the low isolation rate of culture and difficulties in the identification of causative agents by clinical evaluation and direct examination, PCR-based methods have been recommended in different studies (Bialek et al, 2005; Rickerts et al, 2007; Hammond et al, 2011). This study aimed to evaluate the usefulness of novel homemade multiplex PCR capable of simultaneously detecting and identifying the most commonly isolated species of bacteria and fungi directly in the ear aspiration specimens obtained from patients suspected of OE. 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

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