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

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.

Bacterial Etiologies of Ear Infection and Their Antimicrobial Susceptibility Pattern at the University of Gondar Comprehensive Specialized Hospital, Gondar, Northwest Ethiopia: A Six-Year Retrospective Study

“… 40 Different literatures also mentioned S. aureus, P. aeruginosa, Proteus species as the most common isolates from bacterial ear infection. 41–46 Since the natural habitats of most of these bacteria can be skin, environment and soil, ear infection, particularly OE from these isolates 42 , 46 is usually common.…”

Rapid Real-Time Polymerase Chain Reaction for Salmonella Serotyping Based on Novel Unique Gene Markers by Pangenome Analysis

“…Each 20-µl real-time PCR reaction mixture contained 500 nM of each primer pair, 10 µl of 2× Thunderbird SYBR R qPCR 1 http://in~silico.ehu.es/PCR/ Mix (Toyobo, Osaka, Japan), and 20 ng of template DNA. To avoid false-negative results, an internal standard targeting the bacterial 16S rRNA gene fragment was used (Aboutalebian et al, 2021). Amplification was conducted in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States), with an initial denaturation for 2 min at 95 • C, followed by 30 cycles of 95 • C for 5 s and 60 • C for 30 s. The melting curve was generated according to the following conditions: 95 • C for 15 s, 60 • C for 1 min, 95 • C for 30 s, and 60 • C for 15 s. The specificity of the primer pairs developed was tested against each Salmonella strain as well as each non-Salmonella reference strain.…”

“…In contrast, misclassified genome absent the corresponding gene markers, so some serovar had more analyzed genomes than the number of corresponding gene markers. Based on the pangenome analysis, serovar-specific Aboutalebian et al (2021).…”

Multiplex size marker (YEAST PLEX) for rapid and accurate identification of pathogenic yeasts

“…The exceptions were Candida tropicalis and Candida norvegensis , and Rhodotorula mucilaginosa , in which the intergenic spacer (IGS) region and 18S rDNA were used as the target for primer designing, respectively. Primer pairs, except for Candida albicans and Candida parapsilosis which were selected from a previous study, 32 were designed using the Geneious Prime software ( https://www.geneious.com ) by applying a maximum product size limit of 500 bp. Appropriate care was taken to ensure having the primer pairs with similar annealing temperatures to avoid false‐negative results, to reduce the risk of unspecific amplification and to avoid cross‐reaction with other species.…”