RESUMEN
Background: 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. Methods and Materials: 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. 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. Results: 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 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. Conclusion: 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.
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Reacción en Cadena de la Polimerasa Multiplex , Otitis Externa , Bacterias , ADN de Hongos , Hongos/genética , Humanos , Sensibilidad y EspecificidadRESUMEN
PURPOSE: To elucidate the clinical and microbial epidemiology of otomycosis in Isfahan, Iran. METHODOLOGY: From January 2016 to January 2017 all patients clinically suspected of otomycosis at Al-Zahra Hospital, Isfahan, Iran were recruited. Specimens were taken using sterile swabs by an otorhinolaryngologist and subjected to culture and microscopy using potassium hydroxide and Giemsa stain. Isolated fungi were identified based on morphological and molecular characteristics. RESULTS: Otomycosis was confirmed in 97/120 patients (80.8 %). Females (72.2 %) and patients aged 30-39 years (33 %) were more commonly affected than others. Manipulation of ear canal (62.9 %) was the most common predisposing factor. Pruritus was observed in 84.54 â% of the patients followed by hearing impairment (81.4 %), and most episodes were detected over the summer (50.5 %). Culture was positive for 81 (83.5 â%) of confirmed cases and molds were the most prevalent causative agents (n=51, 63 %) followed by yeasts (n=19, 23.4 %) and yeast/mold mixes (n=11, 13.6 %). For the 16 remaining patients, no growth was seen in culture despite a positive result on direct examination. In total, 92 isolates (63 molds and 29 yeasts) were recovered in culture. Application of molecular methods showed 18 fungal species and the vast majority of them belonged to Aspergillus (n=53, 57.6 %) and Candida genus. Among the species involved, Candida parapsilosis (n=22, 22.7 %) and Aspergillus tubingensis (n=15, 15.5 %) were the most encountered species. CONCLUSION: Outcomes from this study showed a different picture of prevalence, where C. parapsilosis and A. tubingensis but not Aspergillus niger were the most species encountered from patients suffering from otomycosis.
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Aspergillus/aislamiento & purificación , Candida/aislamiento & purificación , Otomicosis/diagnóstico , Adulto , Aspergillus/genética , Biodiversidad , Candida/genética , Candida parapsilosis/genética , Candida parapsilosis/aislamiento & purificación , Estudios Transversales , Femenino , Humanos , Irán/epidemiología , Masculino , Epidemiología Molecular , Otomicosis/epidemiología , Otomicosis/microbiología , PrevalenciaRESUMEN
BACKGROUND: Diabetic patients are more susceptible to cutaneous fungal infections. The higher blood sugar levels cause increasing the cutaneous fungal infections in these patients. The main objective of this study was to find the frequency of fungal infections among cutaneous lesions of diabetic patients and to investigate azole antifungal agent susceptibility of the isolates. MATERIALS AND METHODS: In this study, type 1diabetes (n = 78) and type 2 diabetes (n = 44) comprised 47 cases (38.5%) with diabetic foot ulcers and 75 cases (61.5%) with skin and nail lesions were studied. Fungal infection was confirmed by direct examination and culture methods. Antifungal susceptibility testing by broth microdilution method was performed according to the CLSI M27-A and M38-A references. RESULTS: Out of 122 diabetic patients, thirty (24.5%) were affected with fungal infections. Frequency of fungal infection was 19.1% in patients with diabetic foot ulcer and 28% of patients with skin and nail lesions. Candida albicans and Aspergillus flavus were the most common species isolated from thirty patients with fungal infection, respectively. Susceptibility testing carried out on 18 representative isolates (13 C. albicans, five C. glabrata) revealed that 12 isolates (10 C. albicans and two C. glabrata isolates) (66.6%) were resistant (minimum inhibitory concentration [MIC] ≥64 mg/ml) to fluconazole (FCZ). Likewise, eight isolates (80%) of Aspergillus spp. were resistant (MIC ≥4 mg/ml), to itraconazole. CONCLUSION: Our finding expands current knowledge about the frequency of fungal infections in diabetic patients. We noted the high prevalence of FCZ-resistant Candida spp., particularly in diabetic foot ulcers. More attention is important in diabetic centers about this neglected issue.