Comparison of 21-Plex PCR and API 20C AUX, MALDI-TOF MS, and rDNA Sequencing for a Wide Range of Clinically Isolated Yeast Species: Improved Identification by Combining 21-Plex PCR and API 20C AUX as an Alternative Strategy for Developing Countries.

Amir Arastehfar,Weihua Pan,Wanqing Liao,Mohammad Kord,Ferry Hagen,Sadegh Khodavaisy,Kamiar Zomorodian,Mohammad Javad Najafzadeh,Sassan Rezaie,Teun Boekhout,Hamid Badali,Sayed Jamal Hashemi,Wenjie Fang,Maryam Roudbary,Farnaz Daneshnia,Hossein Zarrinfar

doi:10.3389/fcimb.2019.00021

Abstract

Occurrence of non-Candida albicans Candida (NCAC) species that are associated with elevated MIC values and therapeutic failures are increasing. As a result, timely and accurate means of identification to the species level is becoming an essential part of diagnostic practices in clinical settings. In this study, 301 clinically isolated yeast strains recovered from various anatomical sites [Blood (n = 145), other sites (n = 156)] were used to assess the accuracy and practicality of API 20C AUX and 21-plex PCR compared to MALDI-TOF MS and large subunit rDNA (LSU rDNA). MALDI-TOF MS correctly identified 98.33% of yeast isolates, 100% of top five Candida species, 95.7% of rare yeast species, while 1.3% of isolates were misidentified. API 20C AUX correctly identified 83.7% of yeast isolates, 97.2% of top five Candida species, 61.8% of rare yeast species, while 16.2% of yeast isolates were misidentified. The 21-plex PCR, accurately identified 87.3% of yeast isolates, 100% of top five Candida species, 72% of rare yeast species, but it misidentified 1.3% of rare yeast species while 9.9% of whole yeast isolates were not identified. The combination of rapidity of 21-plex PCR and comprehensiveness of API 20C AUX, led to correct identification of 92% of included yeast isolates. Due to expensiveness of MALDI-TOF MS and sequencing, this combination strategy could be the most accurate and inexpensive alternative identification strategy for developing countries. Moreover, by the advent and development of cost-effective, reliable, and rapid PCR machines that cost 130 US dollars, 21-plex could be integrated in routine laboratories of developing and resource-limited countries to specifically identify 95% causative agents of yeast-related infections in human. Databases of MALDI-TOF MS, API 20C AUX, and the number of target species identified by 21-plex require further improvement to keep up with the diverse spectrum of yeast species.

Highlights

Increasing population of immunocompromised patients and administration of broad-spectrum antibiotics etc. (Pappas, 2006), led to a higher occurrence of fungal infections in clinical settings (Yapar, 2014)
We have compared the accuracy of the API 20C AUX and 21-plex PCR methods in the light of MALDI-TOF MS and sequencing of D1/D2 domains of rDNA
MALDI-TOF MS showed a good accuracy for identification of a diverse range of opportunistic yeast species (98.3%)

Summary

Introduction

Increasing population of immunocompromised patients and administration of broad-spectrum antibiotics etc. (Pappas, 2006), led to a higher occurrence of fungal infections in clinical settings (Yapar, 2014). Applying changes to the clinical practices and interventions resulted in epidemiological landscape and emergence of non-Candida albicans Candida (NCAC) species (Pham et al, 2014). Since the introduction of echinocandins as a prophylactic antifungal, selective pressure has aided in emergence of NCAC species that are less susceptible to this class of antifungals (Pham et al, 2014). More frequent isolation of yeast species exhibited inherent less susceptibility/acquired resistance to fluconazole and those with multi-drug resistant traits (MDR) highlight the importance of correct identification (Pfaller et al, 2008; Bizerra et al, 2014; Pham et al, 2014; Chowdhary et al, 2016). Due to the availability of trifle classes of antifungals, monitoring frequency, and epidemiology of yeast species would become an imperative practice in clinical routine laboratories

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Results

Conclusion