Development a hydrolysis probe-based quantitative PCR assay for the specific detection and quantification of Candida auris

Document Type : Original Articles

Authors

1 Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

10.18502/cmm.6.3.4665

Abstract

Background and Purpose: Candida auris is an emerging multidrug-resistant pathogen. The identification of this species with the conventional phenotypic or biochemical mycological methods may lead to misidentification. Molecular-based species-specific identification methods such as quantitative real-time polymerase chain reaction (qPCR) facilitate a more reliable identification of C. auris than mycological methods. Regarding this, the present study aimed to develop a hydrolysis probe-based qPCR assay for the rapid, accurate identification of C. auris.
Materials and Methods: The internal transcribed spacer 2 regions in the nuclear ribosomal DNA of C. auris and other related yeasts were assayed to find a specific PCR target for C. auris. A 123-base-pair target was selected, and primers and a probe were designed for hydrolysis probe-based real-time PCR with TaqMan chemistry. Ten-fold serial dilutions of C. auris ranging from 106 to 100 CFU/mL were prepared to establish a standard curve to quantify the yeast.
Results: The qPCR assay was able to identify and quantify C. auris with a detection limit of 1 C. auris CFU per reaction. Specificity was confirmed by the non-amplification of the sequences belonging to other Candida species, yeasts, molds, bacteria, or human DNAs. The standard curve of the assay showed a highly significant linearity between threshold values and dilution rates (R2=0.99; slope=−3.42).
Conclusion: The applied qPCR assay facilitated the rapid and accurate identification and quantification of emerging opportunistic C. auris. Therefore, considering the promising test validation results, we succeeded to develop a rapid and accurate hydrolysis probe-based qPCR assay for the screening and identification of C. auris.
 

Keywords

References

1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H. Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol. 2009; 53(1):41-4.
2. Lee WG, Shin JH, Uh Y, Kang MG, Kim SH, Park KH, et al. First three reported cases of nosocomial fungemia caused by Candida auris. J Clin Microbiol. 2011; 49(9):3139-42.
3. Chowdhary A, Anil Kumar V, Sharma C, Prakash A, Agarwal K, Babu R, et al. Multidrug-resistant endemic clonal strain of Candida auris in India. Eur J Clin Microbiol Infect Dis. 2014; 33(6):919-26.
4. Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP, et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis. 2017; 64(2):134-40.
5. Emara M, Ahmad S, Khan Z, Joseph L, Al-Obaid I, Purohit P, et al. Candida auris candidemia in Kuwait, 2014. Emerg Infect Dis. 2015; 21(6):1091-2.
6. Al-Siyabi T, Al Busaidi I, Balkhair A, Al-Muharrmi Z, Al-Salti M, Al'Adawi B. First report of Candida auris in Oman: clinical and microbiological description of five candidemia cases. J Infect. 2017; 75(4):373-6.
7. Magobo RE, Corcoran C, Seetharam S, Govender NP. Candida auris–associated candidemia, South Africa. Emerg Infect Dis. 2014; 20(7):1250-1.
8. Morales-López SE, Parra-Giraldo CM, Ceballos-Garzón A, Martínez HP, Rodríguez GJ, Álvarez-Moreno CA, et al. Invasive infections with multidrug-resistant yeast Candida auris, Colombia. Emerg Infect Dis. 2017; 23(1):162-4.
9. Schelenz S, Hagen F, Rhodes JL, Abdolrasouli A, Chowdhary A, Hall A, et al. First hospital outbreak of the globally emerging Candida auris in a European hospital. Antimicrob Resist Infect Control. 2016; 5(1):35.
10. Schwartz IS, Hammond GW. Outbreak detection: first reported case of multidrug-resistant Candida auris in Canada. Canada Commun Dis Rep. 2017; 43(7-8):150-3.
11. Ruiz Gaitan AC, Moret A, Lopez Hontangas JL, Molina JM, Aleixandre Lopez AI, Cabezas AH, et al. Nosocomial fungemia by Candida auris: first four reported cases in continental Europe. Rev Iberoam Micol. 2017; 34(1):23-7.
12. Abastabar M, Haghani I, Ahangarkani F, Rezai MS, Taghizadeh Armaki M, Roodgari S, et al. Candida auris otomycosis in Iran and review of recent literature. Mycoses. 2019; 62(2):101-5.
13. Ku TSN, Walraven CJ, Lee SA. Candida auris: disinfectants and implications for infection control. Front Microbiol. 2018;
9:726.
14. Kumar D, Banerjee T, Pratap CB, Tilak R. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis. J Infect Dev Ctries. 2015; 9(4):435-7.
15. Mizusawa M, Miller H, Green R, Lee R, Durante M, Perkins R, et al. Can multidrug-resistant Candida auris be reliably identified in clinical microbiology laboratories? J Clin Microbiol. 2017; 55(2):638-40.
16. Kathuria S, Singh PK, Sharma C, Prakash A, Masih A, Kumar A, et al. Multidrug-resistant Candida auris misidentified as Candida haemulonii: characterization by matrix-assisted laser desorption ionization–time of flight mass spectrometry and DNA sequencing and its antifungal susceptibility profile variability by Vitek 2, CLSI broth microdilution, and Etest method. J Clin Microbiol. 2015; 53(6):1823-30.
17. Kim TH, Kweon OJ, Kim HR, Lee MK. Identification of uncommon Candida species using commercial identification systems. J Microbiol Biotechn. 2016; 26(12):2206-13.
18. Wattal C, Oberoi JK, Goel N, Raveendran R, Khanna S. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory. Eur J Clin Microbiol Infect Dis. 2017; 36(5):807-12.
19. Sharma C, Singh A, Singh PK, Prakash A, Meis JF, Chowdhary A. Genotyping of multidrug resistant Indian Candida auris isolates by multi locus sequence typing, amplified fragment length polymorphism and MALDI-TOF-MS and their antifungal susceptibility profile: P184. Mycoses. 2015; 58:119-20.
20. Cendejas-Bueno E, Kolecka A, Alastruey-Izquierdo A, Theelen B, Groenewald M, Kostrzewa M, et al. Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts. J Clin Microbiol. 2012; 50(11):3641-51.
21. Chowdhary A, Sharma C, Duggal S, Agarwal K, Prakash A, Singh PK, et al. New clonal strain of Candida auris, Delhi, India. Emerg Infect Dis. 2013; 19(10):1670-3.
22. Sharma C, Kumar N, Meis JF, Pandey R, Chowdhary A. Draft genome sequence of a fluconazole-resistant Candida auris Strain from a candidemia patient in India. Genome Announc. 2015; 3(4):e00722-15.
23. Alizadeh M, Kolecka A, Boekhout T, Zarrinfar H, Nahzag MA, Badiee P, et al. Identification of Candida species isolated from vulvovaginitis using matrix assisted laser desorption ionization-time of flight mass spectrometry. Curr Med Mycol. 2017; 3(4):21-5.
24. Bellanger AP, Gbaguidi‐Haore H, Liapis E, Scherer E, Millon L. Rapid identification of Candida sp. by MALDI‐TOF mass spectrometry subsequent to short‐term incubation on a solid medium. APMIS. 2019; 127(4):217-21.
25. Buil JB, van der Lee HAL, Curfs-Breuker I, Verweij PE, Meis JF. External quality assessment evaluating the ability of dutch clinical microbiological laboratories to identify Candida auris. J Fungi (Basel). 2019; 5(4):94.
26. Kordalewska M, Zhao Y, Lockhart SR, Chowdhary A, Berrio I, Perlin DS. Rapid and accurate molecular identification of the emerging multidrug-resistant pathogen Candida auris. J Clin Microbiol. 2017; 55(8):2445-52.
27. Leach L, Zhu Y, Chaturvedi S. Development and validation of a real-time PCR assay for rapid detection of Candida auris from surveillance samples. J Clin Microbiol. 2018; 56(2):e01223-17.
28. Lima A, Widen R, Vestal G, Uy D, Silbert S. A TaqMan probe-based Real-Time PCR assay for the rapid identification of the emerging multidrug-resistant pathogen Candida auris on the BD max system. J Clin Microbiol. 2019; 57(7):e1604.
29. Khodadadi H, Karimi L, Jalalizand N, Adin H, Mirhendi H. Utilization of size polymorphism in ITS1 and ITS2 regions for identification of pathogenic yeast species. J Med Microbiol. 2017; 66(2):126-33.
30. Zarrinfar H, Mirhendi H, Fata A, Khodadadi H, Kordbacheh P. Detection of Aspergillus flavus and A. fumigatus in bronchoalveolar lavage specimens of hematopoietic stem cell transplants and hematological malignancies patients by Real-Time polymerase chain reaction, nested PCR and mycological assays. Jundishapur J Microbiol. 2015; 8(1):e13744.
31. Borman AM, Szekely A, Johnson EM. Comparative pathogenicity of united kingdom isolates of the emerging pathogen Candida auris and other key pathogenic Candida species. mSphere. 2016; 1(4):e00189-16.
32. Chowdhary A, Sharma C, Meis JF. Candida auris: a rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog. 2017; 13(5):e1006290.
33. Forsberg K, Woodworth K, Walters M, Berkow EL, Jackson B, Chiller T, et al. Candida auris: the recent emergence of a multidrug-resistant fungal pathogen. Med Mycol. 2018; 57(1):1-12.
Current Medical Mycology
Volume 6, Issue 3
September 2020
Pages 50-56

Files

History

  • Receive Date: 21 August 2020
  • Revise Date: 21 September 2020
  • Accept Date: 22 September 2020
  • Publish Date: 01 September 2020

Share

How to cite

Statistics