Using Single Multiplex PCR Reaction to Identify Candida Species in Vaginal isolates: Comparison between Phenotypic and Molecular Methods

Document Type : Original Research

Authors
1 Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran
2 Cell and molecular research center, Birjand University of Medical Sciences, Birjand, Iran
3 Department of Gynecology and Obstetrics, Birjand University of Medical University, Birjand, Iran
4 Student Research committee, Birjand University of Medical University, Birjand, Iran
Abstract
Backgrounds: In this research, an attempt was made to identify Candida isolates collected from women with suspected vulvovaginal candidiasis using single Multiplex PCR reaction as a swift and valid method. Beside, this method was compared with phenotypic methods.

Materials & Methods: In this study, 250 vaginal swabs were collected from patients referring to obstetrics and gynecology specialists. In addition to phenotypic methods, multiplex PCR designed by species-specific primers was performed to identify Candida isolates in a single reaction. Descriptive statistics were analyzed by t-test and Chi-square test in SPSS software (Ver. 22) (p< .05).

Findings: According to the results, 92 positive samples were diagnosed using the culture method. Four species were identified by culturing the specimens on CHROM agar. The most common Candida species isolated was C. albicans (54.3%), followed by C. parapsilosis (28.2%), C. glabrata (17.4%), and C. krusei (1.0%). The most common Candida spp. identified by Multiplex PCR method were C. albicans (50.0%), C. glabrata (33.7%), and C. parapsilosis (6.2%). Also, three mixed infections with C. albicans and C. glabrata as well as C. albicans and C. parapsilosis were identified

Conclusion: In comparison to phenotypic methods, considering the cost-effectiveness of PCR methods, the single multiplex PCR reaction was shown to be efficient in epidemiological studies on pathogenic species.

Keywords

Subjects


Denning DW, Kneale M, Sobel JD, Rautemaa-Richardson R. Global burden of recurrent vulvovaginal candidiasis: a systematic review. Lancet Infec Dis 2018; 18: e339–e349.
2 Dehghan P., Kharazi M, Yazdani M, Zomorodian K, Chadeganipour M, Akbari M. Diagnosis of candida species isolated from patients with vaginal candidiasis and healthy individuals based on clinical symptoms and paraclinical evidences. J Isfahan Med Sch 2012; 30.
3 Bitew A, Abebaw Y. Vulvovaginal candidiasis: Species distribution of Candida and their antifungal susceptibility pattern. BMC Womens Health 2018; 18. [PMID: 29902998 DOI: 10.1186/s12905-018-0607-z]
4 Zeng X, Zhang Y, Zhang T, Xue Y, Xu H, An R. Risk Factors of Vulvovaginal Candidiasis among Women of Reproductive Age in Xi’an: A Cross-Sectional Study. Biomed Res Int 2018; 2018. [PMID: 29977925 DOI: 10.1155/2018/9703754]
5 Ghssein G, Ibrahim J-N, Ghaddar N, El Roz A. Emergence of Vulvovaginal Candidiasis among Lebanese Pregnant Women: Prevalence, Risk Factors, and Species Distribution. Infect Dis Obs Gynecol 2019; 2019.
6 Akbarzadeh M, Bonyadpoure B, Pacshir K, Mohagheghzadeh M. Causes and clinical symptoms of vaginal candidiasis in patients referring to selective clinics of Shiraz University of Medical Sciences. J Arak Uni Med Sci 2010; 13.
7 Ahmad A, Khan AU. Prevalence of Candida species and potential risk factors for vulvovaginal candidiasis in Aligarh, India. Eur J Obstet Gynecol Reprod Biol 2009; 144: 68–71.
8 Esmaeilzadeh S, Omran SM, Rahmani Z. Frequency and Etiology of Vulvovaginal Candidiasis in Women Referred to a Gynecological Center in Babol, lran.
9 Gharaghani M, Rezaei-Matehkolaei A, Zarei Mahmoudabadi A, Keikhaei B. The frequency, antifungal susceptibility and enzymatic profiles of Candida species isolated from neutropenic patients. Jundishapur J Microbiol 2016; 9. [PMID: 28138378 DOI: 10.5812/jjm.41446]
10 Falahati M, Sharifinia S, Foroumadi AR, Bolouri F, Akhlagh L, Yazdan Parast A, Haghani H. Drug resistance pattern in Candida species isolated from vaginitis. Razi J Med Sci 2009; 16: 0.
11 ElFeky DS, Gohar NM, El-Seidi EA, Ezzat MM, AboElew SH. Species identification and antifungal susceptibility pattern of Candida isolates in cases of vulvovaginal candidiasis. Alexandria J Med 2016; 52: 269–277.
12 Abdu AB, Alade T, Omotu C. Azole Resistance and Detection of the ERG11 Gene in Clinical Candida albicans Isolated from Pregnant Women with Vulvovaginitis Attending Federal Medical Centre, Yenagoa, Nigeria. Int STD Res Rev 2019; ISRR: 1–11. [DOI: 10.9734/isrr/2019/v8i230097]
13 Roudbary M, Roudbarmohammadi SH, Bakhshi B, Farhadi Z, Nikoomanesh F. Identification of Candida species isolated form Iranian women eith vaginal candidasis by PCR-RFLP method. EurJ Exp Biol 2013; 3: 365–369.
14 Mahmoudi Rad M, Zafarghandi S, Abbasabadi B, Tavallaee M. The epidemiology of Candida species associated with vulvovaginal candidiasis in an Iranian patient population. Eur J Obstet Gynecol Reprod Biol 2011; 155: 199–203. [PMID: 21194828 DOI: 10.1016/j.ejogrb.2010.11.022]
15 Fouladi B, Yadegari MH, Rajabibazl M, Fazaeli A, HashemzadehChaleshtori M. Identification of candida species in patients with vulvovaginitis presenting different clinical symptomse. J Adv Med Biomed Res 2015; 23: 53–67.
16 Odds FC, Jacobsen MD. Multilocus Sequence Typing of Pathogenic Candida Species. Eukaryot Cell 2008; 7: 1075. [PMID: 18456859 DOI: 10.1128/EC.00062-08]
17 Gandhi TN, Patel MG, Jain MR. Prospective Study of Vaginal Discharge and Prevalence of Vulvovaginal Candidiasis in a Tertiary Care Hospital. Int J Curr Res Rev 2015; 7: 34–38.
18 Bautista-Muñoz C, Boldo XM, Villa-Tanaca L, Hernández-Rodríguez C. Identification of Candida spp. by randomly amplified polymorphic DNA analysis and differentiation between Candida albicans and Candida dubliniensis by direct PCR methods. J Clin Microbiol 2003; 41: 414–420. [PMID: 12517882 DOI: 10.1128/JCM.41.1.414-420.2003]
19 Taverna CG, Bosco-Borgeat ME, Murisengo OA, Davel G, Boité MC, Cupolillo E, Canteros CE. Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species. Mem Inst Oswaldo Cruz 2013; 108: 178–185. [DOI: 10.1590/0074-0276108022013009]
20 Posteraro B, Efremov L, Leoncini E, Amore R, Posteraro P, Ricciardi W, Sanguinetti M. Are the Conventional Commercial Yeast Identification Methods Still Helpful in the Era of New Clinical Microbiology Diagnostics? A Meta-Analysis of Their Accuracy. J Clin Microbiol 2015; 53: 2439. [PMID: 25994160 DOI: 10.1128/JCM.00802-15]
21 Guzel AB, Ilkit M, Akar T, Burgut R, Demir SC. Evaluation of risk factors in patients with vulvovaginal candidiasis and the value of chromID Candida agar versus CHROMagar Candida for recovery and presumptive identification of vaginal yeast species. Med Mycol 2010; 49: 16–25. [PMID: 20608776 DOI: 10.3109/13693786.2010.497972]
22 Hedayati MT, Taheri Z, Galinimoghadam T, Aghili SR, Cherati JY, Mosayebi E. Isolation of different species of Candida in patients with vulvovaginal candidiasis from sari, Iran. Jundishapur J Microbiol 2015; 8. [DOI: 10.5812/jjm.8(4)2015.15992]
23 Nikoomanesh F, Roudbarmohammadi S, Khoobi M, Haghighi F, Roudbary M. Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. Artif Cells, Nanomedicine Biotechnol 2019; 47: 64–72. [PMID: 30663419 DOI: 10.1080/21691401.2018.1543193]
24 Nikoomanesh F, Roudbarmohammadi S, Roudbary M, Bayat M, Heidari G. Investigation of bcr1 Gene Expression in Candida albicans Isolates by RT-PCR Technique and its Impact on Biofilm Formation. Infect Epidemiol Med 2016; 2: 22–24. [DOI: 10.18869/modares.iem.2.1.22]
25 Arastehfar A, Fang W, Pan W, Lackner M, Liao W, Badiee P, Zomorodian K, Badali H, Hagen F, Lass-Flörl C, Boekhout T. YEAST PANEL multiplex PCR for identification of clinically important yeast species: stepwise diagnostic strategy, useful for developing countries. Diagn Microbiol Infect Dis 2019; 93: 112–119. [PMID: 30377018 DOI: 10.1016/j.diagmicrobio.2018.09.007]
26 Arastehfar A, Fang W, Pan W, Liao W, Yan L, Boekhout T. Identification of nine cryptic species of Candida albicans, C. glabrata, and C. parapsilosis complexes using one-step multiplex PCR. BMC Infect Dis 2018; 18: 1–9.
27 Yücesoy M, Marol S. Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species. Ann Clin Microbiol Antimicrob 2003; 2: 8. [PMID: 14613587 DOI: 10.1186/1476-0711-2-8]
28 Mahmoudi Rad M, Zafarghandi S, Abbasabadi B, Tavallaee M. The epidemiology of Candida species associated with vulvovaginal candidiasis in an Iranian patient population. Eur J Obstet Gynecol Reprod Biol 2011; 155: 199–203. [PMID: 21194828 DOI: 10.1016/J.EJOGRB.2010.11.022]
29 Farahyar S, Izadi S, Razmjou E, Falahati M, Roudbary M, Ashrafi-Khozani M, Ansari S, Fattahi A, Ghahri-Mobaser Z, Rahimi M. Low prevalence of antifungal resistant Candida africana, in the C. albicans complex causing vulvovaginal candidiasis. Heliyon 2020; 6: 0–4. [DOI: 10.1016/j.heliyon.2020.e03619]
30 Gharaghani M, Rezaei-Matehkolaei A, Mahmoudabadi AZ, Keikhaei B. The frequency, antifungal susceptibility and enzymatic profiles of Candida species isolated from neutropenic patients. Jundishapur J Microbiol 2016; 9.
31 Nasrollahi Z, Yadegari MH, Mohammadi SR, Roudbari M, Poor MH, Nikoomanesh F, Bazl MR. Fluconazole resistance Candida albicans in females with recurrent vaginitis and Pir1 overexpression. Jundishapur J Microbiol 2015; 8. [DOI: 10.5812/jjm.21468]
32 Carvalho A, Costa-De-Oliveira S, Martins ML, Pina-Vaz C, Rodrigues AG, Ludovico P, Rodrigues F. Multiplex PCR identification of eight clinically relevant Candida species. Med Mycol 2007; 45: 619–627. [PMID: 17885953 DOI: 10.1080/13693780701501787]
33 Taira CL, Okay TS, Delgado AF, Ceccon MEJR, Almeida MTG de, Negro GMB Del. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients. BMC Infect Dis 2014; 14: 406. [PMID: 25047415 DOI: 10.1186/1471-2334-14-406]
34 Mahdavi Omran S., Esmailzadeh S. Comparsion of anti-Candida activity of Thyme, Pennyroyal, and Lemon essential oils versus antifungal drugs against Candida species. Jundishapur J Microbiol 2009; 2: 53–60.
35 Deorukhkar SC, Saini S, Mathew S. Virulence Factors Contributing to Pathogenicity of Candida tropicalis and Its Antifungal Susceptibility Profile. Int J Microbiol 2014; 2014. [PMID: 24803934 DOI: 10.1155/2014/456878]
36 Ghajari A, Lotfali E, Ahmadi NA, Fassihi PN, Shahmohammadi N, Ansari S, Norouzi M, Arab-Mazar Z. Isolation of different species of candida in patients with vulvovaginal candidiasis from Damavand, Iran. Arch Clin Infect Dis 2018; 13. [DOI: 10.5812/archcid.59291]
37 Kim TH, Kweon OJ, Kim HR, Lee MK. Identification of Uncommon Candida Species Using Commercial Identification Systems. J Microbiol Biotechnol 2016; 26: 2206–2213. [PMID: 27713213 DOI: 10.4014/JMB.1609.09012]
38 Ragheb SM, Jimenez L. Polymerase chain reaction/rapid methods are gaining a foothold in developing countries. PDA J. Pharm. Sci. Technol. 2014; 68: 239–255.