Design of species-specific primers for rapid detection and identification of Candida parapsilosis sensu stricto

Authors

  • Monika Novak Babič
  • Nina Gunde-Cimerman

DOI:

https://doi.org/10.14720/abs.63.1.15915

Keywords:

Candida parapsilosis, detection methods, emerging pathogen, novel primers, conventional PCR, species specific

Abstract

Candida species are the cause of approximately two million cases of candidiasis yearly worldwide, and are frequently involved in life-threatening infections. After Candida albicans, the Candida parapsilosis complex is the second most common cause of Candida infections, particularly in patients in intensive care units and in neonates. Contrary to many Candida species, C. parapsilosis sensu stricto is frequently present in water, and on surfaces made of plastic, rubber, and silicone, where it acts as aprimary coloniser for biofilm establishment. Identification methods for the C. parapsilosis complex include culture-dependent methods, MALDI-TOF, and multiplex PCR using ITS region, but remains amongst the most frequently misidentified species, due to the genetic similarity and lack of species-specific primers. In the present study, we developed novel species-specific primers for detection and identification of C. parapsilosis sensu stricto using locus CPAR2_105320, as template for easily accessible and widely used conventional PCR method. Using these primers, we successfully detected and identified C. parapsilosis sensu strictoin pure cultures isolated from clinical specimens and indoor environments. Additionally, this method enables detection of C. parapsilosis sensu stricto in biofilms and tap water samples from which DNA was extracted, and directly from suspensions of washed swab samples. All positive cases showed single clear band with 574 base pairs. Sequencing of the amplicon proved designed primers to be species-specific. In the future, primers can serve as a tool for rapid detection of C. parapsilosis sensustricto in the environment and clinical settings.

References

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J., 1990. Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410. DOI: https://doi.org/10.1016/S0022-2836(05)80360-2

Arastehfar, A., Fang, W., Pan, W., Lackner, M., Liao, W., Badiee, P., et al. 2019. Yeast panel multiplex PCR for identification of clinically important yeast species: stepwise diagnostic strategy, useful for developing countries. Diagnostic Microbiology and Infectious Disease, doi: 10.1016/j.diag- microbio.2018.09.007 DOI: https://doi.org/10.1016/j.diagmicrobio.2018.09.007

Bloomfield, S. F., Exner, M., Nath, K. J., Signorelli, C., Scott, E. A. 2012: The chain of infection transmission in the home and everyday life settings, and the role of hygiene in reducing the risk of infection. International Scientific Forum on Home Hygiene, United Kingdom, 140 pp.

Brown, G. D., Denning, D. W., Gow, N. A. R., Levitz, S., Netea, M., White, T., 2012. Human fungal infections: the hidden killers. Science Translational Medicine, 4 (165), 165rv13. doi: 10.1126/ scitranslmed.3004404. DOI: https://doi.org/10.1126/scitranslmed.3004404

Cantón, E., Pemán, J., Quindós, G., Eraso, E., Miranda-Zapico, I., Álvarez, M., et al. 2011. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrobial Agents and Chemotherapy, 55, 5590-5596. DOI: https://doi.org/10.1128/AAC.00466-11

Clark, A. E., Kaleta, E. J., Arora, A., Wolk, D. M., 2013. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clinical Microbiology Reviews, 26, 547-603. DOI: https://doi.org/10.1128/CMR.00072-12

Cortés, J. A., Corrales, I. F., 2019. Invasive Candidiasis: Epidemiology and Risk Factors. In: Loreto, É. S., Simoni, M. T. J. (eds.): Fungal Infection. IntechOpen, London, pp. 33-56.

Croxatto, A., Prod’hom, G., Greub, G., 2012. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS Microbiology Reviews, 36 (2), 380-407. doi: 10.1111/j.1574- 6976.2011.00298.x. DOI: https://doi.org/10.1111/j.1574-6976.2011.00298.x

de Hoog, G. S., Guarro, J., Gené, J., Ahmed, S., Al-Hatmi, A. M. S., et al., 2019. Atlas of clinical fungi. 3rd e-ed. Westerdijk Fungal Biodiversity Institute, Utrecht / Reus.

Ghelardi, E., Pichierri, G., Castagna, B., Barnini, S., Tavanti, A., Campa, M., 2008. Efficacy of chro- mogenic Candida agar for isolation and presumptive identification of pathogenic yeast species. Clinical Microbiology and Infection, 14 (2), 141-147. DOI: https://doi.org/10.1111/j.1469-0691.2007.01872.x

Guida, A., Lindstädt, C., Maguire, S. L., Ding, C., Higgins, D. G., Corton, N. J., et al., 2011. Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis. BMC Genomics, 12, 628. doi: 10.1186/1471-2164-12-628. DOI: https://doi.org/10.1186/1471-2164-12-628

Hermansen, R. A., Mannakee, B. K., Knecht, W., Liberles, D. A., Gutenkunst, R. N., 2015. Characte- rizing selective pressures on the pathway for de novo biosynthesis of pyrimidines in yeast. BMC Evolutionary Biology, 15, 232. doi: 10.1186/s12862-015-0515-x. DOI: https://doi.org/10.1186/s12862-015-0515-x

Huang, Y. C., Lin, T. Y., Leu, H. S., Wu, J. L., Chang, H. Y., 1998. Yeast carriage on hands of hospital personnel working in intensive care units. Journal of Hospital Infection, 39, 47-51. DOI: https://doi.org/10.1016/S0195-6701(98)90242-0

Koressaar, T., Remm, M., 2007. Enhancements and modifications of primer design program Primer3. Bioinformatics, 23 (10), 1289-1291. DOI: https://doi.org/10.1093/bioinformatics/btm091

Kulesza, K., Biedunkiewicz, A., Nowacka, K., Glinka, P., 2018. Potentially pathogenic fungi of the Candida genus isolated from the Łyna River – a 20-year study. Annals of Parasitology, 64, 217- 223. doi: 10.17420/ap6403.155.

Lass-Flörl, C., 2009. The changing face of epidemiology of invasive fungal disease in Europe. Mycoses, 52 (3), 197-205. doi:10.1111/j.1439-0507.2009.01691.x. DOI: https://doi.org/10.1111/j.1439-0507.2009.01691.x

Liguori, G., Gallé, F., Lucariello, A., Di Onofrio, V., Albano, L., Mazzarella, G., et al., 2010. Com- parison between multiplex PCR and phenotypic systems for Candida spp. identification. New Microbiologica, 33 (1), 63-67.

Luo, G., Mitchell, T. G., 2002. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. Journal of Clinical Microbiology, 40 (8), 2860-2865. doi: 10.1128/jcm.40.8.2860- 2865.2002. DOI: https://doi.org/10.1128/JCM.40.8.2860-2865.2002

Novak Babič, M., Gunde-Cimerman, N., Vargha, M., Tischner, Z., Magyar, D., Veríssimo, C., et al., 2017b. Fungal contaminants in drinking water regulation? A tale of ecology, exposure, purification and clinical relevance. International Journal of Environmental Research and Public Health, 14 (6), 636. doi: 10.3390/ijerph14060636. DOI: https://doi.org/10.3390/ijerph14060636

Novak Babič, M., Zalar, P., Ženko, B., Schroers, H. J., Džeroski, S., Gunde-Cimerman, N., 2015. Candida and Fusarium species known as opportunistic human pathogens from customer-accessible parts of residential washing machines. Fungal Biology, 119 (2-3), 95-113. doi: 10.1016/j.funbio.2014.10.007. Novak Babič, M., Zalar, P., Ženko, B., Džeroski, S., Gunde-Cimerman, N., 2016. Yeasts and yeast- like fungi in tap water and groundwater, and their transmission to household appliances. Fungal DOI: https://doi.org/10.1016/j.funbio.2014.10.007

Ecology, 20, 30-39. https://doi.org/10.1016/j.funeco.2015.10.001. DOI: https://doi.org/10.1016/j.funeco.2015.10.001

Novak Babič, M., Zupančič, J., Gunde-Cimerman, N., Zalar, P., 2017a. Yeast in anthropogenic and polluted environments. In: Buzzini, P., Lachance, M. A., Yurkov, A. (eds.): Yeasts in Natural Ecosystems: Diversity. Springer, Switzerland, pp.145-169. DOI: https://doi.org/10.1007/978-3-319-62683-3_5

Pires, H. R., dos Santos, M. J., Zaia, E. J., Gomes Martins, H. C., Mendes-Giannini, S. J. M., 2011. Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in-vitro evaluation of the use of clinical antifungals. Memórias do Instituto Oswaldo Cruz, 106 (6), 646-654. DOI: https://doi.org/10.1590/S0074-02762011000600002

Posteraro, B., Efremov, L., Leoncini, E., Amore, R., Posteraro, P., Ricciardi, W., et al., 2015. Are the conventional commercial yeast identification methods still helpful in the era of new clinical mi- crobiology diagnostics? A meta-analysis of their accuracy. Journal of Clinical Microbiology, 53 (8), 2439-2450. doi: 10.1128/JCM.00802-15. DOI: https://doi.org/10.1128/JCM.00802-15

Raghupathi, P. K., Zupančič, J., Brejnrod, A. D., Jacquiod, S., Houf, K., Burmølle, M., et al., 2018. Mi- crobial diversity and putative opportunistic pathogens in dishwasher biofilm communities. Applied and Environmental Microbiology, 84 (5), e02755-17. doi: 10.1128/AEM.02755-17. DOI: https://doi.org/10.1128/AEM.02755-17

Sardi, J. C. O., Scorzoni, L., Bernardi, T., Fusco-Almeida, A. M., Mendes Giannini, M. J. S., 2013. Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. Journal of Medical Microbiology, 62 (Pt 1), 10-24. doi: 10.1099/jmm.0.045054-0. DOI: https://doi.org/10.1099/jmm.0.045054-0

Shah, A., Abdelzaher, A., Phillips, M., Hernandez, R., Solo-Gabriele, H., Kish, J., et al., 2011. Indi- cator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site. Journal of Applied Microbiology, 110, 1571-1583. DOI: https://doi.org/10.1111/j.1365-2672.2011.05013.x

Taira, C. L., Okay, T. S., Delgado, A. F., Ceccon, M. E., de Almeida, M. T., Del Negro, G. M., 2014. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients. BMC Infectious Diseases, 14, 406. doi: 10.1186/1471-2334-14-406. DOI: https://doi.org/10.1186/1471-2334-14-406

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 24, 1596-1599. DOI: https://doi.org/10.1093/molbev/msr121

Trofa, D., Gácser, A., Nosanchuk, J. D., 2008. Candida parapsilosis, an emerging fungal pathogen. Clinical Microbiology Reviews, 21 (4), 606-625. doi: 10.1128/CMR.00013-08. DOI: https://doi.org/10.1128/CMR.00013-08

Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., et al., 2012. Primer3 - new capabilities and interfaces. Nucleic Acids Research, 40 (15), e115. doi: 10.1093/nar/gks596. WHO. 2016: Establishment of PCR laboratory in developing countries, 2nd ed. World Health Organization, Geneva, 96 pp. DOI: https://doi.org/10.1093/nar/gks596

Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T., 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13, 134. doi: 10.1186/1471-2105-13-134. DOI: https://doi.org/10.1186/1471-2105-13-134

Zupančič, J., Novak Babič, M., Gunde-Cimerman, N., 2019. High incidence of an emerging opportu- nistic pathogen Candida parapsilosis in water-related domestic environments. In: Loreto, É. S., Simoni, M. T. J. (eds.): Fungal Infection. IntechOpen, London, pp. 79-93. DOI: https://doi.org/10.5772/intechopen.81313

Zupančič, J., Novak Babič, M., Zalar, P., Gunde-Cimerman, N., 2016. The black yeast Exophiala dermatitidis and other selected opportunistic human fungal pathogens spread from dishwashers to kitchens. PLoS ONE, 11 (2), e0148166. doi:10.1371/journal.pone.0148166. DOI: https://doi.org/10.1371/journal.pone.0148166

Downloads

Published

01.07.2020

Issue

Section

Original Research Paper

How to Cite

Novak Babič, M., & Gunde-Cimerman, N. (2020). Design of species-specific primers for rapid detection and identification of Candida parapsilosis sensu stricto. Acta Biologica Slovenica, 63(1), 63-77. https://doi.org/10.14720/abs.63.1.15915

Similar Articles

1-10 of 139

You may also start an advanced similarity search for this article.