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Validation of high resolution melting analysis (HRM) of the amplified ITS2 region for the detection and identification of yeasts from clinical samples : comparison with culture and MALDI-TOF based identification

Hans Duyvejonck UGent, Piet Cools UGent, Johan Decruyenaere UGent, Kristien Roelens UGent, Lucien Noens UGent, Stefan Vermeulen UGent, Geert Claeys UGent, Ellen Decat UGent, Els Van Mechelen UGent and Mario Vaneechoutte UGent (2015) PLOS ONE. 10(8).
abstract
Aim : Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples. Materials and Methods : A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by Nucli-SENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF. Results : For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample. Discussion : Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
DIAGNOSIS, FUNGI, FLUCONAZOLE, PREVALENCE, RESISTANCE, INFECTIONS, SACCHAROMYCES-CEREVISIAE, CANDIDA, TIME
journal title
PLOS ONE
PLoS One
volume
10
issue
8
article number
e0132149
pages
16 pages
Web of Science type
Article
Web of Science id
000359926900004
JCR category
MULTIDISCIPLINARY SCIENCES
JCR impact factor
3.057 (2015)
JCR rank
11/63 (2015)
JCR quartile
1 (2015)
ISSN
1932-6203
DOI
10.1371/journal.pone.0132149
language
English
UGent publication?
yes
classification
A1
additional info
correction published in: PLoS One (2015) 10(9), e0139501 ; DOI 10.1371/journal.pone.0139501
copyright statement
I have retained and own the full copyright for this publication
id
7005315
handle
http://hdl.handle.net/1854/LU-7005315
date created
2015-12-08 15:08:50
date last changed
2017-04-18 13:03:37
@article{7005315,
  abstract     = {Aim : Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples. 
Materials and Methods : A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by Nucli-SENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF. 
Results : For 83.9\% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1\% of the samples or no Candida species were detected in 52.8\% of the samples. In 16.1\% of the clinical samples, discrepant results were obtained, of which only 6.01\% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample. 
Discussion : Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.},
  articleno    = {e0132149},
  author       = {Duyvejonck, Hans and Cools, Piet and Decruyenaere, Johan and Roelens, Kristien and Noens, Lucien and Vermeulen, Stefan and Claeys, Geert and Decat, Ellen and Van Mechelen, Els and Vaneechoutte, Mario},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  keyword      = {DIAGNOSIS,FUNGI,FLUCONAZOLE,PREVALENCE,RESISTANCE,INFECTIONS,SACCHAROMYCES-CEREVISIAE,CANDIDA,TIME},
  language     = {eng},
  number       = {8},
  pages        = {16},
  title        = {Validation of high resolution melting analysis (HRM) of the amplified ITS2 region for the detection and identification of yeasts from clinical samples : comparison with culture and MALDI-TOF based identification},
  url          = {http://dx.doi.org/10.1371/journal.pone.0132149},
  volume       = {10},
  year         = {2015},
}

Chicago
Duyvejonck, Hans, Piet Cools, Johan Decruyenaere, Kristien Roelens, Lucien Noens, Stefan Vermeulen, Geert Claeys, Ellen Decat, Els Van Mechelen, and Mario Vaneechoutte. 2015. “Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples : Comparison with Culture and MALDI-TOF Based Identification.” Plos One 10 (8).
APA
Duyvejonck, H., Cools, P., Decruyenaere, J., Roelens, K., Noens, L., Vermeulen, S., Claeys, G., et al. (2015). Validation of high resolution melting analysis (HRM) of the amplified ITS2 region for the detection and identification of yeasts from clinical samples : comparison with culture and MALDI-TOF based identification. PLOS ONE, 10(8).
Vancouver
1.
Duyvejonck H, Cools P, Decruyenaere J, Roelens K, Noens L, Vermeulen S, et al. Validation of high resolution melting analysis (HRM) of the amplified ITS2 region for the detection and identification of yeasts from clinical samples : comparison with culture and MALDI-TOF based identification. PLOS ONE. 2015;10(8).
MLA
Duyvejonck, Hans, Piet Cools, Johan Decruyenaere, et al. “Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples : Comparison with Culture and MALDI-TOF Based Identification.” PLOS ONE 10.8 (2015): n. pag. Print.