Occurrence and genetic variability of Candida parapsilosis sensu lato in Hungary.
ABSTRACT The occurrence and genetic variability of Candida parapsilosis isolates in two Hungarian hospitals, located in Debrecen and Pécs, were examined. Among the 209 Candida isolates examined, 20 were found to belong to C. parapsilosis sensu lato, based on morphological, physiological and molecular data. The frequency of occurrence of C. parapsilosis isolates (9.6%) was lower than that observed in Europe but higher than that observed previously in Hungary. The genetic variability of C. parapsilosis sensu lato isolates was also examined using random amplified polymorphic DNA (RAPD) analysis and sequence analysis of the intergenic transcribed spacer (ITS) region of the rRNA gene cluster. The genetic variability of the isolates was relatively high, as revealed by RAPD analysis. Two isolates were found to belong to the recently described Candida metapsilosis species (C. parapsilosis group III), based on ITS sequence data, RAPD analysis and phenotypic data. These two isolates could also be distinguished from C. parapsilosis sensu stricto isolates using a primer pair developed for the detection of C. parapsilosis group I isolates. To the best of the authors' knowledge, this is the first report on the identification of C. metapsilosis from bloodstream infection.
- SourceAvailable from: Eduardo Bagagli[Show abstract] [Hide abstract]
ABSTRACT: Inteins are coding sequences that are transcribed and translated with flanking sequences, and then excised by an autocatalytic process. There are two types of inteins in fungi, the mini-inteins and the full-length inteins, both of which present a splicing domain containing well conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes, and may allow for differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the psilosis complex species C. parapsilosis, C. metapsilosis and C. orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work we analyzed inteins present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis and 22 C. orthopsilosis). We describe an accessible and accurate technique, based on a single PCR reaction that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length inteins in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies among C. orthopsilosis.Journal of clinical microbiology 06/2013; 51(9):2830-6. · 4.16 Impact Factor
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ABSTRACT: The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C. metapsilosis and 18 C. orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C. metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C. orthopsilosis and C. metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C. metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleriamellonella larvae. The mortality rate of G. mellonella larvae infected with C. metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C. orthopsilosis strains. Taken together, our findings demonstrate that C. metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.PLoS ONE 01/2013; 8(7):e68704. · 3.73 Impact Factor
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ABSTRACT: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are human fungal pathogens with clinical importance. The recently reclassified three closely related species have significant variation in virulence, clinical prevalence and susceptibility characteristics to different antifungal compounds. The aim of this study was to investigate the in vitro activity of atorvastatin and fluvastatin against C. metapsilosis, C. orthopsilosis and C. parapsilosis. Susceptibility tests showed that C. parapsilosis was the most sensitive while C. orthopsilosis was the least susceptible species to both drugs. On the basis of the differential sensitivity, we developed a simple, reliable and highly cost-effective plate assay to distinguish these closely related species. Applying this method, 54 isolates belonging to the C. parapsilosis sensu lato complex deposited in Szeged Microbial Collection could be sorted into the three species with 100 % probability.Mycopathologia 08/2013; · 1.65 Impact Factor
Occurrence and genetic variability of Candida
parapsilosis sensu lato in Hungary
Sa ´ndor Kocsube ´,1Mo ´nika To ´th,1Csaba Va ´gvo ¨lgyi,1Ilona Do ´czi,2
Miklo ´s Pesti,3Istva ´n Po ´csi,4Judit Szabo ´5and Ja ´nos Varga1,6
Ja ´nos Varga
1Department of Microbiology, Faculty of Sciences, University of Szeged, PO Box 533, H-6701
2Department of Clinical Microbiology, Faculty of Medicine, University of Szeged, PO Box 427,
H-6701 Szeged, Hungary
3Department of General and Environmental Microbiology, University of Pe ´cs, H-7601 Pe ´cs,
4Department of Microbiology and Biotechnology, Medical and Health Science Center, University
of Debrecen, PO Box 63, H-4010, Debrecen, Hungary
5Institute of Medical Microbiology, Faculty of Sciences, University of Debrecen, PO Box 63, H-
4010, Debrecen, Hungary
6CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
Received 17 July 2006
Accepted 13 October 2006
The occurrence and genetic variability of Candida parapsilosis isolates in two Hungarian hospitals,
located in Debrecen and Pe ´cs, were examined. Among the 209 Candida isolates examined, 20
were found to belong to C. parapsilosis sensu lato, based on morphological, physiological and
molecular data. The frequency of occurrence of C. parapsilosis isolates (9.6%) was lower than that
parapsilosis sensu lato isolates was also examined using random amplified polymorphic DNA
(RAPD) analysis and sequence analysis of the intergenic transcribed spacer (ITS) region of the
rRNA gene cluster. The genetic variability of the isolates was relatively high, as revealed by RAPD
analysis. Two isolates were found to belong to the recently described Candida metapsilosis
species (C. parapsilosis group III), based on ITS sequence data, RAPD analysis and phenotypic
data. These two isolates could also be distinguished from C. parapsilosis sensu stricto isolates
using a primer pair developed for the detection of C. parapsilosis group I isolates. To the best of the
authors’ knowledge, this is the first report on the identification of C. metapsilosis from bloodstream
Infections caused by Candida species are widespread
throughout the world. Although Candida albicans is the
most common Candida species encountered as a cause of
human infection, other Candida species have been increas-
ingly associated with disseminated disease since the 1990s.
Among them, C. parapsilosis has been the second most
common yeast species isolated from bloodstream infections
in several surveys (Safdar et al., 2002; Messer et al., 2006;
Bassetti et al., 2006). This species has emerged as an
important nosocomial pathogen, with clinical manifesta-
tions that include fungaemia, endocarditis, endophthalmi-
tis, septic arthritis and peritonitis, and infection usually
occurs in association with invasive procedures or prosthetic
devices (Weems, 1992). In some children’s hospitals, C.
parapsilosis has become the predominant species causing
candidaemia (Levy et al., 1998). This species is more
frequent in bloodstream infections of neonates, in trans-
nutrition or previous antifungal therapy (Almirante et al.,
2006). This species is also frequently associated with
catheter-associated candidaemia and intravenous hyperali-
mentation (Kremery & Barnes, 2002).
Previous studies have clarified that C. parapsilosis sensu lato
isolates can be divided into three groups that can be
Abbreviations: ITS, intergenic transcribed spacer; RAPD, random
amplified polymorphic DNA.
The GenBank/EMBL/DDBJ accession numbers for the ITS sequences
of C. metapsilosis 12821 and Bp57 are DQ786952 and DQ786953,
Neighbour-joining tree-based ITS sequence data of the examined
Candida isolates are available as supplementary data with the online
version of this paper.
19046838 G 2007 SGMPrinted in Great Britain
Journal of Medical Microbiology (2007), 56, 190–195
distinguished based on several criteria, including randomly
amplified polymorphic DNA (RAPD) analysis (Lehmann
et al., 1992), isoenzyme electrophoresis (Lin et al., 1995),
sequences of the internal transcribed spacer (ITS) region of
the rRNA gene cluster (Lin et al., 1995; Pryce et al., 2006),
hybridization to a fingerprinting probe (Enger et al., 2001),
DNA relatedness (Roy & Meyer, 1998), morphotyping
1993), single nucleotide polymorphisms (Fundyga et al.,
2004), mitochondrial DNA sequence differences (Nosek
(Song et al., 2005). Recently, Tavanti et al. (2005) have
recognized C. parapsilosis groups II and III as separate
species, Candida orthopsilosis and Candida metapsilosis,
respectively, based on multilocus sequence typing studies.
Accordingly, C. parapsilosis sensu lato includes C. para-
psilosissensustrictoandthe twonewly describedspecies.The
two latter species are recovered relatively rarely in clinical
samples (Tavanti et al., 2005). In addition, C. parapsilosis
group IV has also been recognized recently among Brazilian
clinical Candida isolates by Iida et al. (2005).
In this study, we examined the occurrence of C. parapsilosis
isolates among Candida isolates collected in Hungarian
hospitals, and examined the genetic variability of these
isolates using sequence analysis of the ITS region and the
Clinical samples. Altogether, 209 Candida isolates from blood sam-
ples from two Hungarian hospitals, located in Debrecen and Pe ´cs,
were examined. All examined isolates came from different patients. C.
parapsilosis isolates were identified by standard morphological and
physiological methods within the hospitals (Barnett et al., 2000).
Phenotypying. The API 20C AUX version 3.0 kit (bioMe ´rieux) was
used to type the species. Isolates from stored slants were streaked
onto nutrient agar and incubated at 30uC for 24 and 48 h. Samples
were then analysed according to the manufacturer’s prescribed
methods. The antifungal susceptibilities of isolates were determined
by the Etest method (AB Biodisk). The Etest was performed in
accordance with the manufacturer’s instructions, with the use of
Casitone agar plates (AB Biodisk, 2003). A 0.5 McFarland turbidity
suspension was used as a standard in the test. The drug concentra-
tions of Etest strips were 0.016–256 mg l21for fluconazole, and
0.002–32 mg l21for itraconazole, voriconazole and amphotericin B.
The incubation time was 24–48 h at 30uC, depending on the growth
characteristics. The MICs for amphotericin B were taken as the drug
concentrations causing 100% inhibition. MICs for azoles were read
at the visually selected end point of 80% inhibition of growth.
Interpretative susceptibility criteria for these antifungal agents were
used as published by the Clinical and Laboratory Standards Institute
(CLSI) and in the literature (National Committee for Clinical
Laboratory Standards, 2002; de Hoog et al., 2000).
Genotyping. Isolates were cultivated in YPD medium (0.5% Bacto
yeast extract, 1% glucose, 0.5% Bacto peptone) and centrifuged,
and DNA was extracted from the cells using the Masterpure yeast
DNA purification kit (Epicentre Biotechnologies) according to
the instructions of the manufacturer. For confirmation of the
species assignment of the isolates, the species-specific primer pair
developed for C. parapsilosis by Luo & Mitchell (2002) was used.
For the identification of C. parapsilosis sensu stricto (group I) iso-
lates, the primer pair developed by Pontieri et al. (2001) was
applied. The ITS region of the isolates was amplified using primers
ITS1 and ITS4 (White et al., 1990). DNA fragments were purified
from the excised agarose blocks using Genelute spin columns
(Sigma-Aldrich). Direct sequencing of the fragments was performed
on an ABI 373A DNA sequencer (Applied Biosystems) using dideoxy
terminator reaction chemistry. Sequences were determined from
both strands using ITS1 and ITS4 as primers. The GenBank acces-
sion numbers for the ITS sequences of C. metapsilosis 12821 and
Bp57 are DQ786952 and DQ786953, respectively.
(Williams et al., 1990). Fungal DNA sequences were amplified by using
the following decamer primers of the Operon random primer
kit (Operon Technology): OPC-02 (59-GTGAGGCGTC-39), OPC-05
(59-GATGACCGCC-39), OPC-07 (59-GTCCCGACGA-39), OPC-09
(59-CTCACCGTCC-39), OPC-12 (59-TGTCATCCCC-39), OPC-14
(59-TGCGTGCTTG-39), OPC-16 (59-CACACTCCAG-39), OPC-18
(59-TGAGTGGGTG-39), OPC-20 (59-ACTTCGCCAC-39), OPD-12
(59-CACCGTATCC-39), OPE-17 (59-CTACTGCCGT-39), OPG-07
(59-GAACCTGCGG-39), OPG-19 (59-GTCAGGGCAA-39), OPH-18
(59-GAATCGGCCA-39), OPK-04 (59-CCGCCCAAAC-39), OPM-18
(59-CACCATCCGT-39), OPR-15 (59-GGACAACGAG-39) and OPW-
17 (59-ACCGGCTTGT-39),andUBC (University ofBritish Columbia)
primersUBC 8(59-CCTGGCGGTA-39),UBC 18(59-GGGCCGTTTA-
39) and UBC 66 (59-GAGGGCGTGA-39).
Data analysis. Sequence alignments were performed by using
CLUSTAL_X (Thompson et al., 1997) and improved manually.
Evolutionary distances between the sequences were calculated by
Kimura’s formula (Kimura, 1980) using the program DNADIST.
Phylogenetic trees were prepared by the neighbour-joining method
(Saitou & Nei, 1987) using the program NEIGHBOR of the PHYLIP
package. Bootstrap values were calculated from 1000 replications of
NEIGHBOR and CONSENSE of the package (Felsenstein, 1995).
RAPD bands were scored visually. Only those bands were taken into
binary matrices of RAPD data were converted to distance matrices
using PhylTools (Buntjer, 1997). RAPD data were analysed by the
neighbour-joining method using the program NEIGHBOR of the PHYLIP
package (Saitou & Nei, 1987; Felsenstein, 1995).
RESULTS AND DISCUSSION
Occurrence of C. parapsilosis in Hungarian
Altogether, 103 and 106 Candida isolates were collected in
two Hungarian hospitals located in Debrecen and Pe ´cs,
respectively, during 2004–2005. Among these, 10 and 16
isolates, respectively, were found to belong to C. parapsilosis
(Table 1). The C. parapsilosis species-specific primer pair
(Luo & Mitchell, 2002) was successfully used to confirm the
identity of eight and 12 isolates as C. parapsilosis sensu lato.
API 20C AUX tests also confirmed that these isolates
belonged to C. parapsilosis sensu lato. Overall, 9.6% of the
collected isolates were found to belong to C. parapsilosis
sensu lato,while the remaining isolates were foundto belong
to C. albicans, Candida lusitaniae (teleomorph Clavispora
lusitaniae; Kurtzman & Fell, 1998) or Candida krusei
Occurrence of Candida parapsilosis in Hungary
(teleomorph Issatchenkia orientalis; Kurtzman & Fell, 1998)
(Table 1). This observation could have been caused by
misidentifications or, more probably, by contamination of
some of the cultures during storage of the isolates after
Previously, C. parapsilosis has been found to be responsible
(Pfaller et al., 2001). More recent surveys have revealed that
C. parapsilosis is responsible for more than 20% of
candidaemias in Italy (Bassetti et al., 2006; Bedini et al.,
2006), Spain (Almirante et al., 2006; Rodriguez et al., 2006;
San Miguel et al., 2006) and South American countries
(Brito et al., 2006; Rodero et al., 2005), while this species is
encountered less frequently in Turkey (12.5%; Yapar et al.,
2006) and Norway (6%; Sandven et al., 2006). In a
worldwide survey, Messer et al. (2006) have found C.
parapsilosis to be responsible for candidaemias in 16.36% of
the 336 cases examined from Europe. In another survey,
Tortorano et al. (2004, 2006) have found that C. parapsilosis
is responsible for 6.9–30% (mean 13.54%) of candidaemias
in different European countries, with the highest incidence
found in Spain. In a previous study, Do ´czi et al. (2002)
found that C. parapsilosis was responsible for 4.9% of
bloodstream infections caused by Candida species in a
university hospital in Hungary. These data indicate that the
frequency of C. parapsilosis infections varies widely among
different institutions and countries (Pfaller et al., 2001). The
9.6% incidence of this species found in this survey is higher
than that observed previously in Hungary, but lower than
the mean observed in European countries.
Genetic variability of C. parapsilosis isolates
RAPD analysis of the isolates was carried out by using 21
random decamer primers. The genetic variability observed
among the isolates was low: most isolates exhibited very
similar or the sameRAPD patterns with most primerstested
(Fig. 1). Similarly low genetic variability has been observed
among C. parapsilosis sensu stricto isolates by Zeng et al.
(1996), Lehmann et al. (1992) and Enger et al. (2001). For
preparation of the similarity matrix, the presence or absence
of 72 DNA bands was taken into account. The dendrogram
form two main clusters which are supported by high
bootstrap values (Fig. 2). One of these includes two isolates,
12821 and Bp57, which could also be distinguished from
other C. parapsilosis isolates using the C. parapsilosis group
I-specific primer pair developed by Pontieri et al. (2001).
This primer pair did not amplify the expected product from
isolates 12821 and Bp57 (data not shown).
Sequences of the ITS region of the isolates were also
Table 1. Origin and species assignment of the Candida isolates examined in this study
Strain numberOrigin Species assignment based on ITS sequences
Throat, Pe ´cs
Stomach, Pe ´cs
Blood, Pe ´cs
Blood, Pe ´cs
Blood, Pe ´cs
Nail, Pe ´cs
Nail, Pe ´cs
Blood, Pe ´cs
Fingernail, Pe ´cs
Nail, Pe ´cs
Sputum, Pe ´cs
Blood, Pe ´cs
Blood, Pe ´cs
Clinical sample, Pe ´cs
Blood, Pe ´cs
Blood, Pe ´cs
192Journal of Medical Microbiology 56
S. Kocsube ´ and others
that two of the isolates that belonged to C. parapsilosis sensu
lato were members of the C. metapsilosis species (see
Supplementary Fig. S1). In other studies, C. metapsilosis has
been identified in the USA, Japan, Brazil, Norway and
Belgium (Enger et al., 2001; Rycovska et al., 2004; Iida et al.,
2005; Tavanti et al.,2005). To thebestof our knowledge, the
present study is the first report on the occurrence of this
species in Central Europe. In addition, in this study, C.
metapsilosis was isolated from a bloodstream infection for
the first time. The results of the API 20C AUX tests also
indicated that these two isolates belonged to the C.
metapsilosis species, since in a similar manner to the results
of Lin et al. (1995), the two isolates could grow on D-xylitol,
in contrast to the C. parapsilosis sensu stricto isolates (data
Antifungal susceptibility tests
All isolates were susceptible to the antifungal drugs tested,
with MICs <1 mg l21for amphotericin B and voricona-
zole, <8 mg l21for fluconazole and <0.125 mg l21for
itraconazole. The lowest MIC values were obtained for
itraconazole and voriconazole. According to earlier studies,
the amphotericin B, fluconazole, itraconazole and vorico-
nazole susceptibilities of C. parapsilosis isolates range from
0.03 to 2 mg l21, 0.06 to 16 mg l21, 0.03 to 2 mg l21and
0.02 to 1 mg l21, respectively (Lin et al., 1995; Rex et al.,
1995; Pfaller et al., 2001; Lu et al., 2004; St-Germain et al.,
MIC values obtained for C. metapsilosis isolates fell within
the range of MICs of C. parapsilosis isolates (data not
shown). C. metapsilosis isolates exhibited lower mean MIC
values for voriconazole and amphotericin B than C.
parapsilosis isolates. Similarly, lower MIC values have
been observed for amphotericin B in C. metapsilosis than
in C. parapsilosis in earlier studies (Lin et al., 1995).
However, further isolates should be examined to draw
In conclusion, 9.6% of Candida infections were found to be
caused by C. parapsilosis sensu lato in our survey of two
Hungarian hospitals. Two of these isolates were found to
belong to the recently described C. metapsilosis species. This
is believed to be the first report on the identification of C.
metapsilosis from bloodstream infection. Further studies are
in progress to identify the rest of the Candida isolates using
molecular and phenotypic data, and to survey other
Hungarian hospitals for the presence of C. metapsilosis.
This work was financially supported by grant GVOP-3.1.1-2004-05-
0471/3.0. The technical assistance of Judit Dea ´k and Ma ´ria Lele is
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Fig. 1. RAPD profiles of the examined C. parapsilosis sensu
lato isolates generated by primer OPR-15. Lanes: 1, 100 bp
DNA ladder; 2, C. parapsilosis 5312; 3, C. parapsilosis 5308;
4, C. parapsilosis Bp73; 5, C. parapsilosis Bp46; 6, C. para-
psilosis 25329; 7, C. parapsilosis Bp42; 8, C. metapsilosis
12821; 9, C. metapsilosis Bp57; 10, C. krusei Bp47; 11, 1 kb
Fig. 2. Neighbour-joining tree based on RAPD profiles of C.
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