Endemic Genotypes of Candida albicans Causing Fungemia Are Frequent in the Hospital

Abstract

Genotyping of Candida albicans strains causing candidemia can uncover the presence of endemic genotypes in the hospital. Using a highly reproducible and
discriminatory microsatellite marker panel, we studied the genetic diversity of 217 C. albicans isolates from the blood cultures of 202 patients with candidemia (from January 2007 to December 2011). Each isolate represented
1 candidemia episode. Multiple episodes were defined as the isolation of C. albicans in further blood cultures taken ≥7 days after the last isolation in blood culture. Of the 202 patients, 188 had 1 episode,
13 had 2 episodes, and 1 had 3 episodes. Identical genotypes showed the same alleles for all 6 markers. The genotypes causing
both episodes were identical in most patients with 2 episodes (11/13; 84.6%). In contrast, 2 different genotypes were found
in the patient with 3 episodes, one causing the first and second episodes and the other causing the third episode (isolated
6 months later). We found marked genetic diversity in 174 different genotypes: 155 were unique, and 19 were endemic and formed
19 clusters (2 to 6 patients per cluster). Up to 25% of the patients were infected by endemic genotypes that infected 2 or
more different patients. Some of these endemic genotypes were found in the same unit of the hospital, mainly neonatology,
whereas others infected patients in different wards.

Full text

Endemic Genotypes of Candida albicans Causing Fungemia Are
Frequent in the Hospital
Pilar Escribano,
a,b,c
Marta Rodríguez-Créixems,
a,b,c
Carlos Sánchez-Carrillo,
a,b,c
Patricia Muñoz,
a,b,c,d
Emilio Bouza,
a,b,c,d
Jesús Guinea
a,b,c,d
Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, Madrid, Spain
a
;
Instituto de Investigación Sanitaria del Hospital Gregorio Marañón, Madrid, Spain
b
; CIBER de Enfermedades Respiratorias (CIBER RES CD06/06/0058), Palma de Mallorca,
Spain
c
; Microbiology Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
d
Genotyping of Candida albicans strains causing candidemia can uncover the presence of endemic genotypes in the hospital. Us-
ing a highly reproducible and discriminatory microsatellite marker panel, we studied the genetic diversity of 217 C. albicans iso-
lates from the blood cultures of 202 patients with candidemia (from January 2007 to December 2011). Each isolate represented 1
candidemia episode. Multiple episodes were defined as the isolation of C. albicans in further blood cultures taken >7 days after
the last isolation in blood culture. Of the 202 patients, 188 had 1 episode, 13 had 2 episodes, and 1 had 3 episodes. Identical geno-
types showed the same alleles for all 6 markers. The genotypes causing both episodes were identical in most patients with 2 epi-
sodes (11/13; 84.6%). In contrast, 2 different genotypes were found in the patient with 3 episodes, one causing the first and sec-
ond episodes and the other causing the third episode (isolated 6 months later). We found marked genetic diversity in 174
different genotypes: 155 were unique, and 19 were endemic and formed 19 clusters (2 to 6 patients per cluster). Up to 25% of the
patients were infected by endemic genotypes that infected 2 or more different patients. Some of these endemic genotypes were
found in the same unit of the hospital, mainly neonatology, whereas others infected patients in different wards.
Candidemia is generally a nosocomial infection, and half of all
cases are caused by Candida albicans (1–5). Studying the ge-
netic relationship between C. albicans causing fungemia in the
hospital can uncover the presence of endemic genotypes, which
may suggest horizontal transmission and enable us to implement
prevention measures.
However, in the absence of genotyping, the potential routes of
infection and the presence of endemic genotypes of C. albicans in
the hospital are unknown. Several procedures are used to geno-
type C. albicans (6–8), and microsatellites in particular have a high
discriminatory power, the ability to detect heterozygote diploid
organisms (codominance), and a high reproducibility (9–12).
Previous studies have shown the presence of endemic geno-
types of C. albicans causing candidemia in specific hospital units,
mostly adult and neonatal intensive care units (ICUs) (8,13,14).
However, it is unknown whether endemic genotypes can be found
in other parts of the hospital. Furthermore, the proportion of
patients infected by endemic C. albicans genotypes has been
poorly studied.
We investigated the genotypic diversity of C. albicans isolates
from patients with candidemia who were admitted to a large ter-
tiary hospital in order to determine the percentage of patients
infected by endemic genotypes and the ward of hospitalization.
(This study was presented in part at the 22nd European Con-
gress of Clinical Microbiology and Infectious Diseases [ECCMID;
P-745], London, United Kingdom, 31 March to 3 April 2012.)
MATERIALS AND METHODS
Hospital description, definition of candidemia episodes, and patients
studied. This study was carried out at a large teaching hospital that serves
a population of approximately 715,000 inhabitants in the city of Madrid,
Spain. The institution cares for all types of patients at risk of acquiring
candidemia, including patients admitted to medical and surgical ICUs,
neonates, patients with hematological malignancies, solid organ trans-
plant recipients, and patients with central venous catheters.
During the study period, blood samples for culture were obtained by
standard procedures and incubated in the automated Bactec-NR system
(Becton-Dickinson, Cockeysville, MD).
From January 2007 to December 2011, 202 patients admitted to the
hospital had 217 episodes of candidemia caused by C. albicans. An episode
of candidemia was defined as the isolation of C. albicans from a blood
culture. In the absence of a consensus for the definition of additional
episodes of candidemia, we arbitrarily defined additional episodes as the
isolation of C. albicans in further blood cultures taken ⱖ7 days after the
last isolation in the previous episode.
Identification of the isolates. Blood cultures with presumptive visu-
alization of yeasts in the Gram stain were subcultured on CHROMagar
Candida plates (CHROMagar, Paris, France) and incubated at 35°C. Iso-
lates were identified by means of the ID 32C system (bioMérieux, Marcy
l’Etoile, France). Identification of C. albicans was confirmed by amplifi-
cation and sequencing of the ITS1-5.8S-ITS2 region (15).
Genotyping procedure. We genotyped 1 C. albicans strain represent-
ing one episode per patient using a panel of 6 short tandem repeats
(STRs), as reported elsewhere (9,11,12). The sizes of the amplified frag-
ments were determined by capillary electrophoresis with a 3130xl analyzer
(Applied Biosystems, Life Technologies Corporation, Carlsbad, CA) us-
ing the GeneScan ROX marker. Electropherograms were analyzed using
GeneMapper v.4.0 software (Applied Biosystems-Life Technologies Cor-
poration, CA). A C. albicans strain was used as a control in each run to
ensure accuracy of the size and to minimize run-to-run variation.
Received 22 February 2013 Returned for modification 3 April 2013
Accepted 17 April 2013
Published ahead of print 24 April 2013
Address correspondence to Jesús Guinea, jguineaortega@yahoo.es.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00516-13
2118 jcm.asm.org Journal of Clinical Microbiology p. 2118 –2123 July 2013 Volume 51 Number 7

Genotypic analysis. As C. albicans is diploid and can be homozygous
or heterozygous for each marker, the allelic composition for each locus
was studied.
The parameters of genetic diversity studied for each locus were as
follows: the number of alleles per locus and the frequency of null alleles (if
a mutation occurs at the annealing site, then the marker can no longer be
used, and the allele becomes a null allele) (16); observed heterozygosity
(Ho) (direct count calculated as the number of heterozygous genotypes
over the total number of genotypes analyzed for each locus); expected
heterozygosity (He) (He ⫽1⫺冱pi
2
, where pi is the frequency of the ith
allele) (17); Wright’s fixation index [F⫽1⫺(Ho/He)], which shows the
relationships between Ho and He and detects an excess or deficiency of
heterozygotes (37); and, finally, the probability of identity for unrelated
individuals [PI ⫽1⫺冱pi
4
⫹冱冱 (2pipj)
2
, where pi and pj are the fre-
quencies of the ith and jth alleles, respectively], which measures the prob-
ability that 2 randomly drawn diploid genotypes will be identical, assum-
ing observed allele frequencies and random assortment (19).
Significant deviations (P⬍0.001) in Hardy-Weinberg equilibrium at
the individual loci were tested using the Markov chain method. The com-
putations were performed using Arlequin version 3.01 (20) and Identity
1.0 (21).
The total allelic composition was converted to binary data by scoring
the presence or absence of each allele. Data were treated as categorical, and
the genetic relationships between the genotypes were studied by con-
structing a minimum spanning tree in BioNumerics version 6.6 (Applied
Maths, St.-Martens-Latem, Belgium). Genotypes showing the same alleles
for all 6 markers were considered identical. Endemic genotypes were de-
fined as identical genotypes infecting ⱖ2 different patients. A cluster was
defined as a group of ⱖ2 patients infected by an endemic genotype.
Endemic genotypes were confirmed after running the isolates in du-
plicate. The patients involved in each cluster were geographically related if
they were admitted to the same ward. In clusters involving patients who
were not geographically related at the time of the blood sample collection,
we studied the wards where patients had been hospitalized during the
previous 2 years.
RESULTS
Distribution of episodes of candidemia. At the time of diagnosis,
the 202 patients were admitted to the medical oncology and on-
cohematology units (n⫽21), adult postsurgical or medical ICUs
(n⫽34), pediatric and neonatology units (n⫽42), and other
adult units (n⫽105). The number of episodes per year ranged
from 32 to 55; the highest numbers of episodes were found in 2007
and 2010 (Fig. 1). The number of episodes recorded in ICUs was
higher in 2007; in contrast, the highest number of episodes in
pediatric units was found in 2010.
Intra-patient genotyping. Of the 202 patients admitted to the
medical oncology and oncohematology units, 188 had 1 episode,
13 had 2 episodes, and 1 had 3 episodes. In most of the patients
with 2 episodes (11/13; 84.6%), the genotypes involved with both
episodes were identical (mean, 10 days between episodes). In the
remaining 2 patients, the second episode occurred 10 and 13 days
after the first episode, respectively. Genotypes from the first and
second episodes differed in 2 and 3 markers, respectively.
In contrast, 2 different genotypes were found in the patient
with 3 episodes, one causing the first 2 episodes (9 days between
the first and the second episodes) and the other causing the third
episode (isolated 6 months later). The genotypes differed in 4
markers.
Genetic diversity and interpatient genotyping. The parame-
ters of genetic diversity are shown in Table 1. We found high
genetic diversity among the 217 C. albicans strains studied, as
shown by the high number of alleles detected, the low frequency of
null alleles, and the high heterozygosity. Despite the high diversity,
we observed heterozygote deficiency, as shown by the positive
values of Wright’s fixation index and the statistically significant
(P⬍0.001) departure from Hardy-Weinberg equilibrium in the
allele frequencies of the 6 loci. The probability of identity index
was 1.05 ⫻10
⫺8
, which showed that the markers with the highest
numbers of different alleles were the most informative.
A total of 174 genotypes were found in the 217 strains studied;
the genotype distribution is shown in Fig. 2. Of the 174 genotypes,
155 were unique and infected 1 patient each; the remaining 19
were endemic and formed 19 clusters (named 1 to 19) that in-
volved 51 patients (2 to 6 patients per cluster) (Fig. 2). Clusters
were classified according to the ward of hospitalization at the time
of blood sample collection.
The patients involved in 10 of the 19 clusters (53%) were geo-
graphically related. The first group accounted for 7 of the 19 clus-
ters and involved patients admitted to the same ward at the time of
blood sample collection, mostly in the neonatology unit (Table 2).
The 5 clusters involving neonates were observed from 2008 to
2010; 3 out of the 5 clusters included patients diagnosed in 2010,
when the highest number of cases of candidemia caused by C.
albicans was found in the unit (Fig. 1). These findings suggest the
TABLE 1 Genetic diversity in the C. albicans isolates studied
STR
a
No. of
different
alleles
Frequency
of null
alleles
b
Observed
heterozygosity
c
Expected
heterozygosity
Wright’s
index
d
Probability
of identity
e
CAI 36 0.082 0.75 0.91 0.17 0.012
CAIII 8 0.008 0.65 0.67 0.02 0.139
CAVI 36 0.113 0.65 0.86 0.24 0.028
CDC3 8 ⫺0.067 0.77 0.66 ⫺0.16 0.164
HIS3 33 0.149 0.57 0.85 0.32 0.035
EF3 20 0.143 0.59 0.85 0.31 0.035
Mean 23.5 0.071 0.67 0.80 0.15 0.07
a
Short tandem repeat. Allele frequencies of the 6 loci differed significantly (P⬍0.001)
from those expected in a population in Hardy-Weinberg equilibrium.
b
A frequency of null alleles of ⬍0.07 was considered nonsignificant.
c
Observed and expected heterozygosities ranged from 0 (no heterozygosity) to 1
(highest heterozygosity).
d
Wright’s index indicates a deficiency of heterozygosity (positive values) or excess
heterozygosity (negative values).
e
Probability of identity values near zero indicate the highest discriminative power of
the STR.
FIG 1 Distribution of episodes of candidemia diagnosed in each year of the
study period. The distribution of patients is also shown grouped by unit of
admission at the time of diagnosis.
C. albicans Endemic Genotypes
July 2013 Volume 51 Number 7 jcm.asm.org 2119

presence of outbreaks of candidemia, as most of the patients in-
volved were in the unit at the same time (Fig. 3). The second group
accounted for 3 of the 19 clusters that involved patients who were
in different wards at the time of the blood sample collection, al-
though they had previously shared a hospital ward (Table 3).
The 27 patients in the remaining 9 clusters (47%) did not show
a geographical relationship either at the time of blood sample
collection or during the previous 2 years. The patients who were
admitted were mainly adults (Table 4).
DISCUSSION
Candidemia caused by C. albicans is generally nosocomial (5).
Although C. albicans is part of the microbiota of patients with
candidemia, the disease can also be caused by exogenous strains
acquired during a hospital stay (10,22,23). Candidemia may be
transmitted horizontally in hospitalized patients when it is caused
by exogenous strains. Genotyping of isolates allows us to under-
stand the role of nosocomial transmission of C. albicans strains in
hospitalized patients (24,25).
We observed that most patients (75%) were infected by differ-
TABLE 2 Clusters of patients admitted to the same ward at the time of
blood sample collection
Cluster
code
No. of
patients
involved Ward of admission
Date of blood
culture collection
(month/day/yr)
1 2 Neonatology 02/14/2009
02/16/2010
6 2 Digestive medicine 12/16/2007
05/06/2008
7 2 Neonatology 03/02/2010
04/15/2010
10 2 General surgery 11/25/2009
12/03/2009
15 4 Neonatology 09/21/2010
09/24/2010
10/05/2010
10/24/2010
18 3 Neonatology 08/02/2010
12/10/2010
12/16/2010
19 2 Neonatology 06/11/2008
06/12/2008
FIG 2 Minimum spanning tree showing the distribution of the 174 genotypes (circles) found in the strains studied and the number of strains belonging to the
same genotype (larger circles indicate higher numbers). The connecting lines between the circles show the similarity between the profiles: the black lines indicate
differences in only 1 marker, and the gray lines indicate differences in 2 or more markers. The numbers represent the cluster codes.
Escribano et al.
2120 jcm.asm.org Journal of Clinical Microbiology

ent genotypes, suggesting an endogenous origin, as reported by
others (26,27). However, we found that up to 25% of patients can
be infected by endemic C. albicans genotypes. Consequently, the
strains might have a common source, such as health care workers,
biomedical devices, parenteral nutrition, and the hospital envi-
ronment (13,28,29). Interestingly, only half of the patients in-
fected by endemic genotypes were or had been admitted to the
same ward at the time of blood sample collection; in these cases,
the patients were usually in the ward at the same time. Genotyping
of the strains from the patients admitted to the neonatology ward
showed that endemic genotypes persisted in the unit for up to
several months, as illustrated by the patients in clusters 1 and 18
(Fig. 3). However, several of the clusters were found in 2010
among patients who were in the unit at the same time, suggesting
the presence of an outbreak of candidemia during that period.
Of note, 13% of the patients were infected by endemic geno-
types, although we were unable to demonstrate any geographical
relationship between them. The patients were mainly adults and
had been admitted to the hospital at different times, as shown in
Table 4. Some patients in these clusters (cluster codes 2, 3, 8, 11,
and 12) were diagnosed with candidemia at similar times, thus
suggesting a common source for the isolates. A potential explana-
tion is the presence of persistent endemic genotypes adapted to
surviving in common areas of the hospital. Patients may become
infected when visiting these areas during their stay, after ingestion
of contaminated food, or even after receiving contaminated med-
ication. Another explanation might be that these genotypes occur
more frequently than others (12,30,31) and can be actively trans-
mitted from person to person, from the environment to patients,
and from health care workers to patients.
The presence of clusters involving patients who are not geo-
graphically related may be a consequence of the limitation of the
genotyping procedure. A lack of discrimination of the STR mark-
ers used was ruled out for different reasons. First, we found
marked diversity, as shown by the total probability of identity of
1.05 ⫻10
⫺8
, which indicates that the probability of finding 2
strains with the same genotype was almost zero. Second, a clonal
nature for the population structure is suggested by the statistically
significant deviation from Hardy-Weinberg equilibrium, proba-
bly owing to heterozygote deficiency. Finally, heterozygote defi-
ciency was not due to a lack of amplification of markers, as shown
by the low frequency of null alleles. Heterozygote deficiency might
be caused by the clonal nature of the C. albicans populations (31–
34), by chromosomal rearrangements such as aneuploidy (a lack
of chromosomes or presence of extra chromosomes), or by a loss
of heterozygosity as a response to antifungal stress (35–37).
Our study has several limitations. We did not determine a po-
tential source of infection or route of transmission in the hospital
because we did not study isolates from the hospital environment,
from health care workers, from other anatomical sites of the pa-
FIG 3 Timeline showing length of stay (months) for the patients involved in the 5 clusters in the neonatology unit. The numbers indicate the date of blood
sample collection for each patient involved in the cluster.
TABLE 3 Clusters involving patients who were admitted to different wards at the time of diagnosis of candidemia but who had a shared ward of
admission in the previous 2 years
Cluster
code
No. of
patients
involved
Ward of admission at time
of diagnosis
Date of blood culture
collection
(month/day/yr)
Ward of hospitalization
in the previous month Month of coincidence
4 2 Pediatric ICU 04/13/2008 Pediatric hematology March 2008
Pediatric hematology 06/16/2008
9 2 General surgery 05/09/2008 General surgery May 2008
Internal medicine 06/19/2008
17 2 Internal medicine 06/28/2007 Internal medicine May 2008
Digestive medicine 05/07/2009
C. albicans Endemic Genotypes
July 2013 Volume 51 Number 7 jcm.asm.org 2121

tients with candidemia, or even from mothers who could colonize
and further infect neonates during delivery. Since strains may
have been commensal fungi in the host, transmission between
patients could be ruled out. Furthermore, we cannot exclude the
possibility that endemic genotypes are a consequence of chromo-
somal rearrangements in the isolates or homoplasy (alleles with
identical sizes but different sequences), so we must therefore ac-
cept them as an intrinsic limitation of microsatellite analysis.
In summary, we found marked genetic diversity among C. al-
bicans isolates causing candidemia. However, up to 25% of the
patients were infected by endemic genotypes detected in 2 or more
patients. Some of these endemic genotypes were found in the same
units, whereas others infected patients in different wards. Future
studies are necessary to clarify the sources and routes of transmis-
sion of endemic genotypes in hospitals.
ACKNOWLEDGMENTS
We thank Thomas O’Boyle for editing and proofreading the article.
This work was supported by grants from the Fondo de Investigación
Sanitaria (grants PI11/00167 and PI10/02868) and Santander-Universi-
dad Complutense de Madrid (GR35/10-A). P. Escribano (CD09/00230)
and J. Guinea (MS09/00055) are supported by the Fondo de Investigación
Sanitaria. Ainhoa Simon Zarate holds a grant from the Fondo de Investi-
gación Sanitaria and provides technical support in the Línea Instrumental
Secuenciación. The 3130xl genetic analyzer was partially financed by
grants from the Fondo de Investigación Sanitaria (IF01-3624 and IF08-
36173).
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TABLE 4 Clusters involving patients who were not admitted to the
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years
Cluster
code
No. of
patients
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Date of blood
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(month/day/yr)
Ward of admission at
time of diagnosis
2 3 7/17/2010 Angiology
11/23/2010 Neonatology
12/16/2010 Neonatology
3 6 4/26/2008 Digestive medicine
7/18/2009 Oncology
4/30/2010 Oncohematology
7/15/2010 Oncology
12/18/2010 Geriatric
8/23/2011 General surgery
5 2 3/20/2008 Pediatric ICU
3/1/2010 Major heart postsurgical
surgery unit
8 2 11/3/2008 General surgery
11/13/2008 Major heart post-surgical
surgery unit
11 4 5/9/2008 Pneumology
9/13/2008 Adult ICU
9/23/2008 Postsurgical ICU
2/24/2010 Geriatric
12 3 5/10/2007 General surgery
8/20/2007 Geriatric
2/25/2010 General surgery
13 2 12/10/2007 Digestive medicine
3/12/2008 Major heart postsurgical
surgery unit
14 3 1/3/2008 Digestive medicine
11/18/2008 Geriatric
2/10/2009 Oncology
16 2 7/7/2010 Angiology
4/20/2011 Internal medicine
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