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Endemic Genotypes of Candida albicans Causing Fungemia 1
Are Frequent in the Hospital 2
3
Pilar Escribano 1, 2, 3 4
Marta Rodríguez-Créixems 1, 2, 3 5
Carlos Sánchez-Carrillo 1, 2, 3 6
Patricia Muñoz 1, 2, 3, 4 7
Emilio Bouza 1, 2, 3, 4 8
Jesús Guinea* 1, 2, 3, 4 9
10 1Clinical Microbiology and Infectious Diseases Department, Hospital General 11
Universitario Gregorio Marañón, Universidad Complutense de Madrid, Madrid, Spain 12 2Instituto de Investigación Sanitaria del Hospital Gregorio Marañón, Madrid, Spain 13 3CIBER de Enfermedades Respiratorias (CIBER RES CD06/06/0058), Palma de 14
Mallorca, Spain 15 4Microbiology Department, School of Medicine, Universidad Complutense de Madrid, 16
Madrid, Spain 17
18
Running title: C. albicans endemic genotypes 19
Key words: Candida albicans, microsatellite, genetic diversity, endemic 20
genotype, cluster, transmission 21
22
Abstract word count: 214 23
Text word count: 2185 24
Correspondence: 25 Jesús Guinea 26 Servicio de Microbiología Clínica y Enfermedades Infecciosas 27 Hospital General Universitario Gregorio Marañón 28 C/Dr. Esquerdo, 46 29 28007 Madrid, Spain 30 Phone: +34 914265104 31 Fax: +34 915044906 32 [email protected] 33 34
This study was presented in part at the 22nd European Congress of Clinical 35
Microbiology and Infectious Diseases (ECCMID; P-745), London, UK, March 31 36
to April 3, 2012. 37
38
Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00516-13 JCM Accepts, published online ahead of print on 24 April 2013
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ABSTRACT 39
Genotyping of Candida albicans strains causing candidemia can uncover the 40
presence of endemic genotypes in the hospital. Using a highly reproducible and 41
discriminatory microsatellite marker panel, we studied the genetic diversity of 42
217 C. albicans isolates from the blood cultures of 202 patients with candidemia 43
(January 2007 to December 2011). Each isolate represented 1 candidemia 44
episode. Multiple episodes were defined as isolation of C. albicans in further 45
blood cultures taken ≥7 days after the last isolation in blood culture. Of the 202 46
patients, 188 had 1 episode, 13 had 2 episodes, and 1 had 3 episodes. Identical 47
genotypes showed the same alleles for all 6 markers. The genotypes causing 48
both episodes were identical in most patients with 2 episodes (11/13; 84.6%). In 49
contrast, 2 different genotypes were found in the patient with 3 episodes, one 50
causing the first and second episodes and the other causing the third episode 51
(isolated 6 months later). We found a marked genetic diversity in 174 different 52
genotypes: 155 were unique, and 19 were endemic and formed 19 clusters (2-6 53
patients per cluster). Up to 25% of patients were infected by endemic genotypes 54
that infected 2 or more different patients. Some of these endemic genotypes 55
were found in the same unit, mainly neonatology, whereas others infected 56
patients in different wards. 57
58
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INTRODUCTION 59
Candidemia is generally a nosocomial infection, and half of all cases are 60
caused by Candida albicans (1, 18, 24, 26, 28). Studying the genetic 61
relationship between C. albicans causing fungemia in the hospital can uncover 62
the presence of endemic genotypes, which may suggest horizontal 63
transmission, and enable us to implement prevention measures. 64
However, in the absence of genotyping, the potential routes of infection and 65
the presence of endemic genotypes of C. albicans in the hospital are unknown. 66
Several procedures are used to genotype C. albicans (4, 10, 32), and 67
microsatellites, in particular, have a high discriminatory power, the ability to 68
detect heterozygote diploid organisms (codominance), and high reproducibility 69
(6, 9, 30, 31). 70
Previous studies have shown the presence of endemic genotypes of C. 71
albicans causing candidemia in specific hospital units, mostly adult and 72
neonatal intensive care units (ICUs) (3, 21, 32). However, it is unknown whether 73
endemic genotypes can be found in other parts of the hospital. Furthermore, the 74
proportion of patients infected by endemic C. albicans genotypes has been 75
poorly studied. 76
We investigated the genotypic diversity of C. albicans isolates from patients 77
with candidemia who were admitted to a large tertiary hospital in order to 78
determine the percentage of patients infected by endemic genotypes and the 79
ward of hospitalization. 80
81
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MATERIALS AND METHODS 82
Hospital description, definition of candidemia episodes, and patients 83
studied. This study was carried out at a large teaching hospital that serves a 84
population of approximately 715,000 inhabitants in the city of Madrid, Spain. 85
The institution cares for all types of patients at risk of acquiring candidemia, 86
including patients admitted to medical and surgical ICUs, neonates, patients 87
with hematological malignancies, solid organ transplant recipients, and patients 88
with central venous catheters. 89
During the study period, blood samples for culture were obtained by 90
standard procedures and incubated in the automated BACTEC-NR system 91
(Becton Dickinson, Cockeysville, Maryland, USA). 92
From January 2007 to December 2011, 202 patients admitted to the 93
hospital had 217 episodes of candidemia caused by C. albicans. An episode of 94
candidemia was defined as the isolation of C. albicans from blood cultures. In 95
the absence of a consensus for the definition of additional episodes of 96
candidemia, we arbitrarily defined additional episodes as the isolation of C. 97
albicans in further blood cultures taken ≥7 days after the last isolation in the 98
previous episode. 99
100
Identification of the isolates. Blood cultures with presumptive visualization 101
of yeasts in the Gram stain were sub-cultured on CHROMagar Candida plates 102
(CHROMagar, Paris, France) and incubated at 35°C. Isolates were identified by 103
means of the ID 32C system (bio-Mérieux, Marcy l'Etoile, France). Identification 104
of C. albicans was confirmed by amplification and sequencing of the ITS1-5.8S-105
ITS2 region (36). 106
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107
Genotyping procedure. We genotyped 1 C. albicans strain representing 108
one episode per patient using a panel of 6 short tandem repeats (STRs), as 109
reported elsewhere (6, 30, 31). The size of the amplified fragments was 110
determined by capillary electrophoresis with a 3130xl analyzer (Applied 111
Biosystems-Life Technologies Corporation, Carlsbad, California, USA) using the 112
GeneScan ROX marker. Electropherograms were analyzed using 113
GeneMapper® v.4.0 software (Applied Biosystems-Life Technologies 114
Corporation, California). A C. albicans strain was used as a control in each run 115
to ensure accuracy of size and to minimize run-to-run variation. 116
117
Genotypic analysis. The allelic composition was studied for each locus, as 118
C. albicans is diploid and can be homozygous or heterozygous for each marker. 119
The parameters of genetic diversity studied for each locus were as follows: 120
number of alleles per locus and frequency of null alleles (if a mutation occurs at 121
the annealing site, then the marker can no longer be used and the allele 122
becomes a null allele) (8); observed heterozygosity (Ho, direct count calculated 123
as the number of heterozygous genotypes over the total number of genotypes 124
analyzed for each locus); expected heterozygosity (He=1 - ∑pi2 where pi is the 125
frequency of the ith allele) (23); Wright’s fixation index (F=1 - Ho/He), which 126
shows the relationships between Ho and He and detects an excess or 127
deficiency of heterozygotes (37); and, finally, the probability of identity for 128
unrelated individuals (PI=1 - ∑pi4 + ∑∑ (2pipj)2, where pi and pj are the 129
frequency of the ith and jth alleles respectively), which measures the probability 130
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that 2 randomly drawn diploid genotypes will be identical assuming observed 131
allele frequencies and random assortment (25). 132
Significant deviations (p<0.001) in Hardy-Weinberg equilibrium at individual 133
loci were tested using the Markov chain method. The computations were 134
performed using Arlequin version 3.01 (14) and IDENTITY 1.0 (35). 135
Total allelic composition was converted to binary data by scoring the 136
presence or absence of each allele. Data were treated as categorical, and the 137
genetic relationship between the genotypes was studied by constructing a 138
minimum spanning tree (BioNumerics version 6.6, Applied Maths, St.-Martens-139
Latem, Belgium). Genotypes showing the same alleles for all 6 markers were 140
considered identical. Endemic genotypes were defined as identical genotypes 141
infecting ≥2 different patients. A cluster was defined as a group of ≥2 patients 142
infected by an endemic genotype. 143
Endemic genotypes were confirmed after running the isolates in duplicate. 144
The patients involved in each cluster were geographically related if they were 145
admitted to the same ward. In clusters involving patients who were not 146
geographically related at the time of blood sample collection, we studied the 147
wards where patients had been hospitalized during the previous 2 years. 148
149
RESULTS 150
Distribution of episodes of candidemia. At the time of diagnosis, the 202 151
patients were admitted to the medical oncology and onco-hematology units 152
(n=21), adult post-surgical or medical ICUs (n=34), pediatric and neonatology 153
units (n=42), and other adult units (n=105). The number of episodes per year 154
ranged from 32 to 55; the highest numbers of episodes were found in 2007 and 155
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2010 (Figure 1). The number of episodes recorded in ICUs was higher in 2007; 156
in contrast, the highest number of episodes in pediatric units was found in 2010. 157
158
Intra-patient genotyping. Of the 202 patients admitted to the medical 159
oncology and onco-hematology units, 188 had 1 episode, 13 had 2 episodes, 160
and 1 had 3 episodes. In most of the patients with 2 episodes (11/13; 84.6%), 161
both genotypes were identical (mean 10 days between episodes). In the 162
remaining 2 patients, the second episode occurred 10 and 13 days after the first 163
episode. Genotypes from the first and the second episodes differed in 2 and 3 164
markers. 165
In contrast, 2 different genotypes were found in the patient with 3 166
episodes, one causing the first 2 episodes (9 days between the first and the 167
second episode) and the other causing the third episode (isolated 6 months 168
later). Both genotypes differed in 4 markers. 169
170
Genetic diversity and inter-patient genotyping. The parameters of 171
genetic diversity are shown in Table 1. We found a high genetic diversity among 172
the 217 C. albicans strains studied, as shown by the high number of alleles 173
detected, the low frequency of null alleles, and the high heterozygosity. Despite 174
the high diversity, we observed heterozygote deficiency, as shown by the 175
positive values of Wright’s fixation index and the statistically significant (p < 176
0.001) departure from Hardy–Weinberg equilibrium in the allele frequencies of 177
the 6 loci. The probability of identity index was 1.05 x 10-8, which showed that 178
markers with the highest number of different alleles were the most informative. 179
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A total of 174 genotypes were found in the 217 strains studied; genotype 180
distribution is shown in Figure 2. Of the 174 genotypes, 155 were unique and 181
infected 1 patient each; the remaining 19 were endemic and formed 19 clusters 182
(named 1 to 19) that involved 51 patients (2-6 patients per cluster) (Figure 2). 183
Clusters were classified according to the ward of hospitalization at the time of 184
blood sample collection. 185
The patients involved in 10 of the 19 clusters (53%) were geographically 186
related. The first group accounted for 7 of the 19 clusters and involved patients 187
admitted to the same ward at the time of blood sample collection, mostly in the 188
neonatology unit (Table 2). The 5 clusters involving neonates were observed 189
from 2008 to 2010; 3 out of the 5 clusters included patients diagnosed in 2010, 190
when the highest number of cases of candidemia caused by C. albicans was 191
found in the unit (Figure 1). These findings suggest the presence of outbreaks 192
of candidemia, as most of the patients involved were in the unit at the same 193
time (Figure 3). The second group accounted for 3 of the 19 clusters that 194
involved patients who were in different wards at the time of the blood sample 195
collection, although they had previously shared a ward of hospitalization (Table 196
3). 197
The 27 patients conforming the remaining 9 clusters (47%) did not show a 198
geographical relationship either at the time of blood sample collection or during 199
the previous 2 years. The patients who were admitted were mainly adults (Table 200
4). 201
202
DISCUSSION 203
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Candidemia caused by C. albicans is generally nosocomial (28). Although 204
C. albicans is part of the microbiota of patients with candidemia, the disease 205
can also be caused by exogenous strains acquired during hospital stay (9, 27, 206
33). Candidemia may be transmitted horizontally in hospitalized patients when it 207
is caused by exogenous strains. Genotyping of isolates allows us to understand 208
the role of nosocomial transmission of C. albicans strains in hospitalized 209
patients (12, 16). 210
We observed that most patients (75%) were infected by different 211
genotypes, suggesting an endogenous origin, as reported by other authors (13, 212
34). However, we found that up to 25% of patients can be infected by endemic 213
C. albicans genotypes. Consequently, the strains could have a common source, 214
such as health care workers, biomedical devices, parenteral nutrition, and the 215
hospital environment (2, 3, 5). Interestingly, only half of the patients infected by 216
endemic genotypes were or had been admitted to the same ward at the time of 217
blood sample collection; in these cases, patients were usually in the ward at the 218
same time. Genotyping of strains from patients admitted to the neonatology 219
ward showed that endemic genotypes persisted in the unit for up to several 220
months, as illustrated by the patients conforming clusters 1 and 18 (Figure 3). 221
However, several of the clusters were found in 2010 among patients who were 222
in the unit at the same time, suggesting the presence of an outbreak of 223
candidemia during that period. 224
Of note, 13% of the patients were infected by endemic genotypes, 225
although we were unable to demonstrate any geographical relationship between 226
them. The patients were mainly adults and had been admitted to hospital at 227
different times, as shown in Table 4. Some patients in these clusters (cluster 228
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codes 2, 3, 8, 11, and 12) were diagnosed with candidemia at similar times, 229
thus suggesting a common source for the isolates. A potential explanation could 230
be the presence of persistent endemic genotypes adapted to surviving in 231
common areas of the hospital. Patients may become infected when visiting 232
these areas during their stay, after ingestion of contaminated food, or even after 233
receiving contaminated medication. Another explanation could be that these 234
genotypes are more frequent than others (11, 20, 31) and could be actively 235
transmitted from person to person, from the environment to patients, and from 236
health care workers to patients. 237
The presence of clusters involving patients who are not geographically 238
related may be a consequence of the limitation of the genotyping procedure. 239
Lack of discrimination of the STR markers used was ruled out for different 240
reasons. First, we found a marked diversity, as shown by the total probability of 241
identity of 1.05 x 10-8, which indicates that the probability of finding 2 strains 242
with the same genotype was almost zero. Second, a clonal nature for the 243
population structure is suggested by the statistically significantly deviation from 244
Hardy–Weinberg equilibrium, probably owing to heterozygote deficiency. And 245
finally, heterozygote deficiency was not due to lack of amplification of markers, 246
as shown by the low frequency of null alleles. Heterozygote deficiency could be 247
caused by the clonal nature of the C. albicans populations (19, 20, 22, 29), by 248
chromosomal rearrangements such as aneuploidy (lack of chromosomes or 249
presence of extra chromosomes), or by loss of heterozygosity as a response to 250
antifungal stress (7, 15, 17). 251
Our study has several limitations. We did not determine a potential source 252
of infection or route of transmission in the hospital because we did not study 253
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isolates from the hospital environment, from health care workers, from other 254
anatomical sites of the patients with candidemia, or even from mothers who 255
could colonize and further infect neonates during delivery. Since strains may 256
have been commensal fungi in the host, transmission between patients could 257
be ruled out. Furthermore, we cannot exclude that endemic genotypes are a 258
consequence of chromosomal rearrangements in the isolates or homoplasy 259
(alleles with identical sizes but with different sequences) and must therefore 260
accept them as an intrinsic limitation of microsatellite analysis. 261
In summary, we found a marked genetic diversity among C. albicans 262
isolates causing candidemia. However, up to 25% of patients were infected by 263
endemic genotypes detected in 2 or more patients. Some of these endemic 264
genotypes were found in the same units, whereas others infected patients in 265
different wards. Future studies are necessary to clarify the source and routes of 266
transmission of endemic genotypes in the hospital. 267
268
ACKNOWLEDGMENTS 269
We would like to thank Thomas O’Boyle for editing and proofreading the 270
article. 271
This work was supported by grants from Fondo de Investigación Sanitaria 272
(grants number PI11/00167 and PI10/02868) and Santander-Universidad 273
Complutense de Madrid (GR35/10-A). 274
P. Escribano (CD09/00230) and J. Guinea (MS09/00055) are supported 275
by the Fondo de Investigación Sanitaria. 276
Ainhoa Simon Zarate holds a grant from the Fondo de Investigación 277
Sanitaria and provides technical support in the Línea Instrumental 278
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Secuenciación. The 3130xl Genetic Analyzer was partially financed by grants 279
from Fondo de Investigación Sanitaria (IF01-3624, IF08-36173). 280
281
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FIGURES AND TABLES 282
Figure 1. Distribution of episodes of candidemia diagnosed in each year of the 283
study period. The distribution of patients is also shown grouped by area of 284
admission at the time of diagnosis. 285
286
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Figure 2. Minimum spanning tree showing the distribution of the 174 genotypes 287
(circles) found in the strains studied and the number of strains belonging to the 288
same genotype (larger circles indicate higher numbers). Circles in white 289
represent unique genotypes. Coloured circles represent clusters of strains 290
isolated within the same ward (light solid), strains from patients with the same 291
admission ward (lines), and strains from patients with no geographic similarities 292
(dark solid). The connecting lines between circles show the similarity between 293
profiles: black lines indicate differences in only 1 marker, and grey lines indicate 294
differences in 2 or more markers. Numbers represent the cluster codes. 295
296
297
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Figure 3. Timeline showing length of stay (months) for the patients involved in the 5 clusters in the neonatology unit. The 298
numbers indicate the date of blood sample collection for each patient involved in the cluster. 299
300
301
302
303
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Table 1. Genetic diversity in the C. albicans isolates studied. 304
305 306
STR* Number of different alleles
Frequency of null alleles
Observed heterozygosity
Expected heterozygosity
Wright’s index
Probability of identity
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
307
*Short tandem repeat. Allele frequencies of the 6 loci differed significantly (p < 0.001) from those expected 308 in a population in Hardy–Weinberg equilibrium. 309 Frequency of null alleles < 0.07 was considered nonsignificant. 310 Observed and expected heterozygosity range from 0 (no heterozygosity) to 1 (highest heterozygosity). 311 Wright’s index indicates deficiency of heterozygosity (positive values) or excess heterozygosity (negative 312 values). 313 Probability of identity values near zero indicates the highest discriminative power of the STR. 314 315 316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
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Table 2. Clusters of patients admitted to the same ward at the time of blood 335
sample collection. 336
337 338
339 Cluster code
No. of patients involved
Ward of admission
Date of blood culture
collection
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
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Table 3. Clusters involving patients who were admitted to different units at the 340
time of diagnosis of candidemia but who had shared a ward of admission in the 341
previous 2 years. 342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
Cluster code
No. of patients involved
Ward of admission at the time
of diagnosis
Date of blood culture collection
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
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Table 4. Clusters involving patients who were not admitted to the same unit at 360
the time of blood sample collection or in the previous 2 years. 361
362
Cluster code
No. of patients involved
Date of blood culture collection (month/day/year)
Ward of admission at the time of diagnosis
2 3
7/17/10 Angiology
11/23/10 Neonatology
12/16/10 Neonatology
3 6
4/26/08 Digestive medicine
7/18/09 Oncology
4/30/10 Oncohematology
7/15/10 Oncology
12/18/10 Geriatric
8/23/11 General surgery
5 2 3/20/08 Pediatric ICU
3/1/10 Major heart post-surgical surgery unit
8 2 11/3/08 General surgery
11/13/08 Major heart post-surgical surgery unit
11 4
5/9/08 Pneumology
9/13/08 Adult ICU
9/23/08 Post-surgical ICU
2/24/10 Geriatric
12 3
5/10/07 General surgery
8/20/07 Geriatric
2/25/10 General surgery
13 2 12/10/07 Digestive medicine
3/12/08 Major heart post-surgical surgery unit
14 3
1/3/08 Digestive medicine
11/18/08 Geriatric
2/10/09 Oncology
16 2 7/7/10 Angiology
4/20/11 Internal Medicine
363
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