Endemic Genotypes of Candida albicans Causing Fungemia Are ...

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

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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

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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

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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


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

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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


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


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 4

5/9/08 Pneumology

9/13/08 Adult ICU

9/23/08 Post-surgical ICU

2/24/10 Geriatric

12 3


8/20/07 Geriatric


13 2 12/10/07 Digestive medicine


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

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Endemic Genotypes of Candida albicans Causing Fungemia Are ...

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