Molecular epidemiology of Candida parapsilosis sepsis from outbreak investigations in neonatal intensive care units

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Infection, Genetics and Evolution 8 (2008) 103–109 www.elsevier.com/locate/meegid

Molecular epidemiology of Candida parapsilosis sepsis from outbreak investigations in neonatal intensive care units Errol Reiss a,*, Brent A. Lasker b, Naureen J. Iqbal a, Michael J. James c, Beth A. Arthington-Skaggs a a

Mycotic Diseases Branch, Division of Foodborne, Bacterial and Mycotic Diseases, National Center for Zoonotic, Vector borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b Bacterial Zoonoses Branch, Division of Foodborne, Bacterial and Mycotic Diseases, National Center for Zoonotic, Vector borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA c Johns Hopkins University School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore MD 21287, USA Received 17 September 2007; received in revised form 15 October 2007; accepted 23 October 2007 Available online 3 December 2007

Abstract The DNA probe, Cp3-13, was used in a Southern blot assay for genotyping Candida parapsilosis (CP) from 3 fungemia outbreaks in neonatal intensive care units (NICUs) in the southeastern U.S. Genotyping, in 2 outbreaks, supplied evidence of horizontal transmission of CP. In the third outbreak, bloodstream isolates (BSIs) of 2 genotypes circulated in the NICU, one was shared by a BSI and a healthcare worker’s hand culture. A fourth cluster of recurrent episodes of fungemia occurred in outpatients of a children’s hospital receiving total parenteral nutrition (TPN) at home. Each child was infected with a different CP genotype which persisted during recurrences. These genotypes were included in a dendrogram from a CDC population-based surveillance for candidemia consisting of 73 clone-corrected Cp3-13 genotypes (overall SAB = 0.36). Analysis revealed a cluster of 11 genotypes (mean SAB = 0.66) including 3 pairs with identical hybridization profiles. A second cluster of 8 genotypes contained clones from 3 outbreaks (mean SAB = 0.76) but no clustering of genotypes specific for neonates was identified. No decreased susceptibility to azole and polyene antifungal agents was detected in this collection of CP. The frequent occurrence of transmission of CP in this vulnerable population underlines the relevance of Cp3-13 subtyping to investigate suspected transmission and persistence of CP strains in the NICU. # 2007 Elsevier B.V. All rights reserved. Keywords: Candida parapsilosis; Neonatal intensive care unit; Genotyping; Genetic relatedness

1. Introduction Septicemia is the most common severe infection encountered in the neonatal intensive care unit (NICU). Depending on the institution, Candida species rank second to fourth as the most frequent cause of late-onset sepsis in very low birth weight (VLBW < 1500 g) infants (Bendel, 2005; Stoll et al., 1996; Yalaz et al., 2006). Occurring 72 h after birth, late-onset sepsis is referred to as nosocomial sepsis to emphasize the possibility of transmission in the NICU. Candida parapsilosis (CP) has emerged as the predominant fungal pathogen causing bloodstream infections (BSIs) among VLBW infants in some

* Corresponding author. Tel.: +1 404 639 3374; fax: +1 404 639 3546. E-mail address: [email protected] (E. Reiss). 1567-1348/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2007.10.007

NICUs and in others ranks second to C. albicans (Benjamin Jr, et al., 2000; Rodriguez et al., 2006; San Miguel et al., 2005). Crude mortality caused by Candida species infections in the NICU was estimated at 25–30% (Bendel, 2005) with an attributable mortality of 13% (Fridkin et al., 2006). CP sepsis of the newborn can have a high case-to-fatality ratio, which in 1 study resulted in death in 42% of 19 infants with CP fungemia (Benjamin Jr et al., 2000). Premature infants are susceptible to Candida infections because of an immature immune system and immature skin which is not an efficient barrier to Candida (Schelonka and Infante, 1998; Siegfried, 1998). Other risk factors are indwelling catheters of all types, broad spectrum antibiotics, and steroid therapy (Chapman, 2003). Transmission of Candida species to infants in the NICU may be via maternal contact, postnatally from healthcare workers (HCW), or from contaminated solutions and fomites. Colonization of the hands of HCWs varies according to the hospital: in

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one study 47% of the nurses and 44% of the physicians in the NICU were colonized with CP (Verduyn Lunel et al., 1999). Gastrointestinal colonization with Candida species in the NICU was estimated at 45.5% in infants who later developed candidemia, compared with 21.9% colonization in non-case infants (Saiman et al., 2000). Genomic Southern blot analysis of CP utilizes the complex DNA probe Cp3-13, a 15 kb cloned DNA fragment distributed on 5 of the 10 CP chromosomes (Enger et al., 2001). This genotyping method is more discriminatory than previous methods including chromosomal electrokaryotyping (EK) (Welbel et al., 1996) and is less prone to reproducibility problems than rapid amplification of polymorphic DNA (RAPD) (Lehmann et al., 1992). We applied Cp3-13 subtyping to 3 clusters of CP fungemia in neonates and 1 cluster in children with short bowel syndrome investigated by CDC epidemiologists in the 1990’s before this method was available. Two of these outbreaks had previously been reported (Cano et al., 2005; Welbel et al., 1996). The objectives were to investigate the role of horizontal transmission of CP in the NICU; contribute to a better understanding of the genetic diversity of CP isolates obtained from neonates; and determine whether decreased susceptibility

to antifungal drugs occurred among isolates causing clusters of CP sepsis. 2. Materials and methods 2.1. Strain collection Thirty-four CP isolates from outbreak investigations were obtained from the culture collection of the Mycotic Diseases Branch, CDC (Table 1). An additional 59 CP isolates came from the 1998–2000 CDC surveillance for candidemia in hospitals of Connecticut (CT) and Maryland (MD) (Sofair et al., 2006); 1 additional genotype (MS) represents the CP outbreak clone from fungemia in adult patients in a Mississippi community hospital (Clark et al., 2004). CP was identified by standard methods: microscopic morphology and assimilation reactions in the API 20C AUX kit (bioMerieux Inc. Durham, NC). 2.2. Outbreak and patient information (Table 1) Outbreak I. NICU Louisiana University Medical Center, University Hospital, Shreveport, LA. Five low birth weight

Table 1 Collection of CP bloodstream and surveillance isolates obtained in 4 outbreaks during 1991–1999 Hospital

Culture no.

Body site

Patient no.

Cp3-13 genotypea

NICU, Louisiana State University Medical Center, 1991 Welbel et al. (1996).

Y532-91 Y542-91 Y546-91

Blood Blood Blood

LA-1 LA-2 LA-3

LA-2 LA-2 LA-2

NICU, Regional Med Ctr., Atlanta. 1997

98000678 876MJ-a 98000675 878MJ-a 98000865a 98000867 98000673b 98000674b 980003245

Blood URT Blood I.V. site Blood, URT, CSF, Blood, Axilla (non-case pt)

ATL-1-1 ATL-1-2 ATL-2-1 ATL-2-2 ATL-3-1 ATL-3-2 ATL-4-1 ATL-5-1 ATL-6-1

ATL1-1 ATL1-1 ATL1-1 ATL1-1 ATL1-1 ATL1-1 ATL1-1 ATL1-1 ATL-6-1

NICU –Regional Med Center, Central GA, 1998-1999

CP Y97-863 CP Y97-864 CP Y97-865 MC-2 MC 6 MC 7 MC 8 MC 9 MC 11 MC 23 MC 40 MC 60

Blood case pt Blood, case pt Blood, case pt Hand medical student Skin, non-case infant Hand, Pharmacist Skin, non-case infant Skin, non-case infant Hand, attending physician Hand, Nurse Hand, Nurse Skin non-case infant.

GA-1 GA-2 GA-3 GA-4 GA-5 GA-6 GA-7 GA-8 GA-9 GA-10 GA-11 GA-12

GA-3 GA-12 GA-3 GA-4 GA-5 GA-6 GA-5 GA-3 GA-9 GA-3 GA-11 GA-12

Outpatients of a Children’s Hospital, Memphis TN, 1998-1999 Cano et al. (2005)

Y0884-99 Y0885-99 Y0886-99 Y0887-99 Y0889-99 Y0890-99 Y0891-99 Y0892-99 2000200060 2000200061

Blood, 2/12/98 Blood, 6/13/99 Blood, 5/17/99 Blood, 8/5/99 Blood, 2/17/99 Blood, 5/13/99 Blood, 3/31/99 Blood, 6/4/99 Hand, caregiver patient 2 Hand, caregiver patient 2

ME-1-1 ME-1-3 ME-1-2 ME-1-4 ME-2-1 ME-2-2 ME-3-1 ME-3-2 ME-2-3 ME-2-4

ME-1-3 ME-1-3 ME-1-3 ME-1-3 ME-2-2 ME-2-2 ME-3-2 ME-3-2 ME-2-3 ME-2-4

a

Cp3-13 genotype as shown in Figs. 1 and 2.

E. Reiss et al. / Infection, Genetics and Evolution 8 (2008) 103–109

(LBW, mean 1791 g; range, 585–3425 g) neonates in a 25 bed NICU had positive blood cultures for CP during July 20–27, 1991 (Welbel et al., 1996). Isolates from 3 of these infants were available for analysis. Outbreak II. NICU, Regional Medical Center, Atlanta, GA. This is a 410 bed acute care hospital in Clayton County, GA. During May to October, 1997 hospital infection control identified 5 cases of CP infections in VLBW infants in their NICU: 4 BSI and a culture from cerebrospinal fluid (CSF) from an infant with a ventriculo-peritoneal shunt implanted for hydrocephalus. Risk factors identified for the cases included birthweight <1000 g, intubation with mechanical ventilation, previous antimicrobial therapy, total parenteral nutrition (TPN), and umbilical catheterization. All 5 infants were successfully treated with amphotericin B. The 5 sterile-site CP isolates and an i.v. site culture from a case infant were available to test along with 3 surveillance cultures from upper respiratory tract (URT) isolates of 2 case infants and an axillary isolate from a non-case infant. Outbreak III. NICU—Regional Medical Center, Central GA. The outbreak occurred during September 1–14, 1998 in a 518 bed tertiary and acute care hospital with a 36 bassinet NICU serving southern and central GA. The hospital NICU reported 3 cases of CP BSI and 1 infant with C. albicans candiduria within a 14-day period. All 4 patients were premature LBW infants in the same 18 bassinet area of the NICU. They were treated successfully with combination antifungal therapy. A handculture survey was conducted on all HCWs in contact with these patients. Twelve isolates were available for Cp3-13 subtyping including 3 BSI isolates from case patients, 5 hand cultures of HCWs, and 4 surveillance cultures (skin) from noncase patients in the NICU during the same period. Outbreak IV. Outpatients of a Children’s Hospital, Memphis TN, 1998-1999 (Cano et al., 2005). Three children (patients 1–3) aged 1-, 9-, and 10 years, developed recurrent CP BSIs while being maintained at home on TPN due to short bowel syndrome. 2.3. Strain typing Yeast cells were grown in broth culture, spheroplasts were produced and lysed and DNA was purified by phenolchoroform extractions (Lasker et al., 2001) or by using Qiagen Genomic tip 100 G columns (Qiagen, Inc, Valencia CA). DNA samples were stored at 70 8C. Each agarose gel (15 cm  25 cm) contained Hind III l DNA as a molecular mass marker and the standard tester strain CP B6194. Genomic Southern blots were hybridized with the digoxigenin-labeled Cp3-13 DNA (Enger et al., 2001; Lasker et al., 2006). Restriction fragments (EcoR 1 and Sal 1 double-digest) containing Cp3-13 DNA were visualized with chemiluminescent detection (CDP-Star, Roche Molecular Diagnostics, Indianapolis, IN). Each panel of 15 samples/blot was repeated 2–3 times to assure reproducibility of hybridization profiles. 2.4. Data analysis Cp3-13 hybridization profiles were analyzed with Bionumerics software v. 4.0 (Applied Maths, Austin TX). The

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similarity coefficient value, SAB, is a measure of genetic similarity or pairwise comparison of 2 strains, such that the higher the coefficient the more closely related are the strains. An SAB of 1 denotes all bands are identical whereas when the SAB = 0 there are no common bands. SAB values were computed using the Dice coefficient with dendrograms displayed using an unweighted pair group analysis with arithmetic mean (UPGMA) with a position tolerance of 1%. Isolates were considered moderately related when the SAB  0.85 and highly related when the SAB  0.90. The average SAB for unrelated CP strains was reported as SAB = 0.45 (Enger et al., 2001). Discriminatory power (D) of the genotyping method was determined according to Hunter, 1990. 2.5. Antifungal susceptibility testing All 34 CP cultures were tested with the broth microdilution method to determine minimum inhibitory concentrations (MICs) of fluconazole, 5-fluorocytosine, amphotericin B, itraconazole, and voriconazole according to the guideline M27-A2 (Clinical Laboratory Standards Institute (CLSI), 2002). MICs were defined as the lowest drug concentration producing a prominent decrease in growth after 48 h incubation with drug. Amphotericin B and caspofungin MICs were also determined using the E-test (AB Biodisk, Solna, Sweden). 3. Results 3.1. Hybridization profiles of C. parapsilosis outbreak collections The Cp3-13 hybridization profiles and genetic relatedness of patient, non-case patient, and HCW isolates are shown in Fig. 1. The genotypes of CP BSIs from 3 LBW infants (LA 1–3, outbreak I), were indistinguishable (SAB = 1), supporting the presence of a common source. Cp3-13 hybridization profiles obtained from outbreak II indicated a single Cp3-13 genotype was shared by BSIs from 3 case patients (ATL 1-1, ATL 2-1, ATL 5-1) and 1 CSF isolate from a 4th patient (ATL 4-1). Moreover, CP isolates from i.v. sites (ATL 2-2) and a URT specimen from 1 case patient (ATL 3-2) also had the same genotype as the bloodstream and CSF isolates. An URT isolate from another patient (ATL-1-2) was a minor genotype variant of the clone responsible for fungemia. A CP isolate from a noncase patient’s axilla (ATL-6-1) treated in the NICU during the same time period was an unrelated genotype. Cp3-13 hybridization profiles obtained from outbreak III indicated that the blood culture from 1 infant (GA-3) was highly related (SAB  0.90) to the hand culture of a nurse (GA-10), and the skin culture from a non-case infant (GA-8). The hand culture of another nurse (GA-11) was moderately related (SAB, 0.85) to the hand culture of a pharmacist (GA-6). Blood cultures of 2 additional case patients (GA-1, GA-2) were indistinguishable (SAB = 1) as was the skin culture from another non-case infant (GA-12) concurrently being treated in the NICU. Analysis of outbreak IV showed that each patient maintained her/his CP strain during repeated episodes of fungemia. The CP

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Fig. 1. Dendrogram depicts 34 CP genotypes from 4 outbreak investigations of fungemia. Louisiana State University Children’s Hospital Shreveport NICU, LA-1 to LA-3: BSI from 3 neonates; Regional Medical Center, Atlanta, GA, NICU, ATL-1 to ATL 6: BSI, a CSF isolate, and surveillance cultures from 6 LBW infants; NICU of a Regional Medical Center in Central GA (shaded areas) GA-1 to GA-3: BSI from 3 infants; GA4 to GA-10: surveillance cultures from HCWs and non-case infants; Outpatients of a Children’s Hospital, Memphis TN receiving TPN at home for short bowel syndrome, ME-1 to ME-3. *CP B6194 is the standard tester strain; SAB = similarity coefficient determined at a position tolerance of 1%. Size markers shown are interpolated with respect to the molecular mass standard. Table 1 provides further descriptions of the isolates.

strain responsible for fungemia in patient ME-2 was different from CP isolated from the hands of that patient’s caregiver: ME-2–3, ME-2–4. Treatment of episodes of fungemia did not eradicate the responsible CP strain. 3.2. Clone-corrected database of C. parapsilosis Cp 3-13 genotypes Cp3-13 hybridization profiles from 34 neonatal isolates were added to the database of 59 CP BSI genotypes from the 1998– 2000 surveillance for candidemia at hospitals in CT and MD (Sofair et al., 2006), also including CP strain ‘‘MS’’, responsible for candidemia in 15 adult patients at a community hospital in Mississippi (Clark et al., 2004) (Fig. 2). This display of Cp3-13 profiles was done to observe any cluster of genotypes associated with neonates, as well as to determine the overall genotype diversity of the collection. Clone-corrected genotypes from each outbreak are included so that only unique outbreak genotypes are displayed, resulting in a dendrogram of genotypes from 73 unrelated CP isolates as shown in Fig. 2.

Two clusters of genotypes are present in the combined collection: Group 1 consisted of 11 CP genotypes with similar hybridization profiles from different hospitals and geographic areas with a mean SAB = 0.66. Another group (‘‘2’’) with a mean SAB = 0.76 contained genotypes from outbreak strains from Mississippi, Memphis, and Central GA (illustrated by genotypes ‘‘MS’’, ‘‘ME1–3’’, and ‘‘GA-3’’). A relatively high degree of discriminatory power (D = 0.98) was observed for the Cp3-13 analysis. Otherwise genotypes representative of 3 neonatal outbreak investigations were dispersed in the dendrogram. Four pairs of isolates from the 1998–2000 surveillance had indistinguishable Cp3-13 genotypes as described in Table 2. 3.3. Antifungal drug susceptibility tests These 34 neonatal and children’s CP isolates were fully susceptible to all the antifungal agents tested with available breakpoints. MIC results for fluconazole were in the range of 0.25–4 mg/ml; for itraconazole: 0.015–0.125 mg/ml; for amphotericin B: 0.5–0.1 mg/ml; for voriconazole: 0.007–0.03 mg/ml;

E. Reiss et al. / Infection, Genetics and Evolution 8 (2008) 103–109

Fig. 2. The composite clone-corrected dendrogram shows 73 Cp3-13 genotypes including those (arrows) from outbreak investigations in NICUs in Atlanta (ATL), Central Georgia (GA), Louisiana State University Hospital (LA), pediatric outpatients of a Memphis TN hospital (ME), and the outbreak of CP fungemia among adults in a community hospital in Mississippi (MI) (Clark et al., 2004). The remaining Cp3-13 genotypes are from the 1998–2000 CDC surveillance for candidemia in CT and MD (Sofair et al., 2006). ‘‘98’’, ‘‘99’’, ‘‘00’’ refer to the years in which the bloodstream isolates were obtained. *Isolate B6194 is the C. parapsilosis standard tester strain. Culture designations enclosed in boxes indicate a clone where ‘‘n’’ = the number of indistinguishable genotypes included. SAB = similarity coefficient determined at a position tolerance of 0.5%.

and for 5-fluorocytosine: 0.125–0.25 mg/ml. E-test MICs for amphotericin B ranged from 0.025 to 0.75 mg/ml and for caspofungin, 0.25–1.0 mg/ml after 48 h incubation. 4. Discussion Horizontal transmission of CP was detected by Cp3-13 genotyping in outbreaks at the Louisiana State University

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hospital (outbreak I), and at a Regional Medical Center in Atlanta GA (outbreak II) wherein identical Cp3-13 hybridization profiles were observed among case patients in each outbreak. Two different CP genotype strains were responsible for concurrent BSIs in the NICU in a 14-day period at a Regional Medical Center in Central GA (outbreak III). Genotyping confirmed the persistence of each child’s individual CP strain despite hospitalization and antifungal therapy while they were maintained at home on TPN (outbreak IV). Either the infection was not eradicated or re-infection with the same strain occurred from an unidentified source. Proof or inference of an environmental reservoir and/or HCW colonizing strain was obtained in 2 outbreaks for which Cp3-13 genotyping was applied: a glycerin bottle used for suppositories in the NICU of Louisiana State University Hospital (outbreak I) was implicated by the epidemiologic investigation (Welbel et al., 1996), and for the Regional Medical Center in Central GA (outbreak III), where a genotype of CP isolated from a hand-culture of a HCW was highly related to that from an infant’s CP BSI. The Cp3-13 probe binds weakly to the 2 phenotypically indistinguishable ‘‘sister’’ species C. orthopsilosis and C. metapsilosis (Tavanti et al., 2005). Those species display 4–7 weak bands in Cp3-13 Southern blots and lack the 3.4 kb doublet characteristic of CP (Enger et al., 2001). On that basis, none of the isolates in the current collection were the sister species. The dendrogram of the combined genotypes from outbreaks and those from the 1998–2000 surveillance for candidemia in CT and MD (Hajjeh et al., 2004, Sofair et al., 2006) (Fig. 2) had an overall SAB of 0.36, with a discriminatory power, D = 0.98. Both of these measures indicate a good ability to identify genetically related strains. The SAB of 0.50 of CP isolates from infants and children (Fig. 1) and their dispersion in the dendrogram of the larger collection (Fig. 2) gave no evidence for neonatal-specific CP strains. We did, however, observe 2 clusters of genotypes in the larger collection ‘‘Group 1’’, and ‘‘Group 2’’ in Fig. 2. Such clustering, of geographically and temporally separated CP isolates, suggests that certain genotypes may be more prone to cause candidemia, suggesting a need for further investigation. The collection of surveillance isolates included 4 pairs of indistinguishable genotypes; 3 pairs localized to Group 1 (Fig. 2). They are denoted in Table 2 according to the year of isolation, geographic location, and designation of candidemia as inpatient- (3 days) or outpatient- (3 days) associated. We denote the 4 pairs of isolates with indistinguishable genotypes as ‘‘common’’ genotypes because each was associated with a single CP BSI obtained from a different patient, on a different date, from a different hospital. None of these common genotypes were observed among the outbreak strains. Because of its relatively high degree of discriminatory power and reproducibility of hybridization profiles, Cp3-13 subtyping is capable of delineating a common source or persistent clone of CP in the hospital environment (Clark et al., 2004, Sarvikivi et al., 2005). CP fungemia is associated with horizontal transmission in the nursery and NICU (Bakir et al.,

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Table 2 Four pairs of indistinguishable Cp3-13 genotypes from CDC 1998–2000 surveillance for candidemia at hospitals in Maryland (MD) and Connecticut (CT)a Pair no.

CP strain no. b

Year of isolation

Hospital location (state) and designationc

Candidemia acquired as inpatient (I) or out patient (O)

1

CAS 00-0144 CAS 99-0518

2000 1999

MD, A3 CT, 24

O I

2

CAS 99-0420 CAS 99-0463

1999 1999

MD, A18 MD, A55

I I

3

CAS00-0009 CAS98-0150

2000 1998

CT, 11 CT, 35

I I

4

CAS99-0282 CAS99-0297

1999 1999

CT, 11 CT, 35

I O

a b c

As depicted in Fig. 2. CAS refers to ‘‘Candidemia Surveillance’’. Hospital number.

2006; Bendel, 2005; Benjamin Jr et al., 2000; Chapman, 2003; Fridkin et al., 2006; Huang et al., 2000; Lupetti et al., 2002; Pfaller et al., 1998) and CP strains can persist in individual wards for long periods, even years (Sarvikivi et al., 2005). Isolation of CP from a blood culture is a signal to hospital infection control to increase vigilance in hand-hygiene and catheter care, and to consider fluconazole prophylaxis in NICUs where CP occurs in clusters (Pfaller and Diekema, 2007). Molecular subtyping is an important adjunct to these infection control measures in the NICU. We produced a database consisting of CP genotypes drawn from surveillance projects and outbreak investigations. Continued application of genotyping for CP sepsis is needed and may contribute to a better understanding of whether clusters correspond to CP strains with increased pathogenic potential. Disclaimer The findings and conclusions in this publication are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Acknowledgment The gift of Cp3-13 DNA from David R. Soll, University of Iowa, is gratefully appreciated. References Bakir, M., Cerikcioglu, N., Barton, R., Yagci, A., 2006. Epidemiology of candidemia in a Turkish tertiary care hospital. APMIS 114, 601–610. Bendel, C.M., 2005. Nosocomial neonatal candidiasis. Pediatric Infect. Dis. J. 24, 831–832. Benjamin Jr., D.R.K., McKinney Jr., R.E., Benjamin, D.K., Auten, R., Fisher, R.G., 2000. When to suspect fungal infection in neonates: a clinical comparison of Candida albicans and Candida parapsilosis fungemia with coagulase-negative staphylococcal bacteremia. Pediatrics 106, 712–718. Cano, M.V., Perz, J.F., Craig, A.S., Liu, M., Lyon, G.M., Brandt, M.E., Lott, T.J., Lasker, B.A., Barrett, F.F., McNeil, M.M., Schaffner, W., Hajjeh, R.A., 2005. Candidemia in pediatric outpatients receiving home total parenteral nutrition. Med. Mycol. 43, 219–225. Chapman, R., 2003. Candida infections in the neonate. Curr. Opin. Pediatrics 15, 97–102.

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