Molecular typing of Candida albicans isolates ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Molecular typing of Candida albicans isolates from patients and health care workers in a neonatal intensive care unit J. Ben Abdeljelil1, F. Saghrouni1, N. Emira2, E. Valentin-Gomez3, N. Chatti4, J. Boukadida1, M. Ben Saı¨d1 and L. Del Castillo Agudo3 1 2 3 4

UR 02SP13 Research Unit, Ministry of Public Health, Tunis, Tunisia De´partement de Microbiologie, Faculte´ de Pharmacie, Monastir, Tunisie Departamento de Microbiologia y Ecologia, Facultade de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain Laboratoire de Ge´ne´tique, Biodiversite´ et Environnement, Institut Supe´rieur de Biotechnologie de Monastir, Monastir, Tunisie

Keywords Candida albicans, health care workers, neonates, pulsed-field gel electrophoresis, randomly amplified polymorphic DNA, Tunisia. Correspondence Jihene Ben Abdeljelil, Laboratoire de Parasitologie-Mycologie, Hoˆpital Farhat Hached 4000, Sousse, Tunisie. E-mail: [email protected]

2010 ⁄ 1913: received 25 October 2010, revised 15 June 2011 and accepted 30 July 2011 doi:10.1111/j.1365-2672.2011.05121.x

Abstract Aims: The aim of this study was to investigate the genetic relatedness between Candida albicans isolates and to assess their nosocomial origin and the likeliness of cross-transmission between health care workers (HCWs) and hospitalized neonates in a neonatal intensive care unit (NICU). Methods: We retrospectively analysed 82 isolates obtained from 40 neonates and seven isolates from onychomycosis of the fingers of five HCWs in a Tunisian NICU by using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis with CA1 and CA2 as primers. Results: In RAPD analysis, the discriminatory power (DP) of CA1 and CA2 primers was 0Æ86 and 0Æ81, respectively. A higher DP was achieved by combining patterns generated by both primers (0Æ92), while PFGE karyotyping exhibited the lowest DP (0Æ62). The RAPD-CA1 ⁄ CA2 analysis revealed that 65Æ8% of isolates obtained from neonates derived from a limited number (6) of groups of genetically identical strains, that five temporal clusterings occurred during the study period and that three HCWs’ isolates and 11 isolates obtained from six neonates were identical. Conclusions: These findings argue for the nosocomial transmission of C. albicans in our NICU and for the transfer of strains from HCWs to patients. Significance and Impact of the Study: Identification of relatedness between Candida species obtained from neonates and health care workers by using molecular techniques with high discriminatory power is essential for setting up specific control measures in order to reduce the incidence of nosocomial candidiasis.

Introduction Nosocomial candidiasis is a major problem in neonatal intensive care units (NICUs) where it remains an important cause of morbidity and mortality (Zaoutis et al. 2005; Benjamin et al. 2006; Fridkin et al. 2006). Critically ill neonates represent a population at high risk because of their immature immune system, the greater permeability of their mucosal barriers and the aggressive care they require in NICUs (Herruzo-Cabrera et al. 1995; Stamos and Rowley 1995; Smith et al. 2005; Benjamin et al.

2006). Candida albicans is still the most common cause of invasive candidiasis despite the increase in nonalbicans species reported since several years (Manno et al. 2004; Benjamin et al. 2006). The increase in nosocomial candidiasis and its association with a high mortality rate, despite the introduction of novel antifungal agents, underline the need of prevention. Candida spp. are known to be common commensal organisms, and nosocomial candidiasis has been classically considered to be endogenous and caused by strains initially colonizing the patient, their dissemination being mainly facilitated by

ª 2011 The Authors Journal of Applied Microbiology 111, 1235–1249 ª 2011 The Society for Applied Microbiology

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Genotyping of Candida albicans in neonates

J.B. Abdeljelil et al.

the mucosal barriers’ injury and the use of broadspectrum antibiotics. Nevertheless, exogenous origin of invasive candidiasis is well documented; and outbreaks caused by strains originating from hospital staff, biomedical devices, parenteral nutrition, environment or from the other patients have been reported (Burnie et al. 1985; Fraser 1990; Sherertz et al. 1992; Faix et al. 1995; Reagan et al. 1995; Ruiz-Diez et al. 1997). As the candidosic colonization in the newborn is less frequent than in adult, the exogenous origin of the neonatal candidiasis is even more likely. The molecular typing of the hospital strains is of a big contribution in understanding the population dynamics of pathogenic micro-organisms and the identification of the source of the infection on which depends the preventive approach. Over the last decades, a great variety of strain-typing techniques have become available. They include electrophoretic karyotyping, southern blot hybridization, restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA (RAPD) analysis (Reiss et al. 1998; Trtkova and Raclavsky 2006). The purpose of the present study was to investigate the molecular relationship and the epidemiological links among C. albicans isolates collected from neonates and health care workers (HCWs) in a central Tunisian NICU, by pulsed-field gel electrophoresis (PFGE) and RAPD analysis and to compare the contribution of both techniques in the molecular typing of C. albicans strains. Materials and methods Strains and patients We tested 89 C. albicans isolates: 82 isolates from 40 neonates (23 males and 17 females) hospitalized in the NICU of Farhat Hached university hospital in Sousse region, Tunisia, and seven additional isolates obtained from onychomycosis of the fingers of five HCWs of the same NICU. The 82 neonates’ isolates were collected between December 2002 and April 2007. Forty-seven isolates were obtained from deep sites, 19 isolates from implanted medical devices and 16 isolates from urine samples. The number of isolates obtained from a single neonate ranged from one to six isolates per patient. All the neonates had been hospitalized for more than 3 days prior to the collection of the first isolate. In addition, three epidemiologically unrelated strains were tested as controls: one urine isolate from an adult patient admitted at the Haematology service of the same hospital (in September 2005) and two reference strains (ATCC90028 and SC5314). The sequence of clinical isolates, their anatomical origin and the time of isolation are presented in Table 1. NICU is a ward composed of two open rooms with 6–9 beds each, close to each other. 1236

Techniques Candida albicans identification. The 90 clinical isolates were initially identified as C. albicans by routine procedures used in our laboratory, based on phenotypical methods including characteristic growth on Candida ID medium (bioMe´rieux, Marcy l’Etoile, France), formation of chlamydospores on PCB (Bio-Rad, Marnes-la-Coquette, France), the pattern of sugar assimilation in ID 23C panel (bioMe´rieux) and the agglutination in the Bichro-albicans test (Fumouze, Paris, France). The isolates were then stored at )80C in cryobilles tubes (Laboratoire AES, Combourg, France) until use. Before molecular analysis, isolates were subcultured onto Candida ID chromogenic medium to assess strain viability and species homogeneity. The identification to the species level was confirmed by C. albicans-specific PCR using the primers INT1 (5¢AAGTATTTGGGAGAAGGGAAAGGG-3¢) and INT2 (5¢-AAAATGGGCATTAAGGAAAAGAGC-3¢), deduced from the CaYST1 gene sequence of C. albicans (EMBL accession number AJ251858), described by Baquero et al. (2002). One unit of EcoTaq polymerase (Ecogen, Madrid, Spain) was added to 49Æ7 ml of a solution consisting of 16Æ6 mmol l)1 (NH4)2SO4, 2Æ5 mmol l)1 MgCl2, 67 mmol l)1 Tris–HCl (pH 8Æ8), 0Æ01% v ⁄ v Tween-20, 0Æ2 mmol l)1 each of dATP, dCTP, dGTP, dTTP and unless otherwise stated, 500 ng of target DNA and 0Æ3 mmol l)1 of each primer (INT1 and INT2). DNA was amplified in a PCR thermal cycler (Gene Amp PCR System 2400; Perkin Elmer, Norwalk, USA) by using one cycle at 95C for 3 min, and then 40 cycles as follows: 60 s of denaturation at 94C, 30 s of annealing at 55C and 45 s of primer extension at 72C. At the final cycle, an additional 10 min of incubation at 72C was carried out to ensure complete polymerization of any remaining PCR products. All strains tested yielded the expected 310 bp DNA fragment. Molecular typing i Karyotyping by PFGE Preparation of DNA. The preparation of DNA plugs of C. albicans was carried out according to the procedure described by Chu et al. (1993) with minor modifications. Strains were subcultured onto Sabouraud dextrose agar for 48 h at 28C. Colonies were pelleted by centrifugation at 4000 g for 5 min. The pellet was harvested and washed twice in 1 ml of ET (0Æ05 mol l)1 EDTA, 0Æ01 mol l)1 Tris–HCl, pH 7Æ5) and resuspended in 150 ll of ET containing 10 U of lyticase (Sigma, Paris, France) and heated to 40C. Then, 150 ll of 1% agarose (low-melting-point agarose; Promega, Paris, France) in 0Æ125 mol l)1 EDTA (pH 7Æ5) was added. After mixing, the solution was




Table 1 Description of 93 Candida albicans isolates investigated and summary of results of PFGE, RAPD-CA1, RAPD-CA2 and RAPD-CA1 ⁄ CA2 analysis Patient code

Isolate code

PFGE pulsotype

RAPD-CA1 genotype

RAPD-CA2 genotype

RAPD-CA1 ⁄ CA2 genotype

Anatomical site of isolate

Date of sampling

Time of hospitalization

N1

I I II I I I I I I III III IV IV IV III III III I V VI III III IV IV IV III III III III VII VIII VII I I I I IX IX IV IV I I IV III III

A A A A A A A A A B C D D D E E E F F G C C H H I B B B B B I B A A A A H A J J H H H K B

a a b c c c c c c d e c c c d d d f f d g g h h d d d d d d i d c c c c j k l l d d m n d

1 1 2 3 3 3 3 3 3 4 5 6 6 6 7 7 7 8 8 9 10 10 11 11 12 4 4 4 4 4 13 4 3 3 3 3 14 15 16 16 17 17 18 19 4

Blood urine Blood Blood Endotracheal tube Postoperative wound Hepatic abscess Drain Postoperative wound Blood Umbilical catheter Umbilical catheter Urine Joint fluid Postoperative wound urine urine Blood Blood Blood Mediastinal fluid Blood Urine Blood Umbilical catheter Blood Umbilical catheter Umbilical catheter Blood Blood Urine Endotracheal tube Umbilical catheter Urine Urine Urine Urine Endotracheal tube Urine Endotracheal tube Postoperative wound Urine Fingers’ nails Fingers’ nails Blood

5 November 2005 5 November 2005 23 December 2005 11 September 2006 13 September 2006 26 September 2006 27 September 2006 4 October 2006 4 October 2006 30 November 2006 9 December 2006 2 April 2005 9 April 2005 7 May 2005 12 July 2006 15 July 2006 9 August 2006 9 August 2006 12 August 2006 15 September 2006 19 September 2006 11 October 2006 26 September 2006 29 September 2006 5 October 2006 29 September 2006 5 October 2006 16 October 2006 16 October 2006 28 February 2006 28 February 2006 4 March 2006 19 June 2005 13 July 2005 17 July 2005 1 October 2005 12 August 2006 12 August 2006 8 April 2006 10 April 2006 1 May 2006 5 May 2006 26 October 2006 27 October 2006 13 October 2006

A: 1 November 2005 D: 29 December 2005

HCW5 HCW1 N14

1 2 3 4 5 6 7 8 43 9 10 11 12 13 14 15 16 17 52 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 P6 P7 44

N15

45

III

B

d

4

Umbilical catheter

4 October 2006

N16

46

I

I

o

20

Blood

6 April 2007

N17

47

X

B

d

4

Blood

18 April 2007

N2

N3 N4

N5

N6

N7

N8

N9

N10

N11 N12 N13


A: 1 September 2006 D: 7 November 2006

A: 23 November 2006 Death: 19 December 2006 A: 24 March 2005 D: 13 May 2005 A: 28 June 2006 Death: 12 August 2006

A: 10 September 2006 D: 8 November 2006 A: 16 September 2006 D: 8 November 2006 A: 20 September 2006 Death: 16 October 2006

A: 28 February 2006 Death: 17 March 2006 A: 19 June 2005 D: 15 October 2005

A: 28 July 2006 Death: 18 August 2006 A: 8 April 2006 D: 15 April 2006 A: 29 April 2006 D: 15 May 2006 – – A: 10 October 2006 D: 10 November 2006 A: 21 September 2006 D: 21 October 2006 A: 22 March 2007 Death: 16 April 2007 A: 16 April 2007 Death: 19 April 2007

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Table 1 (Continued) Patient code

Isolate code

PFGE pulsotype

RAPD-CA1 genotype

RAPD-CA2 genotype

N18

48

I

A

c

N19

49

V

F

N20

50

I

N21

51

N22



Date of sampling


3

Urine

12 August 2006

f

21

Blood

12 August 2006

F

p

22

Urine

12 August 2006

V

F

f

21

Endotracheal tube

5 August 2006

53

V

F

f

21

Urine

14 August 2006

HCW1 HCW2 HCW2 N23

P1 P2 P3 57

I I I I

I I I I

q q q q

23 23 23 23

Fingers’ nails Fingers’ nails Fingers’ nails Blood

10 12 25 20

N24

N31

58 59 60 61 62 63 A 66 67 68 69 70 71 72 73 74 75 76

I I I I I I XI I I I I I I I I I I I

I I I I I I A I I I I D D D D D D D

q q q q q q r q q q q d d d d d d d

23 23 23 23 23 23 24 23 23 23 23 25 25 25 25 25 25 25

Blood Abdominal abscess Blood Umbilical catheter Blood Blood Urine Blood Umbilical catheter Blood Umbilical catheter Blood Cerebrospinal fluid Umbilical catheter Cerebrospinal fluid Cerebrospinal fluid Blood Cerebrospinal fluid

20 January 2003 1 February 2003 21 January 2003 4 February 2003 23 January 2003 30 January 2003 17 September 2005 2 February 2003 2 February 2003 7 February 2003 13 February 2003 2 October 2003 2 October 2003 9 October 2003 14 October 2003 10 October 2003 11 October 2003 15 October 2003

A: 23 July 2006 D: 25 August 2006 A: 31 July 2006 D: 22 August 2006 A: 23 July 2006 D: 25 August 2006 A: 1 August 2006 D: 23 August 2006 A: 10 August 2006 D: 30 August 2006 – – – A: 20 January 2003 D: 30 January 2003 A: 19 January 2003 D: 5 February 2003 A: 20 January 2003 D: 10 February 2003 A: 23 January 2003 D: 10 February 2003 – A: 1 February 2003 D: 20 February 2003 A: 1 February 2003 D: 21 February 2003 A: 26 September 2003 Death: 10 November 2003

N32

77

I

D

d

25

Umbilical catheter

10 November 2003

N33

78 79 80

I I I

D D D

d d d

25 25 25

Blood Urine Blood

7 November 2003 9 November 2003 10 November 2003

P4 83

I I XII

L L M

j j e

26 26 27

Fingers’ nails Fingers’ nails Blood

30 April 2004 6 May 2004 26 February 2004

N37

84 85 86 87

I I I I

D D D D

d d d d

25 25 25 25

Blood Blood Postoperative wound Blood

18 April 2004 1 May 2004 7 May 2004 21 April 2004

N38

88

I

D

d

25

Blood

22 April 2004

N39

89 90

I I

D D

d j

25 28

Endotracheal tube Blood

7 May 2004 7 May 2004

N25 N26 Adult N27 N28 N29

N30

N34 HCW3 HCW4 N35 N36

1238

December 2002 December 2002 December 2002 January 2003

A: 27 September 2003 Death: 11 October 2003 A: 11 October 2003 D: 30 October 2003 A: 9 November 2003 D: 20 November 2003 A: 26 October 2003 D: 21 December 2003 A: 7 November 2003 Death: 10 November 2003 – – A: 25 February 2004 D: 15 March 2004 A: 18 April 2004 D: 19 May 2004 A: D: A: D: A: D:

20 19 20 10 29 15

April 2004 May 2004 April 2004 May 2004 April 2004 May 2004




Table 1 (Continued) Patient code

Isolate code

PFGE pulsotype

RAPD-CA1 genotype

RAPD-CA2 genotype



Date of sampling


N40

91

I

D

j

28

Blood

24 November 2004

ATCC90028 SC5314

92 93

XIII –

N D

c c

29 6

– –

– –

A: 20 November 2004 D: 3 December 2004 – –

A, admission; D, discharge; RAPD, randomly amplified polymorphic DNA; PFGE, pulsed-field gel electrophoresis; HCW, health care worker.

poured into the wells of plug moulds and kept at 4C for 15 min. Upon solidification, the agarose plugs were incubated overnight at 37C in 400 ll of LET (0Æ5 mol l)1 EDTA, 0Æ01 mol l)1 Tris–HCl, pH 7Æ5) and then transferred in 400 ll of NDS (0Æ5 mol l)1 EDTA, 0Æ01 mol l)1 Tris–HCl, pH 7Æ5, 1% N-lauroyl sarcosine, 2 mg of proteinase K per ml) for an overnight incubation in a shaker water bath at 50C. The plugs were washed once with 0Æ5 mol l)1 EDTA (pH 9Æ0) and stored at 4C in 0Æ5 mol l)1 EDTA (pH 9Æ0). PFGE analysis. Chromosomal DNA bands were separated on 1% agarose gels in 0Æ5· TBE buffer (0Æ045 mol l)1 Tris–HCl, 0Æ045 mol l)1 borate, 1 mol l)1 EDTA) in contour-clamped homogeneous electric field (CHEF) electrophoresis system (CHEF-DRII; Bio-Rad). Electrophoresis was performed at 150 V for 24 h with a 180 s ramping switch interval and then at 110 V for 22 h with a 360 s ramping switch interval. The temperature of the running buffer was maintained at 14C. A ladder of Saccharomyces cerevisiae chromosomal DNA (Bio-Rad) was used as a molecular weight marker. Gels were stained with ethidium bromide (0Æ5 lg ml)1) for 30 min and photographed under UV transluminator. The SC5314 reference strain was not tested in PFGE. ii RAPD Preparation of DNA. Strains were cultured onto solid Sabouraud agar plates at 28C for 48 h. A single colony was then subcultured overnight onto YPD broth (1% yeast extract, 2% peptone, 2% dextrose) at 28C with shaking at 200 rev min)1. The culture suspension was then harvested by centrifugation and the pellet was vortexed with glass beads (Sigma). The pellet was resuspended in TE (50 mmol l)1 Tris–HCl, 20 mmol l)1 EDTA, pH 7Æ5) for PCR analysis. RAPD analysis. The RAPD analysis was carried out as described by Del Castillo et al. (1997). Two arbitrary primers were used separately: the CA1 (8-mers 5¢-GCT GGTGG-3¢) and CA2 (10-mers 5¢-GCGATCCCCA-3¢) (TIB MOLBIOL, Berlin, Germany). Amplification was achieved in a total reaction volume of 25 ll. The PCR mixture consisted of 20 ng of yeast genomic DNA, 2Æ5 U of DNA polymerase (Ecotaq; Ecogen, Sigma), 10· buffer for Taq DNA Polymerase (Ecogen, Sigma), 200 lmol l)1

of each dNTP (Sigma), 50 mmol l)1 MgCl2 and 0Æ4 lmol l)1 of the primer. After initial denaturation of DNA at 95C for 5 min, 34 cycles of amplification were performed on a thermocycler (PTC-150 Minicycler, Peltier Thermal cycler; MJ Research Inc., San Francisco, CA, USA). Each cycle consisted of a denaturation step at 95C for 30 s, an annealing step at 37C for 30 s and an extension step at 72C for 1 min 30 s, with a final 10 min extension at 72C for the last cycle. Amplified DNA fragments were separated by electrophoresis through 2% agarose gels run in Tris–acetate EDTA buffer at 75 V for 4 h. The gels were then stained with ethidium bromide (0Æ5 lg ml)1), photographed under UV transluminator and analysed. Amplified products from DNA of reference strains of C. albicans (SC5314 and ATCC90028) and molecular mass markers (100 bp DNA ladder and lambda DNA ⁄ EcoRI + HindIII Marker 3) (Fermentas, Madrid, Spain) were run in each gel. iii Clustering analysis For both PFGE and RAPD data analysis, the bands were detected and their size evaluated by using Quantity One 1D analysis software (Bio-Rad). The genetic relationships among isolates were established by cluster and ordination analysis performed on the matrix of genetic similarities. Cluster analysis was performed on the genetic distance matrix with the unweighted paired group method using arithmetic average (UPGMA) (Sneath and Sokal 1973) and the Jaccard’s correlation coefficient calculated on the basis of ERIC-2 patterns by using the mvsp 3.1 software. The Jaccard’s coefficient ranges from 1Æ00 (the two patterns are identical) to 0Æ00 (no common bands between both patterns). We considered that (i) a Jaccard’s coefficient of 1Æ00 is indicative of strains of the same clone, (ii) a Jaccard’s coefficient ranging between 0Æ90 and 0Æ99 is indicative of highly similar but nonidentical strains, (iii) a Jaccard’s coefficient ranging from 0Æ80 and 0Æ89 represents less related isolates and (iv) a Jaccard’s coefficient below 0Æ79 represents unrelated isolates (Lockhart et al. 1995, 1996; Pujol et al. 1997). We calculated the numerical index of discriminatory power (DP) for each typing method used in our study according to the formula proposed by Hunter and Gaston (1988). To achieve maximum strain discrimination, the results of


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RAPD-CA1 and RAPD-CA2 analysis were combined to carry out a composite DNA type. Results PFGE analysis The 91 tested isolates generated 13 different pulsotypes of 6–9 bands each. The molecular size of generated bands ranged from 945 to 2155 bp. Examples of the pulsotypes for isolates 1 through 10 are shown in Fig. 1. The genetic relatedness of isolates determined by PFGE analysis is illustrated by the dendrogram given in Fig. 2. The Jaccard’s coefficient values ranged from 0Æ41 to 1Æ00. At Jaccard’s coefficient of 0Æ41, all the tested isolates, but one, were clustered into a single general cluster. At Jaccard’s coefficient of 1Æ00, 13 different karyotypes (labelled I–XIII) were generated. At this level of similarity, isolates grouped into the same cluster are considered as individuals of the same clone. PFGE analysis allowed identification of six individual clones including 84 isolates, which represented 92Æ3% of the collection. The adult isolate (isolate A) and the ATCC reference strain generated each one specific karyotype (karyotypes XI and XIII, respectively) unrelated to the remaining isolates. The calculated index of DP of PFGE analysis was 0Æ62. RAPD-CA1 analysis CA1 primer generated 14 different patterns of 8–12 bands each. The molecular size of the bands ranged from 347 to 1945 bp. Representative patterns obtained by RAPD-CA1 for isolates 81 through 91 are shown in Fig. 3. The similarity values between isolates ranged from 0Æ65 to 1Æ00. At

M

1

N1 2 3

4

5

N2 6 7

8

9

N3 10 ATCC

2200

1600 1125 1020 785 450 225 Figure 1 Examples of pulsotypes obtained in pulsed-field gel electrophoresis karyotyping. M: size marker (in base pairs). 1–10: isolates 1 through 10 obtained from neonates N1, N2 and N3. ATCC: ATCC90028 reference strain.

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Jaccard’s coefficient of 0Æ65, RAPD-CA1 analysis clustered all of the 92 tested isolates into two general clusters. The first cluster included three (3Æ3%) isolates, whereas the second cluster included the 89 (96Æ7%) remaining isolates. At Jaccard’s coefficient of 1Æ00, 14 genotypes (labelled A–N) were generated with the 92 isolates and 10 individual clones identified including 88 isolates, which represented 95Æ7% of the collection. Each clone included between 2 and 23 isolates. The adult’s isolate (isolate A) generated genotype A shared by five neonates, and the SC reference strain generated the genotype D shared by 12 neonates. However, the ATCC reference strain generated a specific genotype (genotype N). The calculated index of the DP of RAPD-CA1 analysis was 0Æ86. RAPD-CA2 analysis RAPD-CA2 generated 18 different patterns of 10–14 bands each. The molecular size of DNA fragments amplified ranged from 375 to 2272 bp. Examples of the patterns for isolates 81 through 91 are shown in Fig. 4. The genetic relatedness of isolates evidenced by CA2-RAPD analysis. The similarity values between isolates ranged from 0Æ64 to 1Æ00. At Jaccard’s coefficient of 0Æ64, all of the tested isolates were clustered into two general. The first cluster included 17 (18Æ5%) isolates, and the second cluster included the 75 (81Æ5%) remaining isolates. At Jaccard’s coefficient of 1Æ00, 17 different genotypes (labelled a–q) were observed and ten clones identified, including 84 isolates, which represented 91Æ4% of the collection. Each clone included between 2 and 34 isolates. The two reference strains generated the genotype c shared by four neonates. The adult isolate (isolate A) generated a specific genotype (genotype r) unrelated to the remaining isolates. The calculated index of the DP of RAPD-CA2 analysis was 0Æ81. Combined RAPD-CA1 ⁄ CA2 analysis The genetic relatedness of isolates determined by combined RAPD-CA1 ⁄ CA2 analysis is illustrated by the dendrogram given in Fig. 5. The Jaccard’s coefficient values ranged from 0Æ69 to 1Æ00. At Jaccard’s coefficient of 0Æ69, the tested isolates were clustered into two general clusters, cluster I which included 31 (33Æ7%) isolates, and cluster II which included 61 (66Æ3%) isolates. At Jaccard’s coefficient of 1Æ00, 29 genotypes (labelled 1–29) were observed and 78 (84Æ8%) isolates were clustered into 15 clones of 2–17 isolates each. The adult isolate (isolate A) and the ATCC reference strain generated each one specific genotype (genotypes 24 and 29) unrelated to the remaining isolates. The SC reference strain generated genotype 6 shared by one neonate. The calculated index of the DP was at 0Æ92.




UPGMA

0·4

0·5

0·6

18 83 29 36 35 30 28 47 53 51 49 52 45 44 P7 27 26 25 24 20 19 16 15 14 10 9 ATCC P6 38 37 23 22 21 13 12 11 3 A 91 90 89 88 87 86 85 84 P5 P4 80 79 78 77 76 75 74 73 72 71 70 69 68 67 66 63 62 61 60 59 58 57 P3 P2 P1 50 48 46 40 39 34 33 32 31 17 43 8 7 6 5 4 2 1

0·7

0·8

0·9

1

Jaccard's coefficient

Figure 2 Dendrogram showing the genetic relatedness of 90 Candida albicans isolates from neonates and health care workers and ATCC90028 reference strain as determined by pulsed-field gel electrophoresis karyotyping.

Molecular relatedness of Candida albicans isolates To investigate genetic relatedness of tested isolates, we mainly considered the typing results obtained by the combined RAPD-CA1 ⁄ CA2 analysis owing to its highest DP.

The relationship among isolates determined by this composite DNA type and their temporal distribution showed clusterings of many genotypes (Fig. 6). The genotype 25 was shared by 17 isolates collected from 10 neonates between October 2003 and May 2004 (Table 1).


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2027 1904

M1


P4 P5 N35 N36 N37 N38 N39 N40 81 82 83 84 85 86 87 88 89 90 91 SC ATCC M2

1584 1375

947 831

1000 900 800 700

564

600 500 400 300

Figure 3 Examples of patterns obtained in RAPD-CA1 analysis. M1 and M2: size markers (in base pairs). 81 and 82: isolates 81 and 82 obtained from health care workers P4 and P5, respectively. 83–91: isolates 83 through 91 obtained from neonates N35–N40. SC: SC5314 reference strain. ATCC: ATCC90028 reference strain. RAPD, randomly amplified polymorphic DNA.

M1

P4 P5 N35 N36 N37 N38 N39 N40 81 82 83 84 85 86 87 88 89 90 91

SC ATCC M2

2027 1904 1584 1375

947

1000 900

831

800 700 600

564

500 400 300

Figure 4 Examples of patterns obtained in RAPD-CA2 analysis. M1 and M2: size markers (in base pairs). 81 and 82: isolates 81 and 82 obtained from health care workers P4 and P5, respectively. 83–91: isolates 83 through 91 obtained from neonates N35 to N40. SC: SC5314 reference strain. ATCC: ATCC90028 reference strain. RAPD, randomly amplified polymorphic DNA.

The analysis of the chronological distribution of these isolates showed two clusterings: the first one was represented by 11 isolates collected from six neonates (N29, N30, N31, N32, N33 and N34) in October–November 2003 and the second clustering by the six remaining isolates obtained from four neonates (N36, N37, N38 and N39) in April–May 2004 (Table 1). The first clustering corresponds to six cases of invasive candidiasis that had 1242

occurred over a 38-day period with an incidence average of one case per 6 days of hospital setting. The second temporal clustering corresponds to three cases of invasive candidiasis and one case of medical device-related infection. These neonates’ infections had arisen within a 19-day period. The incidence average was of one case per 5 days of hospital setting. These results suggest that the strain represented by the genotype 25 is endemic in the




UPGMA A 20 19 52 17 53 51 49 83 10 18 16 15 14 50 46 29 69 68 67 66 63 62 61 60 59 58 57 P3 P2 P1 23 40 39 89 88 87 86 85 84 80 79 78 77 76 75 74 73 72 71 70 47 45 44 30 28 27 26 25 24 9 ATCC SC5314 13 12 11 36 48 34 33 32 31 43 8 7 6 5 4 3 P7 38 37 P6 22 21 P5 P4 91 90 35 2 1

Cluster II

Cluster I

0·7

0·75

0·8

0·85

0·9

0·95

1

Jaccard's coefficient

Figure 5 Dendrogram showing the genetic relatedness of 90 Candida albicans isolates from neonates and health care workers and ATCC90028 and SC5314 reference strains as determined by combined RAPD-CA1 ⁄ CA2 analysis. RAPD, randomly amplified polymorphic DNA.

NICU. The strain persisted into circulation in the service over at least 8 months and emerged occasionally as a cause of neonates’ infections occurring in close temporal proximity. The association of this genotype with cerebral candidiasis is noteworthy. Indeed, it was identified in the cerebrospinal fluid of three patients.

The genotype 23 was shared by 11 isolates collected from six neonates (N23 through N28) and three isolates (P1, P2 and P3) from two HCWs (Table 1). The HCWs’ isolates were obtained very close to the time of neonates’ infections: the first neonates’ isolates (57 and 58) and the last nurse’s isolate (P3) were collected 25 days apart


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Figure 6 Temporal distribution of the genotypes (G1–G23, G25–G28) of Candida albicans isolated from 40 neonates (N1–N40) and five health care workers (HCW1–HCW5) according to the combined RAPD-CA1 ⁄ CA2 analysis. RAPD, randomly amplified polymorphic DNA; HCW, health care worker.

(Table 1). The 11 neonates’ isolates were collected over a 25-day period. The strain represented by this genotype caused bloodstream infection in all of the six patients and contaminated umbilical catheter in three of them. The incidence average of bloodstream infection was one case per 4 days of hospital setting. These data indicate that the isolates exhibited temporal clustering and suggest that an endemic strain had cross-contaminated the hands of two service co-workers and had caused bloodstream and catheter-related infections in six patients over a period of approximately 2 months. The genotype 4 was shared by ten isolates collected from six neonates: (i) two isolates collected from the same patient (N9) in February–March 2006, (ii) seven isolates collected from four neonates (N3, N8, N14 and N15) in September–November 2006 and (iii) one isolate collected from neonate N17 in April 2007 (Table 1). However, according to the PFGE analysis, the two isolates collected from the neonate N9 in February–March 2006 were clustered into an individual clone represented by karyotype VII and were unrelated to the other collection isolates. The seven isolates collected within September– 1244

November 2006 were identical and were clustered with seven other isolates into the same clone represented by the karyotype III. The last isolate collected in April 2007 generated an individual pattern (karyotype X) in PFGE and was dramatically different from the other collection isolates (Table 1 and Fig. 2). Subsequently, according to both PFGE and RAPD-CA1 ⁄ CA2 analysis, only the seven isolates collected on September–November 2006 were identical. These isolates (obtained from four neonates) caused candidemia in three neonates and contaminated three central catheters. They were collected within a 2-month period suggesting their temporal clustering. The incidence average of bloodstream infection was one case per 15 days of hospital setting. The genotype 21 was shared by three isolates obtained from three neonates (N19, N21 and N22) over a 9-day period. The isolates (49, 51 and 53) were collected from an endotracheal tube, blood and an urine sample (7 and 2 days apart, respectively) (Table 1). These findings indicate the temporal clustering of isolates and the emergence of the strain represented by this genotype as a cause of various nosocomial candidiasis: invasive candidiasis, urinary



colonization or contamination of implanted medical devices. The genotype 3 was shared by 11 isolates collected from three neonates (N2, N10 and N18) between June 2005 and October 2006 (Table 1). This suggests that this strain is endemic in NICU and a long-lasting one as it persisted up to 15 months. The genotype 28 was shared by two blood isolates obtained from neonates N39 and N40, six and a half months apart, suggesting the persistence of this endemic strain in the service over this period. It is important to specify that there was no overlapping between hospitalization periods of both patients. To assess if strain transmission between neonates and medical staff occurred in NICU, we tested seven isolates collected from five HCWs working in the unit and harbouring fingers’ onychomycosis. As mentioned earlier, three isolates (P1, P2 and P3) collected from two HCWs within a 15-day period were identical and mixed in the same clone (represented by the genotype 23) together with 11 additional isolates obtained from six neonates over a 2-month period (Table 1 and Fig. 5). The HCWs’ isolates were obtained prior to the collection of the neonates’ isolates. These results suggest that this strain is an endemic one and had cross-contaminated the hands of service co-workers who subsequently might have contaminated neonates. Two isolates (P4 and P5), obtained from two HCWs 6 days apart, were identical and clustered into an individual clone (represented by the genotype 26) unrelated to the neonates’ isolates collected over the same period (Table 1 and Fig. 5). This finding suggests cross-contamination between NICU co-workers and excludes the likeliness that the HCWs’ strain caused infections in neonates. The two remaining isolates (P6 and P7) collected from two HCWs, 1 day apart, generated two individual genotypes (genotypes 18 and 19) and were unrelated to the other collection isolates (Table 1 and Fig. 5). Among the five HCWs, two had two isolates each. The isolates (P2 and P3) obtained 13 days apart from the first HCW were identical, whereas the isolates (P1 and P7) collected, 4 years apart from the second HCW, were quite different suggesting a strain replacement (Table 1 and Fig. 5). Among the 40 neonates included in our study, 23 have had two or more isolates. A total of 55 isolates were collected from these 23 neonates (Table 1). In 15 (65Æ2%) of the 23 neonates, all isolates were identical. For the eight remaining patients, various scenarios were observed. Two observations are noteworthy: (i) in neonate N1, two isolates were obtained from blood and one isolate from urine. The two isolates collected on the same day from blood (isolate 1) and urine (isolate 2) were identical (genotype 1), but less related to the second isolate


obtained from blood (isolate 3, genotype 2) 48 days apart (Jaccard’s coefficient at 0Æ82) (Table 1 and Fig. 5). These results suggest that the patient had two different episodes of bloodstream infection (caused by genotypes 1 and 2) and that the first episode originated from the urine strain. (ii) In neonate N6, three isolates were collected from blood (isolates 18 and 20) and mediastinal fluid (isolate 19) (Table 1). The second blood isolate (isolate 20) and the mediastinal isolate obtained 22 days apart were identical, forming an individual clone represented by the genotype 10, and were less related (Jaccard’s coefficient at 0Æ80) to the first blood isolate (genotype 9) (Table 1 and Fig. 5). This suggests invasive strain replacement. It is important to specify that none of the three techniques used in our study detected any microevolution of C. albicans isolates. Comparison of urine isolates and invasive isolates from the same patient For seven patients, 1–2 urine isolates and 1–2 invasive isolates were collected (Table 1). These patients had eight episodes of invasive candidiasis. In six (75%) of these episodes, the urine and the invasive isolates were identical. The isolates were collected on the same day in one case (N1). For the remaining episodes, the time between collection of the urine isolates and the invasive isolates ranged between 2 and 28 days with an average of 8 days. In the remaining two episodes (25%), the urine isolate and the invasive isolate were unrelated (Jaccard’s coefficient at 0Æ73 and 0Æ79). For both patients (N5 and N9), the urine isolate and the invasive isolate were collected on the same day (Table 1). Comparison of isolates contamining medical devices and the other isolates from the same patient In our study, we tested 16 isolates obtained from different implanted medical devices removed from 14 neonates. They included ten umbilical catheters, five endotracheal tubes and one drain (Table 1). The catheter isolate was identical to the invasive isolate in five neonates, identical to the urine isolate in two neonates, unrelated to the invasive isolate in two neonates and unrelated to the urine isolate in one neonate. The endotracheal tube isolate was identical to the invasive isolate in two neonates, identical to the urine isolate in one neonate, unrelated to the invasive isolate in one neonate and unrelated to the urine isolate in two neonates. Three observations are noteworthy: (i) in neonate N2, two isolates collected from endotracheal tube and drain were identical to four invasive isolates obtained from blood (isolate 4), postoperative wound (isolates 6 and 43) and hepatic abscess (isolate 7). The six isolates were collected within a 23-day period. The blood isolate (isolate 4) was obtained 2 days


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prior to the collection of the first medical device isolate (isolate 5) (Table 1). (ii) In neonate N4, three isolates were collected sequentially from umbilical catheter (isolate 11), urine sample (isolate 12) and joint fluid (isolate 13), 7 and 28 days apart, respectively (Table 1). The three isolates were identical suggesting that the same strain contaminated the central catheter, colonized the urinary tract and next caused invasive infection. (iii) In neonate N8, four isolates collected from blood (isolates 24 and 27) and two umbilical catheters (isolates 25 and 26), within a 17-day period, were identical. The first blood strain (isolate 24) was isolated prior to the removal of the first catheter. The isolation of the same strain in the second catheter (isolate 26) suggests that the bloodstream infection was the source of the second catheter contamination. Discussion To investigate the genetic relatedness between C. albicans isolates in NICU and to assess the likeliness of crosstransmission between HCWs and hospitalized neonates, we tested 92 C. albicans isolates by using two different molecular-based typing methods: PFGE and RAPD with CA1 and CA2 as primers (Del Castillo et al. 1997). The estimated DP of PFGE, RAPD-CA1, RAPD-CA2 and combined RAPD-CA1 ⁄ CA2 (composite DNA type) was found to be 0Æ62, 0Æ86, 0Æ81 and 0Æ92, respectively. The tested isolates generated 13 PFGE karyotypes, 14 genotypes in RAPD-CA1, 18 genotypes in RAPD-CA2 and 29 genotypes in combined RAPD-CA1 ⁄ CA2. In RAPD, the typing results were markedly improved when the patterns yielded by both primers were combined (DP at 0Æ92). The use of the combined tests (composite DNA type) is recommended if a greater discrimination is required. Yet, techniques with a high DP (>0Æ9) are desirable when the investigation of epidemiological relatedness between isolates is required, mainly in the setting of outbreaks (Pfaller 1995). RAPD is one of the most widely used typing methods in clinical mycology. It has been shown to be very convenient for C. albicans strains typing owing to its high DP (Lehmann et al. 1992; Pujol et al. 1997; Lopez-Ribot et al. 2000; Trtkova and Raclavsky 2006). In addition, it is rapid, easy to design and to perform relatively inexpensive and very suitable for the typing of a large number of isolates. Its main drawback lies in its lack of reproducibility when results need to be compared between laboratories (Power 1996; Pujol et al. 1997; Trtkova and Raclavsky 2006). Herein, we used this technique for the study of the local epidemiology of C. albicans isolates and the interlaboratory comparison was not required. PFGE is expected to be a valid and suitable tool for the measurement of genetic distance between isolates as it 1246

targets the entire genome. In addition, it is a relatively highly reproducible molecular typing method (Mahrous et al. 1990; Pujol et al. 1997; Trtkova and Raclavsky 2006). However, it is time-consuming and is not appropriate for analysis of a large number of isolates as sample preparation is laborious. PFGE analysis has a limited value in terms of differentiation between C. albicans isolates when compared to the RAPD analysis and as confirmed by own findings. We used PFGE analysis despite its predictable low DP as a complementary technique to improve the overall typing results. Indeed, it is recommended for this purpose to combine at least two techniques based on different molecular targets to ascertain results and improve discrimination between C. albicans strains (Pfaller 1995; Trtkova and Raclavsky 2006). Moreover, PFGE may be contributive in differentiating isolates shown to be identical in RAPD. Indeed, PFGE analysis bursted the RAPD-CA1 ⁄ CA2 clone formed by ten isolates and represented by the genotype 4 into three different karyotypes (karyotypes III, VII and X). According to RAPD-CA1 ⁄ CA2 results, the majority (65Æ8%) of isolates obtained from the 40 neonates derived from a limited number (6) of groups of genetically identical strains. This suggests the spread of clonal groups on hospitalized patients and provides evidence (albeit indirectly) for nosocomial transmission of C. albicans in our NICU. In addition, the analysis of the temporal distribution of the neonates’ isolates showed five clusterings caused by four genotypes (genotypes 4, 21, 23 and 25). The occurrence of outbreaks further supports the nosocomial transmission of C. albicans which seems not to be uncommon in our NICU. The five outbreaks consisted mainly of invasive candidiasis (19 cases) and medical devices contamination (seven umbilical catheters and one endotracheal tube). The likeliness of nosocomial transmission of C. albicans in our NICU is reinforced by the two following findings. First, two isolates (P4 and P5), obtained from two HCWs 1 week apart, were identical to each other and formed an individual clone. The identity of isolates taken from different HCWs suggests that an endemic strain had crosscontaminated the hands of NICU co-workers. Similar findings have previously been reported (Marco et al. 1999). Second, three isolates (P1, P2 and P3) collected from two HCWs were identical to each other and to 11 additional isolates obtained from six neonates over the same period. This finding suggests the transfer of the strain between HCWs and patients. Many previous studies demonstrated that isolates obtained from the HCWs’ hands were genetically related to the patients’ isolates, although most HCWs were asymptomatic carriers and in most cases the direction of the strains’ transfer could not be accurately determined (Burnie et al. 1985, 1987;



Fraser 1990; Strausbaugh et al. 1994). In our study, the three HCWs’ isolates mentioned earlier were obtained from two nurses harbouring fingers’ onychomycosis for at least 25 days prior to the collection of the neonates’ isolates. These findings indicate that the ungual lesions of the HCWs were the source of the neonates’ contamination. In addition, the same strain caused bloodstream infection in all of the six neonates and contaminated umbilical catheter in three of them over a 25-day period. This suggests that the outbreak was subsequent to the contamination of catheters by the HCWs’ strain. Unfortunately, because of the retrospective character of our study, we were unable to collect additional samples from potentially colonized HCWs in the unit. We therefore could not further ascertain the mode of transmission of C. albicans in the four remaining outbreaks. However, the isolation of C. albicans from three catheters during one outbreak and from one endotracheal tube during an other outbreak suggests that transmission may have originated from the contaminated implanted medical devices. On the other hand, we did not test mothers’ isolates; thus, we were unable to estimate the importance of the vertical transmission of C. albicans in neonates. It was previously reported that some strains could concentrate in a defined geographical location and that some strains could be endemic in some hospitals and subsequently responsible for a significant proportion of nosocomial candidiasis (Pfaller et al. 1998). Our results indicate that six endemic and ⁄ or epidemic strains were responsible for infection or colonization cases that occurred in our NICU during the study period. One of these strains persisted up to 15 months. Among our patients, 23 have had two or more isolates. RAPD-CA1 ⁄ CA2 analysis showed that: (i) multiple isolates from the same neonate were identical in 65Æ2% of cases, (ii) the same patient can harbour the same strain in different and noncontiguous sites, (iii) unrelated strains can be simultaneously isolated from different body sites at the same period, (iv) the same patient can harbour the same strain over a long period of time as illustrated by the neonate’s N10 case who harboured the same strain over at least 3 months, (v) invasive infection is most often caused by the colonizing strain. Indeed, in six (75%) of the eight episodes of invasive candidiasis, the urine isolate was identical to the invasive one. However, the endogenous origin of the candidosic infection in neonates does not exclude its nosocomial origin because the colonization can be owing to hospital strains (WaggonerFountain et al. 1996; Huang et al. 1998), (vi) the bloodstream strains can contaminate the intravascular catheters, (vii) replacement of strains can occur in recurrent infections as shown in neonates N1 and N6 from whom two unrelated strains were isolated in blood 48 and 22 days


apart, respectively. Strain replacement was also observed in HCW1 from whom two unrelated strains were isolated in ungual lesion 4 years apart. Neither the PFGE nor the RAPD-CA1 ⁄ CA2 revealed any microevolutionary changes within infecting or colonizing strains taken over time from the same patient. This may be explained by the relatively short stay of neonates in NICU. On the other hand, strains had not enough time to diversify and undergo microevolution in these recently colonized neonates. Conclusion Our results confirmed that RAPD analysis is an useful method to assess the genetic relatedness of C. albicans isolates in a relatively large epidemiological study and that the PFGE analysis in contrast has a limited capacity to discriminate highly related isolates. The results of molecular typing of isolates obtained from neonates and HCWs argue for the nosocomial transmission of C. albicans in our NICU and for the transfer of strains from HCWs to patients, the fingers’ onyxis of HCWs being the potential reservoir of hospital strains and the implanted medical devices the route of strains’ transmission. Attention to hand-washing procedures and rigorous care of medical devices would reduce the risk of nosocomial transmission and the incidence of candidiasis in the NICU. In addition, HCWs harbouring an ongoing patent candidiasis should be temporarily discarded. Acknowledgements This work was supported by the UR02SP13 Research Unit, from the Ministry of Health, Tunisia and supported by a grant from the Spanish Ministry of Science and Technology (BFU2006-08684) and University of Valencia (UV-AE-10-24011). This work was partially carried out in the ‘Unidad de Investigacio´n de Genetica Molecular de Candida albicans’ of the department of Microbiology of the University of Valencia. Our gratefulness to the GMCA team. References Baquero, C., Montero, M., Sentandreu, R. and Valentin, E. (2002) Identification of Candida albicans by polymerase chain reaction amplification of a CaYST1 gene intron fragment. Rev Iberoam Micol 19, 80–83. Benjamin, D.K. Jr, Stoll, B.J., Fanaroff, A.A., McDonald, S.A., Oh, W., Higgins, R.D., Duara, S., Poole, K. et al. (2006) Neonatal candidiasis among extremely low birth weight infants: risk factors, mortality rates, and neurodevelopmental outcomes at 18 to 22 months. Pediatrics 117, 84–92.


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Burnie, J.P., Odds, F.C., Lee, W., Webster, C. and Williams, J.D. (1985) Outbreak of systemic Candida albicans in intensive care unit caused by cross infection. Br Med J 290, 746–748. Burnie, J.P., Matthews, R., Lee, W., Philpott-Howard, J., Brown, R., Damani, N., Breuer, J., Honeywell, K. et al. (1987) Four outbreaks of nosocomial systemic candidiasis. Epidemiol Infect 99, 201–211. Chu, W.S., Magee, B.B. and Magee, P.T. (1993) Construction of a SfiI macrorestruction map of the Candida albicans genome. J Bacteriol 175, 6637–6651. Del Castillo, L., Bikandi, J., Nieto, A., Quindos, G., Sentandreu, R. and Ponton, J. (1997) Comparison of morphotypic and genotypic methods for strain delineation in Candida. Mycoses 40, 445–450. Faix, R.G., Finkel, D.J., Andersen, R.D. and Hostetter, M.K. (1995) Genotypic analysis of a cluster of systemic Candida albicans infections in a neonatal intensive care unit. Pediatr Infect Dis J 14, 1063–1068. Fraser, C.A.M. (1990) Cross-infection and diversity of Candida albicans strain carriage in patients and nursing staff on an intensive care unit. J Med Vet Mycol 28, 317–325. Fridkin, S.K., Kaufman, D., Edwards, J.R., Shetty, S. and Horan, T. (2006) Changing incidence of Candida bloodstream infections among NICU patients in the United States: 1995–2004. Pediatrics 117, 1680–1687. Herruzo-Cabrera, R., De-Lope, C., Ferna¢ndez-Arjona, M. and Rey-Calero, J. (1995) Risk factors of infection and digestive tract colonization by Candida spp. in a neonatal intensive care unit. Eur J Epidemiol 11, 291–295. Huang, Y.C., Lin, T.Y., Peng, H.L., Wu, J.H., Chang, H.Y. and Leu, H.S. (1998) Outbreak of Candida albicans fungaemia in a neonatal intensive care unit. Scand J Infect Dis 30, 137–142. Hunter, P.R. and Gaston, M.A. (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 26, 2465–2466. Lehmann, P.F., Lin, D. and Lasker, B.A. (1992) Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA. J Clin Microbiol 30, 3249–3254. Lockhart, S.R., Fritch, J.J., Meier, A.S., Schroppel, K., Srikantha, T., Galask, R. and Soll, D.R. (1995) Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing. J Clin Microbiol 33, 1501–1509. Lockhart, S.R., Reed, B.D., Pierson, C.L. and Soll, D.R. (1996) Most frequent scenario for recurrent Candida vaginitis is strain maintenance with ‘‘Substrain Shuffling’’: demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2. J Clin Microbiol 34, 767–777. Lopez-Ribot, J.L., McAtee, R.K., Kirkpatrick, W.R., Perea, S. and Patterson, T.F. (2000) Comparison of DNA-based typ-

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