Use of Random Amplified Polymorphic DNA as a Typing Method for ...

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1995, p. 2366–2371 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 9

Use of Random Amplified Polymorphic DNA as a Typing Method for Candida albicans in Epidemiological Surveillance of a Burn Unit F. ROBERT,1* F. LEBRETON,2 M. E. BOUGNOUX,3 A. PAUGAM,1 D. WASSERMANN,2 M. SCHLOTTERER,2 C. TOURTE-SCHAEFER,1 AND J. DUPOUY-CAMET1* ˆle´s,2 Ho ˆpital Cochin, Paris, and Laboratoire Laboratoire de Parasitologie1 and Unite´ des Bru de Microbiologie, Ho ˆpital Ambroise Pare´, Boulogne-Billancourt,3 France Received 23 March 1995/Returned for modification 6 May 1995/Accepted 17 June 1995

Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hoˆpital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. albicans strains isolated from various anatomical sites of unrelated patients, and the strains showed 22 different patterns. Random amplified polymorphic DNA was then used in the epidemiological surveillance of the patients in the burn unit over a 9-month period. Seven patterns were identified among 84 isolates from 18 patients. One pattern (pattern A) corresponding to isolates from 7 of the 18 patients (68% of isolates) predominated throughout the 9-month study, while some strains with other profiles were isolated only once. Some profiles appeared to show a particular geographic pattern within the unit, suggesting transmission from room to room. These results underline the importance of fungal surveillance in such patients and the need to inform nursing staff of measures to prevent the spread of Candida spp. from patient to patient. 27, 29, 37), pulsed-field gel electrophoresis (10, 18, 21, 35), and polymorphic DNA amplification (random amplified polymorphic DNA [RAPD]) (3, 16), vastly improved strain differentiation. However, despite their high discriminatory powers and reproducibilities, restriction fragment length polymorphism analysis with hybridization and karyotyping are not suitable for routine epidemiological studies because they are laborious and require specialized equipment. The RAPD method, first described by Williams et al. (38, 39), does not have these drawbacks. It is based on PCR amplification of DNA fragments with arbitrary short primers (9 to 10 bases) with a low annealing temperature (368C). The fragments generated are separated through an agarose gel and are stained with ethidium bromide. Here we report the results of a prospective epidemiological surveillance of C. albicans over a 9-month period in a closed burn unit in which we used the RAPD typing method. The data obtained were analyzed with the aim of drawing epidemiological conclusions and therefore increasing prevention.

Nosocomial infections caused by Candida species are a growing problem and now account for 8% of all nosocomial bloodstream infections, according to the Centers for Disease Control (Atlanta) (22). Candida albicans is the most frequent pathogenic species, and the mortality rate from deep-seated infections ranges from 38 to 50% (1, 23). In most cases, colonization of the skin or mucosa occurs before invasion and systemic infection. Burn patients are particularly susceptible to Candida infections because they have most of the risk factors for hospital-acquired fungal infections, i.e., cutaneous and vascular portals of entry (indwelling catheters, parenteral nutrition), broad-spectrum antimicrobial therapy (which suppresses the normal bacterial flora), and altered immunological status, which is amplified by malnutrition (12, 17). The prevalence of Candida colonization of burn patients varies from 13 to 31.8% (8, 12, 25, 31), and it could be higher in children (17). In adults, this prevalence correlates with older age, larger burn surface area, and longer hospital stays (12, 25, 31). Because of the gravity of fungal infections in such patients, Candida contamination must be detected at an early stage, with a view to starting specific treatment as quickly as possible (36). A particularly high prevalence of C. albicans infections in the burn unit of the Ho ˆpital Cochin (Paris, France) led us to investigate the sources involved by the use of a DNA typing method. C. albicans subtyping methods have been reviewed and compared by Hunter (13), Merz (20), and Pfaller (23). The first methods which were described were based on phenotypic characteristic and, with the exception of isoenzyme analysis, were usually poorly discriminatory (7, 15). Genotyping methods, including restriction fragment length polymorphism analysis with or without hybridization to a DNA probe (11, 24, 26,

MATERIALS AND METHODS Patients and isolates of the burn unit. The burn unit of Ho ˆpital Cochin is composed of an intensive care unit (ICU), where severely burned patients are strictly isolated, and an open unit, for less severely burned patients. The ICU consists of seven single rooms; six rooms have airfluidized beds and a separate bathtub and the seventh room has a conventional bed that is reserved for polytraumatic patients or is used for patients awaiting the release of an airfluidized bed. The ICU nursing staff wear special clothing in the patients’ rooms (gowns, gloves, and hat) and wash their hands when leaving each room. The following data were recorded for each patient: date of injury, total burn surface area, date of admission, age, sex, treatment, room number in the ICU, date of transfer to the open unit, and date of discharge. All patients had local treatment consisting of daily applications of silver sulfadiazine with or without cerium nitrate. Full-thickness burns were excised and grafted as early as possible. Deepfrozen allografts were used to cover large full-thickness burns. Bandages were removed under a shower. All patients received oral amphotericin B prophylaxis. Specimens for fungal culture (wound swabs or biopsy specimens, mouth swabs, urine, and blood) were obtained from all patients in the ICU on their arrival (when possible) and then weekly. Cultures were done on Sabouraud-chloramphenicol agar for deep specimens and on Cand-ID medium (Biome ´rieux, Marcy

* Corresponding author. Mailing address: Laboratoire de Parasitologie, Ho ˆpital Cochin, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France. Phone: 0033.1.42.34.14.97. Fax: 0033.1.42.34.14.96. 2366

RAPD TO TYPE C. ALBICANS ISOLATES

VOL. 33, 1995

2367

TABLE 1. Characteristics of the burn patients Time (days) before: Patient group and no.a

% TBSAb

First specimen collection

Positive culture

No. of isolates

Duration of carriage (wk)

RAPD profile

Mouth

Other site

A A B A C A D B A E E B

4 1 1 5 4 1 2 1 6 6 1 4

0 0 0 0 0 0 0 0 0 0 0 0

A F G B D F D A D

2 3 3 8 0 8 4 4 1

1 1 1 3 3 2 1 1 2

Colonized 1 2c

22 59

6 6

6 6

3 4d

30 53

5 6

5 6

5 6 7 9 10 12

27 13 29 50 85 40

,2 4 4 0 3 0

,2 4 4 0 24 0

3 1 1 6 3 1 2 1 13 6 1 3

Infected 8 11 13 14e

60 63 11 56

,2 4 3 ,2

,2 4 3 9

1 2 7 7

15 16 17 18

57 30 52 36

5 0 1 3

5 7 1 10

10 4 4 2

a Patients were considered colonized in case of digestive tract carriage only and were considered infected in case of digestive tract carriage and culture positivity of a specimen from another site. b TBSA, total burn surface area. c Profile A and then profile B; 1 week of carriage of each and one isolate of each. d Profile C (3 weeks of carriage; four isolates) and then profile A (1 week; one isolate). e Both profile B (11 isolates) and profile D (3 isolates).

l’Etoile, France) for superficial ones. The dates and sites of positive yeast cultures were recorded. Only patients whose specimens grew C. albicans in culture were included in the study. Isolates were identified by assimilation of carbon sources with the API 20C gallery system (Biome´rieux) or growth of blue colonies on Cand-ID medium (Biome´rieux) and were confirmed by chlamydospore formation in potato, carrot, and bile medium (Sanofi-Diagnostic Pasteur, Marnes la Coquette, France) at 278C. Unrelated isolates. Thirty-two isolates from various anatomical sites of 32 unrelated inpatients in distinct units of five different hospitals in the Paris area (see Table 2) were collected and identified as described above. This typing phase was carried out to evaluate the discriminatory power of our technique and the distribution of the characterized profiles in the general patient population. Spheroplast formation and DNA extraction. Spheroplasts were prepared by the technique described by Carruba et al. (6), with modifications. One colony of each isolate was plated on Sabouraud-chloramphenicol agar, and the plate was incubated for 48 h at 308C. One colony was then grown to the stationary phase in 30 ml of YPD broth (1% yeast extract, 2% peptone, 2% dextrose; Difco Laboratories), and the mixture was incubated for 18 h at 358C with agitation and aeration. For spheroplast formation, cultures were washed with 1 M sorbitol (Sigma, Le Perray-en-Yvelines, France) and the pellets were suspended in 3 ml of SE medium (1.2 M sorbitol, 0.1 M EDTA [pH 7.5]) containing 3 mg of Zymolyase 20T (ICN Biomedicals Inc., Costa Mesa, Calif.) and 10 ml of 2-mercaptoethanol, and then the mixture was incubated at 378C for 1 h. The spheroplasts were then distributed into two Eppendorf tubes and washed with SE medium. For the extraction of whole-cell DNA, spheroplasts were incubated with lysis buffer consisting of 500 ml of TE (10 mM Tris HCl [pH 8], 1 mM EDTA [pH 8]) and 100 ml of 10% sodium dodecyl sulfate for 30 min at 658C. Proteins were precipitated with 100 ml of 5 M potassium acetate (19) and were kept on ice for 1 h. The tubes were then centrifuged (at 12,000 3 g for 10 min), and the supernatant was treated with RNase A (10 mg/ml, previously boiled for 5 min; Sigma) at 378C for 1 h. Two phenol extractions were then performed; this was followed by a purification step with chloroform-isoamyl alcohol (24:1 [vol/vol]); the resulting purified DNA was precipitated with 5 M NaCl adjusted to a final concentration of 0.1 M, completed with 95% ethanol, and kept overnight at 2208C (19). After centrifugation (12,000 3 g, 30 min, 48C), the DNA was washed twice with 70% ethanol. The supernatant was discarded, and the pellets were allowed to dry. The DNA was then resuspended in 100 ml of sterile water, and the concentration was determined after electrophoresis in a 1% (wt/vol) agarose gel stained with ethidium bromide and comparison with a molecular weight

marker (bacteriophage l DNA-EcoRI plus HindIII; marker III; Boehringer Mannheim, Meylan, France). RAPD. Twelve oligonucleotides (Genset, Paris, France) containing various percent GC contents were tested for primary screening of 18 unrelated isolates (data not shown). The primer generating the largest number of patterns was used to type all 32 unrelated isolates. This primer (R4) was selected for use in epidemiological typing of isolates from patients in the burn unit. The sequence of R4 is 59-TGGTCGCGGC-39. PCR was carried out with about 25 ng of DNA; 100 mM (each) dATP, dCTP, dTTP, and dGTP; 100 pmol of oligonucleotide; 0.5 U of Taq polymerase (PerkinElmer Cetus, St. Quentin-en-Yvelines, France); and PCR buffer consisting of 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% (wt/vol) gelatin (final concentrations). The final volume of the reaction mixture was 25 ml. The DNA thermal cycler (Perkin-Elmer Cetus) was programmed by using the conditions described by Williams and collaborators (38, 39), i.e., 45 cycles of 1 min at 948C, 1 min at 368C, and 2 min at 728C. The final extension step was prolonged to 10 min at 728C. The resulting DNA fragments were separated through a 2% (wt/vol) agarose gel (SeaKem; Tebu, Le Perray-en-Yvelines, France) with 13 TAE buffer (40 mM Tris-acetate, 1 mM EDTA), stained with ethidium bromide, and then visualized over a source of UV light and photographed (Polaroid 667 film). The sizes of the DNA fragments were determined by comparison with a molecular weight marker (pBR328 DNA-BglI, plus HinfI; Boehringer Mannheim). All isolates were typed twice to assess the reproducibility of the technique. RAPD patterns were read blindly with regard to the patient’s identity, so that the observer’s expectations could not bias the choice of pattern.

RESULTS Burn patients and isolates. During a 9-month period (November 1992 to July 1993), 41 patients were admitted to the ICU. Despite routine oral amphotericin B prophylaxis, 18 of these 41 patients (43.9%) carried C. albicans (Table 1). The mean age of these 18 patients was 36 years (range, 15 to 80 years), and the mean burn surface area was 43% (range, 13 to 85%). The mean time between injury and admission was 5.2 days. Eighty-four C. albicans isolates were obtained from the

2368

ROBERT ET AL.

J. CLIN. MICROBIOL.

FIG. 1. RAPD profiles of 32 unrelated C. albicans isolates (PG1 to PG32, as listed in Table 2) obtained with primer R4. Electrophoresis of the RAPD products was done in a 2% agarose gel. Lanes 1 to 12, first migration; lanes 13 to 29, second migration; lanes 30 to 32, third migration. wm, molecular weight marker (molecular weight marker VI; pBR328 DNA-BglI, HinfI; Boehringer Mannheim).

18 patients. Sixty-nine isolates were isolated from the mouth (82%), 12 were isolated from burn wounds (14.3%), and 1 each was isolated from urine, blood, and an intravenous catheter. The patients were divided into those who were colonized and those who were infected. Patients whose mouth specimens alone were positive for C. albicans on culture were considered colonized, while those with C. albicans in both the mouth and at another site (wound, blood, or urine) were considered infected. Ten patients (55.5%) had only digestive tract colonization, and eight patients were infected, with positive cultures of specimens from both the digestive tract and a normally sterile site. Four patients (patients 10, 14, 16, and 18) were uncolonized on admission and acquired C. albicans in the burn unit. Patients 14 and 16 had positive mouth specimen cultures 1 week after their arrival and subsequently became positive for wound cultures. One patient (patient 14) acquired C. albicans septicemia. Seven patients (patients 8, 11, and 14 to 18) acquired deep-seated C. albicans infections, as determined by positive cultures, while in the ICU; their wound specimen cultures became positive within a mean of 11 days after mouth colonization. Two infected patients (patients 8 and 17) and three colonized patients (patients 5, 9, and 12) had digestive tract colonization on admission. Patients 8 and 17 later acquired the same strain in their wounds. RAPD profiles. Primer R4 yielded 22 different patterns among the 32 isolates from unrelated patients (Fig. 1). Eight couples of strains and one triplet of strains each shared the same pattern (Table 2). Twenty-seven of the 32 isolates (77%) showed two common bands at 1,800 and 760 bp. Some groups of patterns (patterns 3, 9, 16, 17, and 18 and patterns 1, 2, 4, and 6) were very close to one another and may have corresponded to genetically related strains. However, these isolates originated from different hospitals and various anatomical sources. The discriminatory power of primer R4 in this series calculated with the formula of Hunter and Fraser (14) was 0.97. Among the patients in the ICU, RAPD analysis with primer R4 yielded seven profiles (profiles A to G) (Fig. 2). Profile A was the most frequent one and was present among isolates from 7 of 18 patients (38.8%). Primer R4 produced three to seven polymorphic fragments. The examination of the RAPD fragments demonstrated the presence of the two high-intensity bands at 1,800 and 760 bp in six of the seven profiles (A, B, D, E, F, and G). Profile C showed an intense fragment at 550 bp and had no bands in common with any of the other profiles. The similarity coefficients (29, 30) between profiles A, B, D, E,

F, and G were calculated and ranged from 61.5 to 90.9%. Profiles B, C, E, F, and G were clearly different from the 22 previously identified profiles. Although profiles A and D were quite similar to profiles 19 and 14 of strains isolated from the general patient population, there was no clear evidence of identity. Distribution of RAPD profiles. The distributions of the dif-

TABLE 2. Characteristics and primer R4 RAPD patterns of 32 C. albicans isolates from unrelated patients Isolate no.

Site of recoverya

PG1 PG2 PG3 PG4 PG5 PG6 PG7 PG8 PG9 PG10 PG11 PG12 PG13 PG14 PG15 PG16 PG17 PG18 PG19 PG20 PG21 PG22 PG23 PG24 PG25 PG26 PG27 PG28 PG29 PG30 PG31 PG32

Blood Oral cavity Oral cavity Blood Oral cavity BAL Oral cavity Blood BAL Oral cavity Blood BAL Oral cavity NA Urine Skin BAL Aqueous humor Stools BAL Glans NA Vagina Sputum Vagina Oral cavity Vagina Oral cavity Blood Sputum Oral cavity Oral cavity

a

Hospital and unitb

H3, H2, H3, H3, H3, H3, H3, H3, H3, H3, H3, H3, H2 H3 H1 H1, H1, H1, H1, H1, H1, H3 H4 H5 H5 H1 H1, H1, H1 H1 H3, H3,

ICU Med Med Surg Med ICU Med Med Med Med Med Surg

OP OP Med Med ICU OP

OP Surg Med Med

Profile no.

1 1 2 3 2 4 5 5 6 7 8 9 10 3 11 9 12 13 14 15 16 12 17 18 16 14 16 19 20 20 21 22

BAL, bronchoalveolar lavage; NA, not available. Isolates were from hospitals A. Pare´ (H1), R. Poincare´ (H2), Cochin (H3), Villejuif (H4), and Saint-Louis (H5); Med, medicine unit; ICU, intensive care unit; Surg, surgical unit; OP, outpatient department. b

VOL. 33, 1995

FIG. 2. Seven different profiles (profiles A to G) of isolates from the burn unit obtained with primer R4. Electrophoresis of the RAPD products was done in a 2% agarose gel. Lane wm, molecular weight marker III (bacteriophage l DNA-EcoRI plus HindIII; Boehringer Mannheim).

ferent profiles over the 9-month period are reported in Table 1. Profile A was isolated from 7 of 18 patients (25 of 84 isolates; 29.8%). The other profiles were distributed as follows: B from four patients (17 of 84 isolates; 20.2%), D from four patients (13 of 84 isolates; 15.5%), E from two patients (7 of 84 isolates; 8.3%), F from two patients (14 of 84 isolates; 16.7%), and C (4 of 84 isolates; 4.8%) and G (4 of 84 isolates, 4.8%) from one patient each. The strains isolated from different body sites of the same patient (superficial and deep sites) at different times had identical profiles. Isolates of profile A dominated during the first 4 months (six of eight patients) and was carried by patients 3 and 7 for a long period. It reappeared in patient 17 3 weeks after discharge of the last carrier (patient 7). Patient 4 carried an isolate of profile C for 3 weeks and then acquired an isolate of profile A. Patient 2 carried an isolate of profile A and then one of profile B (1 week each). Patient 14 carried isolates

RAPD TO TYPE C. ALBICANS ISOLATES

2369

of both profile B (mouth, wound, catheter) and profile D (wound, blood). The chronological carriage of the different patterns is reported in Fig. 3 according to the ICU room number. Patients 1, 8, and 17 were consecutively hospitalized in the same room, and all carried isolates of profile A. Patients 2, 4, and 7 (from whom isolates of profile A were also isolated) were hospitalized either in the same room or in adjacent rooms. The only two patients carrying isolates of profile F were hospitalized consecutively in the same room. In addition, isolates of profile E were found in only two patients, who were hospitalized in adjacent rooms during the same period. Isolates of other profiles appeared to be randomly distributed among the different rooms. The analysis of the distribution of RAPD patterns according to body site shows that isolates of profile A were indifferently isolated from the mouth and other sites (Table 1). In contrast, isolates of profile D were most frequently isolated from deep sites (wound, blood; 6 of 15 isolates, 40%). DISCUSSION In the present prospective epidemiological study, 18 of the 41 patients admitted to the burn unit during a 9-month period carried C. albicans, despite oral amphotericin B prophylaxis. Among the 18 patients (84 isolates), only 7 different types were identified (with a predominance of one type), whereas 22 types were identified among 32 unrelated patients in other hospital units. None of the profiles of organisms isolated in the burn unit was clearly present in the unrelated patients, although profiles A and D resembled some of the profiles of isolates from unrelated patients. Analysis of the 32 unrelated isolates confirmed the discriminatory value of RAPD, with an index of 0.97, which is quite similar to those of the most powerful genotyping methods, i.e., restriction fragment length polymor-

FIG. 3. Chronological carriage of C. albicans in the different rooms of the burns unit. All rooms are adjacent and are accessed by a corridor. The RAPD profiles are indicated for each patient (each letter corresponds to one positive culture).

2370

ROBERT ET AL.

phism analysis and then hybridization (28–30) and pulsed-field gel electrophoresis (2, 6, 18, 21, 35). It is noteworthy that whatever typing method was used, the patterns obtained among C. albicans isolates are very close to one another (35). However, RAPD has some advantages over these methods: it is rapid and simple, and DNA extraction procedures can be further simplified. These data confirm that our technique is a valuable typing method for epidemiological investigations. Nosocomial transmission of Candida spp. in the ICU was suggested by several observations. Although five patients already had digestive tract colonization on admission, four patients who were uncolonized on admission acquired C. albicans in the ICU. During the first 4 months of the study, isolates of profile A were largely predominant (6 of 8 patients): four patients carried this type, one patient acquired it transiently, and one patient (patient 4) acquired it in the ICU, suggesting dissemination through the unit. Moreover, these six patients were hospitalized either consecutively in the same room or in adjacent rooms during the same period. Isolates of profile F were observed in only two patients; it could have been brought into the unit by patient 11 and was further found in the subsequent occupant of room 3 (patient 15), who remained infected with this strain for 3 months. This observation points to spread as a result of environmental factors. Isolates of profile E were also recovered from two patients who were hospitalized during the same period. Several arguments point to the nosocomial transmission of isolates of profile E: (i) patient 9 was colonized on admission and was the first patient to carry an isolate with this profile, (ii) patient 10 was negative for C. albicans culture on admission and acquired the profile E strain 25 days later, and (iii) these two patients were in adjacent rooms. Isolates of profile D were isolated from three infected patients (37.5%) (patients 14, 16, and 18), who all acquired the corresponding strain in the unit during the same period (Fig. 3). Although the concept of cross infection is not widely accepted, similar observations have been made by other investigators (4, 5, 9) by DNA restriction fragment analysis. Vaudry et al. (33) also characterized a strain that was probably responsible for cross infections between infants who were linked in both time and space in a nursery. This concept was also supported by Vazquez et al. (34), who found identical strains of C. albicans in patients in adjacent beds in a 10-month prospective study in a bone marrow transplant unit and an ICU. In our study, dissemination could have been due to manual transport from patient to patient by the nursing staff or via the environment, although the nursing staff are supposed to change their gloves and wash their hands after leaving each room. No swabs of the nurses’ hands were taken, but it is noteworthy that isolates of profile A were present in the ICU throughout the study. Gut colonization favors subsequent burn infection by Candida spp. (38.8%). In our study, 75% of the patients with Candida burn wound infections had previous digestive tract colonization. In every case, the strains isolated from different anatomical sites of a given patient were identical. These results are in agreement with published data (11, 22, 26, 32), but they do not support the hypothesis of Soll et al. (30) that some strains could have tropism for particular anatomical sites. In our study, all of the patients had digestive tract colonization, and 44.4% had burn wound infections caused by C. albicans. Among five patients already colonized on admission, the endogenous strain later extended to the burn wound in two cases, in keeping with the findings of Reagan et al. (26), who identified the same strain in blood and in superficial sites. Although isolates of profile A were found slightly more frequent than the isolates of other profiles in the oral cavities

J. CLIN. MICROBIOL.

of the patients (6 of 18 patients; 33.3%), this may simply have reflected its overall frequency (29.7% of all isolates), since the oral cavity was the most frequently positive site, as noted previously by Grube et al. (12). In contrast, isolates of profile D tended to be isolated more often from deep specimens, including those from two patients who were uncolonized on admission. In six patients (patients 8, 11, and 14 to 17), colonization of the digestive tract preceded infection of the burn wounds. In conclusion, given its rapidity and simplicity, RAPD appears to be a valuable typing method for epidemiological studies of C. albicans. Although the mechanism of C. albicans acquisition in these burn patients is not clear, our results suggest that (i) cross infection may have occurred in a few cases, (ii) strains of some profiles (profiles A and D) are frequent and may correspond to epidemic strains, and (iii) strains responsible for gut colonization often spread to the burn wounds, as proven by the identical patterns of strains obtained from different sites. These results underline the importance of fungal surveillance in burn units, with a view to preventing wound infection by means of chemoprophylaxis and reinforced asepsis. ACKNOWLEDGMENTS This study was supported by ADERMEPT. We thank D. Young for the English revision of the manuscript. REFERENCES 1. Beck-Sague, C. M., W. R. Jarvis, and the National Nosocomial Surveillance System. 1993. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J. Infect. Dis. 167:1247–1251. 2. Bostock, A., M. N. Khattak, R. Matthews, and J. Burnie. 1993. Comparison of PCR fingerprinting by random amplification of polymorphic DNA, with other molecular typing methods for Candida albicans. J. Gen. Microbiol. 139:2179–2184. 3. Bougnoux, M. E., F. Robert, S. Beria, B. Cassinat, M. H. Nicolas, and J. Dupouy-Camet. 1994. Use of randomly amplified polymorphic DNA markers to distinguish strains of Candida. J. Mycol. Med. 4:3–8. 4. Burnie, J. P., R. Matthews, W. Lee, J. Philpott-Howard, R. Brown, N. Damani, J. Breuer, K. Honeywell, and Z. Jordans. 1987. Four outbreaks of nosocomial systemic candidiasis. Epidemiol. Infect. 99:201–211. 5. Burnie, J. P., F. C. Odds, W. Lee, C. Webster, and J. D. Williams. 1985. Outbreak of systemic Candida albicans in intensive care unit caused by cross infection. Br. Med. J. 290:746–748. 6. Carruba, G., E. Pontieri, F. de Bernardis, P. Martino, and A. Cassone. 1991. DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis. J. Clin. Microbiol. 29:916–922. 7. Caugant, D. A., and P. Sandven. 1993. Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis. J. Clin. Microbiol. 31:215–220. 8. Desai, M. H., D. N. Herndon, and S. Abston. 1987. Candida infection in massively burned patients. J. Trauma 27:1186–1188. 9. Doebbeling, B. N., R. J. Hollis, H. D. Isenberg, R. P. Wenzel, and M. A. Pfaller. 1991. Restriction fragment analysis of a Candida tropicalis outbreak of sternal wound infections. J. Clin. Microbiol. 29:1268–1270. 10. Doebbeling, B. N., P. F. Lehmann, R. J. Hollis, L. C. Wu, A. F. Widmer, A. Voss, and M. A. Pfaller. 1993. Comparison of pulsed-field gel electrophoresis with isoenzyme profiles as a typing system for Candida tropicalis. Clin. Infect. Dis. 16:377–383. 11. Fox, B. C., H. L. T. Mobley, and J. C. Wade. 1989. The use of a DNA probe for epidemiological studies of candidiasis in immunocompromised hosts. J. Infect. Dis. 159:488–494. 12. Grube, B. J., J. A. Marvin, D. M. Heimbach. 1988. Candida: a decreasing problem for the burned patients? Arch. Surg. 123:194–196. 13. Hunter, P. R. 1991. A critical review of typing methods for Candida albicans and their application. Crit. Microbiol. Rev. 17:417–434. 14. Hunter, P. R., and C. A. Fraser. 1989. Application of a numerical index of discriminatory power to a comparison of four physiochemical typing methods for Candida albicans. J. Clin. Microbiol. 27:2156–2160. 15. Lehmann, P. F., B. J. Kemker, C. B. Hsiao, and S. Dev. 1989. Isoenzyme biotypes of Candida species. J. Clin. Microbiol. 27:2514–2521. 16. Lehmann, P. F., D. Lin, and B. A. Lasker. 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. 17. MacMillan, B. G., E. J. Law, and I. A. Holder. 1972. Experience with Candida infections in the burn patient. Arch. Surg. 104:509–513.

VOL. 33, 1995 18. Mahrous, M., T. J. Lott, S. A. Meyer, A. D. Sawant, and D. G. Ahearn. 1990. Electrophoretic karyotyping of typical and atypical Candida albicans. J. Clin. Microbiol. 28:876–881. 19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. Merz, W. G. 1990. Candida albicans strain delineation. Clin. Microbiol. Rev. 3:321–334. 21. Merz, W. G., C. Connelly, and P. Hieter. 1988. Variation of electrophoretic karyotypes among clinical isolates of Candida albicans. J. Clin. Microbiol. 26:842–846. 22. Pfaller, M. A. 1990. The use of biotyping and DNA fingerprinting in typing Candida albicans from hospitalized patients. Diagn. Microbiol. Infect. Dis. 13:481–489. 23. Pfaller, M. A. 1992. Epidemiological typing methods for mycoses. Clin. Infect. Dis. 14(Suppl):S4–S10. 24. Powderly, W. G., K. Robinson, and E. J. Keath. 1992. Molecular typing of Candida albicans isolated from oral lesions of HIV-infected individuals. AIDS 6:81–84. 25. Prasad, J. K., and I. Feller. 1987. A ten-year review of Candida sepsis and mortality in burned patients. Surgery 101:213–216. 26. Reagan, D. R., M. A. Pfaller, R. J. Hollis, and R. P. Wenzel. 1990. Characterization of the sequence of colonization and nosocomial candidemia using DNA fingerprinting and a DNA probe. J. Clin. Microbiol. 28:2733–2738. 27. Scherer, S., and D. A. Stevens. 1987. Application of DNA typing methods to epidemiology and taxonomy of Candida species. J. Clin. Microbiol. 25:675– 679. 28. Schmid, J., M. Rotman, B. Reed, C. L. Pierson, and D. R. Soll. 1993. Genetic similarity of Candida albicans strains from vaginitis patients and their partners. J. Clin. Microbiol. 31:39–46. 29. Soll, D. R. 1993. DNA fingerprinting of Candida albicans. J. Mycol. Med. 3:37–44.

RAPD TO TYPE C. ALBICANS ISOLATES

2371

30. Soll, D. R., R. Galask, J. Schmid, C. Hanna, K. Mac, and B. Morrow. 1991. Genetic dissimilarity of commensal strains of Candida spp. carried in different anatomical locations of the same healthy women. J. Clin. Microbiol. 29:1702–1710. 31. Spebar, M. J., and B. A. Pruitt. 1981. Candidiasis in the burned patients. J. Trauma 21:237–239. 32. Stevens, D. A., F. C. Odds, and S. Scherer. 1990. Application of DNA typing methods to Candida albicans epidemiology and correlations with phenotype. Rev. Infect. Dis. 12:258–266. 33. Vaudry, W. L., A. J. Tierney, and W. M. Wenman. 1988. Investigation of a cluster of systemic Candida albicans infections in a neonatal intensive care unit. J. Infect. Dis. 158:1375–1379. 34. Vazquez, J. A., V. Sanchez, C. Dmuchowski, L. M. Dembry, J. D. Sobel, and M. J. Servos. 1993. Nosocomial acquisition of Candida albicans: an epidemiologic study. J. Infect. Dis. 168:195–201. 35. Vos, A., R. J. Hollis, M. A. Pfaller, R. P. Wenzel, and B. N. Doebbeling. 1994. Investigation of the sequence of colonization and candidemia in non-neutropenic patients. J. Clin. Microbiol. 32:975–980. 36. Wassermann, D., M. N. Hoang, J. Dupouy-Camet, F. Lebreton, M. Schlotterer, and C. Tourte-Schaefer. 1991. Inte´reˆt de la surveillance mycologique chez le grand bru ˆle´. Ann. Mediterr. Burns Club 4:94–96. 37. Whelan, W. L., D. R. Kirsch, K. J. Kwon-Chung, S. M. Wahl, and P. D. Smith. 1990. Candida albicans in patients with the acquired immunodeficiency syndrome: absence of a novel or hypervirulent strain. J. Infect. Dis. 162:513–518. 38. Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalsky, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531–6535. 39. Williams, J. G. K., A. R. Kubelik, J. A. Rafalsky, and S. V. Tingey. 1991. Genetic analysis with RAPD markers, in more gene manipulations in fungi, p. 431–439. In J. W. Bennett and L. L. Lasure (ed.). Academic Press, Inc., New York.