INTRODUCTION

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During the period June 1990 through February 1993, personnel from the Centers for Disease Control and Prevention (CDC) investigated seven unusual outbreaks of postoperative infections of the bloodstream and surgical sites and acute febrile episodes involving a variety of microorganisms after surgical procedures in hospitals in multiple states (2). In these outbreaks, the cause of infection was extrinsic contamination of a lipid-based anesthetic medication, propofol. We report the findings of an investigation of an outbreak of Candida albicans infections in one hospital.

Hospital A is a 444-bed tertiary care center with an active surgical program. Approximately 793 surgical procedures are performed there each month; of these, 56% are performed on an outpatient basis and 44% are performed on inpatients. Hospital A has 10 operating rooms (ORs) where all surgeries are performed. In June 1990, the infection control nurse from this hospital reported to the Hospital Infections Program, National Center for Infectious Diseases, CDC, that three patients had developed C. albicans wound infection with fungemia or endophthalmitis following surgeries performed during April 1990. The state Department of Public Health was notified, and CDC was invited to assist in a further assessment of this apparent outbreak.

(Presented in part at the 91st Annual General Meeting of the American Society for Microbiology, Dallas, Tex., 5 to 9 May 1991 [8a].)

	MATERIALS AND METHODS

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Epidemiological methods. (i) Case definition. A case patient was defined as any hospital A patient with a positive culture for C. albicans from a wound and a positive blood or intraocular culture following a surgical procedure performed between 16 and 30 April 1990. This epidemic period was chosen to include all patients who had surgical procedures performed 1 week before the date of surgery of the first case patients through the end of April 1990.

(ii) Case ascertainment. To identify case patients, we reviewed reports from infection control practitioners at other hospitals that had admitted patients with serious postoperative infections following surgery in hospital A. In addition, we reviewed hospital A's microbiology records from 1 January to 8 June 1990 for C. albicans-positive cultures, and infection control personnel at hospital A notified surgeons so that other potentially infected patients could be identified. We reviewed the medical charts of all case patients identified.

(iii) Review of case patients. We reviewed the medical records of all case patients and abstracted information on age, sex, race, duration of hospitalization, underlying disease, nature and date of surgical procedures, duration of fever, receipt of therapeutic and/or prophylactic antimicrobial agents or steroids, room assignment, other procedures, infections, and outcome.

(iv) Cohort study. To determine whether this cluster of case patients represented an outbreak, we compared the rate of C. albicans wound and blood or eye infections in the epidemic and preepidemic (1 January to 15 April 1990) periods. To assess potential risk factors for C. albicans postsurgical infections, a cohort study was performed. Since patients who had surgery on one of two particular days appeared to be at greater risk of becoming infected, we limited the cohort study to patients who had surgery on those days, i.e., 23 April and 27 April 1990. The risk factors analyzed included the patient's demographic and underlying health status characteristics, exposure to intraoperative intravenous anesthesia medications and solutions, exposure to different preoperative skin preparations (povidone-iodine scrub or paint and shaving), exposure to surgical drains, receipt of surgical prophylactic antimicrobial therapy, and exposure to surgeons and anesthesia and OR nursing personnel. During the epidemic period, 86 surgeons, 16 anesthesiologists (14 on the hospital staff), and 68 ancillary OR nursing personnel (surgical assistants and scrub and circulating nurses) participated in surgical procedures at hospital A.

(v) Statistical methods. All data were collected on standardized forms and entered into a microcomputer for analysis. Student's t test or Fisher's exact test were used to test for the significance of associations. When the rate ratio for a presumed risk factor was undefined, the algorithm procedure of Thomas and Gart was used to obtain confidence limits and a P value (15).

Molecular subtyping methods. (i) Purification of genomic DNA. All isolates of C. albicans were grown for 20 h at 30°C on a rotatory shaker at 150 rpm in 10 ml of YPD broth (1% [wt/vol] glucose, 1% [wt/vol] yeast extract [Difco Laboratories, Detroit, Mich.], and 2% [wt/vol] Bacto Peptone [Difco]) in 50-ml flasks. The cells were harvested by centrifugation, and then spheroplasts were produced by the method of Scherer and Stevens (12), using Zymolyase-20T (ICN ImmunoBiologicals, Costa Mesa, Calif.). Genomic DNA was purified by repeated phenol-chloroform extractions of cleared lysates as described by Mason et al. (8).

(ii) CARE-2 Southern blot analysis. One microgram of genomic DNA was digested with 10 U of EcoRI (New England Biolabs, Beverly, Mass.) or HincII at 37°C in the buffered solutions recommended by the manufacturer. DNA fragments were separated on a 0.7% (wt/vol) agarose gel (International Biotechnologies Inc., New Haven, Conn.) in Tris-borate buffer (7), stained with ethidium bromide, and transferred to nitrocellulose filters (type BA85; Scheicher and Schuel, Keene, N.H.) by standard protocols (7). Methods for labeling C. albicans repetitive element 2 (CARE-2) DNA nonisotopically with digoxigenin-11-dUTP by random priming, hybridization and washing conditions, and color development with the Genius kit (Boehringer Mannheim, Indianapolis, Ind.) were described previously by Lasker et al. (3). Filters were photographed wet with Polaroid type 55 film.

(iii) Electrophoretic karyotype. Yeast DNA plugs were prepared as previously described (6). Transverse-alternating field electrophoresis (TAFE) was preformed as previously described (5), using a Geneline 1 (Beckman Instruments, Fullerton, Calif.) chamber. Briefly, the conditions were as follows: a run time of 72 h; a 5-min pulse time; 100 V; 1× TAFE buffer (10 mM Tris base, 0.5 mM EDTA, 0.025% acetic acid), 1% agarose; 14°C. Following electrophoresis, the gels were stained with ethidium bromide and photographed.

Procedure and personnel investigation. To evaluate the potential role of procedural factors, we conducted a review of the anesthesia care of the patients by interviewing several of the anesthesiologists and observing two surgical procedures with general anesthesia administered by two different anesthesiologists. Hand cultures were obtained from all 14 hospital staff anesthesiologists. All individuals wiped their hands with individually wrapped sterile handiwipes presoaked in 10 ml of 0.02% Tween 80 in water, which were then placed into separate sterile jars containing 250 ml of Tween 80 solution in water. These specimens were then transported to CDC. After being shaken, separate aliquots (100, 10, 1, and 0.1 ml) of each hand culture solution were filtered through a 0.45-µm-pore-size filter. The filters were then placed on Trypticase soy agar plates and incubated at 30°C for 24 to 48 h, and total viable counts were determined.

Environmental investigation. Before our on-site investigation, hospital infection control personnel had made cultures of swabs from equipment and solutions from the anesthetic cart of one anesthesiologist, anesthesiologist 9. These included anesthetic agents (two different lots of unopened ampules of propofol [Diprivan; Zeneca Pharmaceuticals, Wilmington, Del.], vecuronium, gallamine, and epinephrine); anesthetic equipment (forceps, medicine holder, pressure gauge towel, paper label, plastic tubing, rubber glove, tourniquet, lubricant, and inhalant mask); intravenous electrolyte solutions (saline, dextrose, and Ringer's solution); remaining propofol and saline solutions from patient intravenous administration sets; and nonsterile water. At the time of our investigation, we repeated environmental cultures of anesthetic equipment and medications from anesthesiologist 9's cart, including propofol drawn up in a mock-up intravenous pump administration set by anesthesiologist 9.

	RESULTS

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Case patient characteristics. From 1 January through 8 June 1990, specimens from 62 surgical patients from hospital A were culture positive for C. albicans: sputum (38 patients), urine (13 patients), sputum and urine (5 patients), wound (3 patients), and other (3 patients). No eye isolates yielded C. albicans. Review of the microbiology laboratory records identified only patient 1 (with blood and wound cultures positive for C. albicans). The three other case patients were identified only after they were readmitted to other hospitals for management of C. albicans endophthalmitis. Case ascertainment, therefore, may have been incomplete.

Description of case patients. The case patients ranged in age from 28 to 72 years; three (75%) were female (Table 1). One case patient was an inpatient at hospital A at the time of surgery, and three underwent outpatient surgical procedures. All case patients underwent clean or clean-contaminated surgical procedures, and all had 24-h maximum postoperative axillary temperatures of 101°F (range, 101 to 103°F). The mean interval between the surgical procedure and the first C. albicans-positive culture was 18 days (range, 1 to 45 days).

Cohort study. When we compared case patients with non-case patients, exposure to surgical drains (3 of 4 versus 14 of 67; relative risk (RR), 9.5; P = 0.04), receipt of propofol by infusion pump (3 of 4 versus 15 of 67; RR, 8.8; P = 0.048), and higher mean 24-h maximum postoperative temperature (102 ± 1 versus 98.7 ± 1°F; P < 0.001) were identified as risk factors (Table 2). When we compared the exposures of case patients and non-case patients to the surgical, anesthesia, and OR nursing personnel, anesthesiologist 9 and OR nurses 6 and 10 were significantly more often associated with case patients than non-case patients (Table 3). The independent effects of these two OR nurses could not be estimated, since their exposures were all to case patients who also received propofol by infusion pump administered by anesthesiologist 9. However, the magnitude of the lower bound of the confidence interval and the calculated P value indicate that the strength of association for exposure to these two OR nurses was less than that for anesthesiologist 9. 

Results of procedural and laboratory investigations. The anesthesiologists had separate anesthetic carts, which when not in use were kept in the corridor outside the anesthetic office-supply room in the OR suite. Anesthetic medications for intravenous administration were routinely prepared in this corridor before surgical procedures. Propofol in 20-ml ampules was administered for induction (bolus injection) and/or maintenance (continuous infusion by a syringe pump) anesthesia. Anesthesiologists routinely opened medication ampules with ungloved hands and used either a 30-ml syringe for bolus administration of propofol or a 60-ml syringe fitted with extension tubing for continuous propofol infusion.

Molecular subtype analysis. To determine if case patient isolates were genetically related, and thus if there was a common-source outbreak, we analyzed the isolates for restriction fragment length polymorphisms (RFLPs) by using the CARE-2 probe. CARE-2 hybridizes specifically to genomic DNA of C. albicans and not to that of other Candida species (3). CARE-2 hybridized to 12 to 17 EcoRI restriction endonuclease fragments per strain, ranging in size from 23 to 1.0 kb (Fig. 1). CARE-2 hybridization profiles were identical for case patient 3 (Fig. 1, lane 3) and case patient 2 (Fig. 1, lane 4), but these clearly differed from the CARE-2 hybridization profiles observed for patients 1 and 4 (Fig. 1, lanes 5 and 6, respectively), the four nonoutbreak reference isolates (Fig. 1, lanes 7 to 10), a C. albicans isolate from a randomly chosen hospital A patient (i.e., unassociated with the outbreak) (Fig. 1, lane 1), and a C. albicans isolate from anesthesiologist 1 (Fig. 1, lane 2). The patterns of CARE-2 hybridization bands for case patient 1 and case patient 4 also were identical but were clearly different from the CARE-2 hybridization patterns observed for C. albicans isolates from case patients 2 and 3 and from those of the other isolates in the panel. To confirm this observation, genomic DNA was digested with a second restriction endonuclease, HincII. CARE-2 hybridization profiles observed for HincII-digested samples for case patients 2 and 3 and for case patients 1 and 4 were identical, but the case patient profiles were significantly different from the profiles observed for the other isolates analyzed in the panel (data not shown).

View larger version (100K): [in this window] [in a new window]   FIG. 1.   Hybridization of digoxigenin-labeled CARE-2 DNA probe to EcoRI-digested genomic DNA. Lane 1, control patient, hospital A, C. albicans isolate; lane 2, anesthesiologist 1, hand C. albicans isolate; lane 3, case patient 3; lane 4, case patient 2; lane 5, case patient 1; lane 6, case patient 4; lane 7, control 1 C. albicans isolate; lane 8, control 2 C. albicans isolate; lane 9, control 3 C. albicans isolate; lane 10, bacteriophage lambda DNA digested with HindIII used as a molecular size marker (sizes are shown on the right of the gel).

View larger version (108K): [in this window] [in a new window]   FIG. 2.   Ethidium bromide-stained gel following TAFE. Lane 1, anesthesiologist 1, hand C. albicans isolate; lane 2, case patient 3; lane 3, case patient 2; lane 4, case patient 1; lane 5, case patient 4; lane 6, control 1 C. albicans isolate; lane 7, control 2 C. albicans isolate; lane 8, control 3 C. albicans isolate.

	DISCUSSION

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The data from our investigation suggest that this unusual cluster of postsurgical C. albicans infections in patients undergoing clean and clean-contaminated surgery was due to extrinsic contamination of the intravenous anesthetic propofol. The findings of our analysis that are consistent with this hypothesis are the associations of C. albicans postsurgical infection with the receipt of propofol by the infusion pump and with preparation and administration of the infusion pump by a single anesthesiologist, anesthesiologist 9.

Cultures of this anesthesiologist's hands, performed at the time of our investigation 2 months after the case-patients' surgical procedures, were negative for C. albicans, although coagulase-negative Staphylococcus species, Aspergillus fumigatus, and Candida parapsilosis were isolated. The large proportion of anesthesiologists in this hospital colonized with Candida species, compared with rates from other hospitals, suggests that there was an increased risk of transient hand colonization with Candida species at hospital A. Other studies have shown wide variation (15 to 54%) in Candida sp. colonization on hands of health care workers (10, 11).

The likely mechanism for contamination of propofol was via the hands of anesthesiologist 9 during manipulation of the anesthetic agent. Reports from our interviews with anesthesiologists suggest that during the administration of anesthesia, aseptic technique and infection control practices were not always followed. In particular, anesthesiologist 9 was reported to prepare multiple syringes of propofol before the first surgical procedure of the day, to be used throughout the day. In addition, he reused multiple syringes for propofol administration on the same patient during the surgical procedure.

Propofol is an intravenous hypnotic anesthetic agent which received Food and Drug Administration (FDA) approval in 1989. Propofol is a sterile, nonpyrogenic, white soybean-oil-in-water emulsion to be used by intravenous delivery for induction (by bolus administration) and/or maintenance (by drip infusion) anesthesia. The product in use in hospital A had no preservative, and refrigeration was not recommended by the manufacturer. Therefore, the association of postsurgical infections with receipt of propofol by infusion could be explained by the longer duration of administration, allowing extrinsically contaminating microorganisms from the hands of the anesthesiologist to proliferate during the infusion interval. Growth studies performed at CDC showed that when propofol is inoculated with low numbers (<10¹ CFU/ml) of C. albicans, these microorganisms rapidly proliferate to high numbers at 30°C (86°F): 10¹ CFU/ml within 5 h and 10⁴ CFU/ml within 24 h (1). Since 11 June 1996, a preservative (disodium edetate [0.005%] has been added to the propofol parenteral emulsion specifically to retard the rate of growth of microorganisms in the event of accidental extrinsic contamination.

This study represents one outbreak of postsurgical infections and endotoxemia associated with extrinsic contamination of propofol investigated by CDC and the first to involve C. albicans infections. The other propofol-related outbreaks were reported from multiple states and included postsurgical infections (surgical wound or bloodstream) with different species of microorganisms (2). The investigation of these clusters of postsurgical infections suggests that patients may experience severe life-threatening complications as a consequence of breaks in aseptic technique in combination with a drug that is capable of supporting the rapid growth of microorganisms.

As a result of these investigations, the manufacturer of propofol, in conjunction with the FDA, revised the label and package inserts and sent letters to all anesthesiologists, nurse anesthetists, and registered pharmacists in the United States emphasizing the importance of using aseptic technique in the preparation and administration of propofol; an additional warning was added to inform practitioners that multidosing from single vials may result in contamination and subsequent infection and that each vial was for a single use only. Subsequently, a preservative was added to the drug formulation which acts to retard but not entirely eliminate the growth of microorganisms and to reduce the risk of nosocomial infections. Although the relative contributions of such control measures are unknown, there have been no recent outbreak reports. However, it is entirely possible that outbreaks are not reported because they indicate a failure of good infection control practices in the hospital. Propofol is very widely used in the United States and many other countries, and yet these infectious outbreaks have predominantly been reported from the United States. In addition, this is one of only two drugs to hold FDA approval for use as a sedative in intensive care unit patients, and consequently many patients may be receiving prolonged intravenous infusions of the drug, which may put them at significant risk for infection.

We applied two highly discriminatory and reproducible molecular subtyping methods, CARE-2 RFLP and electrophoretic karyotype analysis, to the case and noncase isolates in an attempt to identify a common source by determining the clonality of C. albicans that infected four patients. Identical CARE-2 hybridization profiles derived from two different restriction endonucleases, EcoRI and HincII, were observed for patients 1 and 4 and for patients 2 and 3 (Fig. 1), and these profiles were clearly different from the profiles observed for the other isolates examined. The results of our CARE-2 RFLP and electrophoretic karotype analyses were in agreement. Moreover, CARE-2 hybridization profiles for strains from patients 1 and 4 and patients 2 and 3 differed significantly, suggesting that these two strains were not minor variants from a common clone or different due to microevolution (4). One interesting epidemiological question remains: how was nosocomial transmission possible for two different strains of C. albicans, on two different days, to each of two different patients per day? This finding most likely suggests that multiple strains of C. albicans were carried on the hands of anesthesiologist 9 during the time of the outbreak and that these two strains were present in approximately equal numbers (i.e., transient monoclonal or stable polyclonal colonization). Colonization of healthy individuals by multiple strains at the same anatomic site has been reported previously (9, 13, 14). However, this is the first report of multiple-strain carriage causing nosocomial infections. The possibility of transmission by multiple exogenous sources cannot, however, be entirely ruled out, since C. albicans was not isolated from anesthesiologist 9's hand cultures. This investigation reinforces the benefits of a combined epidemiological and molecular analysis.

Our investigation emphasizes that all personnel administering anesthesia in hospitals should receive in-service training in infection control and aseptic preparation of intravenous medications and solutions. The infection control quality assurance program should include periodic observation and review of anesthesia practice. Strict aseptic technique must always be maintained during handling of propofol; however, its earlier capability of supporting the rapid growth of microorganisms may have been modified by the recent addition of preservative to the drug's formulation. Propofol should be drawn into sterile syringes immediately after the ampules are opened, and administration should then begin without extended delay. Propofol should be prepared for single-patient use only, and any unused portion should be discarded at the end of the surgical procedure.