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Cross-Canada Spread of Methicillin-Resistant Staphylococcus aureus via Transplant Organs Lynn Johnston, Linda Chui, Nicholas Chang, Sheila Macdonald, Margaret McKenzie, William Kennedy, David Haldane, Robert Bethune, Geoff Taylor, Martha Hanakowski, and Gregory Tyrrell
From the Queen Elizabeth II Health Sciences Centre, the IWK-Grace Health Centre for Children, Women, and Families, and the Departments of Medicine, Pathology and Laboratory Medicine, and Pediatrics, Dalhousie University, Halifax, Nova Scotia; and Provincial Laboratory of Public Health, University of Alberta Hospital Microbiology Laboratory, Department of Laboratory Medicine and Pathology, Division of Infection Control, and Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
Infectious complications represent a significant cause of morbidity and deaths among transplant recipients. The potential sources of pathogens are several [1, 2]. Transplant recipients are at risk for acquiring organisms that may be responsible for active or latent infection in the donor at the time of organ retrieval. Organs and tissues may become microbially contaminated during harvesting, processing, preservation, and transportation. Transplant recipients are also at risk of reactivation of their own latent infections. Very frequently it is difficult, if not impossible, to determine the origin of the infectious agent: the donor, exogenous sources, or the latently infected recipient. However, it has been suggested that donor-to-recipient transmission of bacterial infection most commonly is due to organ/tissue contamination during processing and preservation [3]. Recently, molecular techniques have enabled us to determine the relatedness of bacterial isolates and whether the bacteria cultured as part of an epidemiological investigation are derived from a common source [4]. Staphylococcus aureus has been documented as a bacterial contaminant of donor kidneys and corneas [3, 5, 6] and has been linked to infection of kidney and cornea recipients [6, 7].
Received 1 December 1998; revised 17 May 1999. Reprints or correspondence: Dr. Lynn Johnston, QEII Health Sciences Centre, Room 5014 ACC, 1278 Tower Road, Halifax, Nova Scotia, B3H 2Y9 (
[email protected]). Clinical Infectious Diseases 1999;29:819 –23 © 1999 by the Infectious Diseases Society of America. All rights reserved. 1058 – 4838/99/2904 – 0016$03.00
Methicillin-resistant S. aureus (MRSA), although an increasingly frequent and serious nosocomial pathogen, has been reported to be the cause of donor-to-recipient infection only once [8]. Microbial contamination of donor organs and tissues appears to occur considerably more commonly than subsequent recipient infection [1, 2]. When kidney recipients have developed transplantation-transmitted bacterial infection, the outcome has tended to be poor, with death or transplant nephrectomy occurring in the vast majority of cases [1]. This study describes cross-country donor-to-recipient transmission of MRSA, confirmed by molecular typing of the donor and recipient isolates. Background On 10 February 1997 the Infection Control Department at the Queen Elizabeth II Health Sciences Centre (QEII), in Halifax, Nova Scotia, was notified by its microbiology laboratory of an MRSA-positive culture from a donor’s corneal ring. On 3 February 1997 the Infection Control Department at the University of Alberta Hospital (UAH; Edmonton, Alberta, Canada) had been notified by its microbiology laboratory of an MRSA-positive culture of blood that had been obtained immediately postoperatively (on 30 January) from a liver transplant recipient. Investigations were undertaken to determine the source of the MRSA and whether nosocomial transmission had occurred. Organ and tissue retrieval from the donor had taken place at the IWK-Grace Health Centre for Children, Women, and Families (IWK), also in Halifax. The kidneys and corneas were transplanted in Halifax and the liver was sent to the UAH.
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We report our investigation of the transmission of methicillin-resistant Staphylococcus aureus (MRSA) through transplantation. The kidneys, liver, and corneas were harvested from a child who died in Nova Scotia. Several days postmortem it was learned that culture of a premortem endotracheal tube aspirate from the donor yielded MRSA. Both kidneys were transplanted into a child in Nova Scotia and the liver into a child in Alberta. Both recipients subsequently became blood culture–positive for MRSA. One corneal ring from the donor was MRSA-positive. All four MRSA isolates were mecA-positive by polymerase chain reaction (PCR). The relatedness of the MRSA isolates was examined by restriction fragment length polymorphism (RFLP) analysis, a 16S–23S ribosomal PCR typing method, and comparison of antibiograms. Results were identical for all four MRSA isolates. These findings indicate that MRSA from the donor was transferred to recipients during implantation of harvested organs in Alberta and Nova Scotia, a cross-Canada spread.
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Materials and Methods
MRSA target DNA was extracted by means of standard procedures. ITS-PCR was performed as previously reported [11, 12]. Amplification was carried out with an automated thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). All amplicons were analyzed on a 1.5% agarose gel. In preparation of samples for PFGE, bacteria were embedded in 1.5% low-melting-point (LMP) agarose [13]. The agarose plugs were lysed in a lysostaphin lysing solution (0.25 mol of EDTA [pH, 9.0], 1% lauroylsarcosine, and lysostaphin [10 mg/mL]) for 30 minutes at 37°C. The plugs were then incubated in ESP solution (0.5 mol of EDTA [pH, 9.0], 0.5% lauroylsarcosine, and proteinase K [0.5 mg/mL]) for 24 hours at 50°C. After this, the plugs were rinsed twice with TE buffer containing 1 mM of phenylmethylsulfonylfluoride, followed by three washes in TE buffer. They were then preincubated in Sma I restriction buffer (Gibco BRL, Burlington, Ontario, Canada) for 35 minutes, after which time the buffer was removed and replaced with 100 mL of fresh buffer containing 20 units of Sma I (Gibco BRL). Digestion was carried out at 25°C for 1.5 hours. Plugs were then subjected to PFGE with use of 1% agarose and the CHEF-DRIII system (BioRad Laboratories, Mississauga, Ontario, Canada) with an initial ramping time of 5 seconds and a final ramping time of 25 seconds for 24 hours at 10°C. Results Organ and tissue retrieval and transplantation took place over a 6-day period between 29 January and 3 February 1997. Donor. A 23-month-old girl was transferred from her local hospital to the IWK on 26 January 1997 with severe ischemic encephalopathy secondary to foreign body aspiration. She was stabilized at her local hospital and transported by air within 12 hours. At the time of intubation she was noted to reflux gastric contents and aspirate. Chest radiography revealed bilateral pneumothoraces, pneumomediastinum, and progressive pulmonary infiltrates, felt due to pulmonary hemorrhage. She continued to do poorly neurologically and was pronounced braindead on 29 January. Liver, both kidneys, and both corneas were then harvested for transplantation. Throughout her hospitalization she was afebrile and infection was not suspected clinically. However, a blood culture performed 27 January later yielded Klebsiella pneumoniae (reported 30 January), and an endotracheal tube aspirate culture done 29 January yielded both K. pneumoniae and MRSA. She had previously been in good health, with no prior hospitalizations. She was an only child whose parents were not employed in the health care field. Organ recipient number 1. A 14-year-old girl from New Brunswick with chronic renal failure secondary to glomerulosclerosis received both kidneys. She had been dialysisdependent since 1995, and most recently her condition had been managed with hemodialysis. She was admitted to the IWK on 29 January and underwent transplantation several hours later, on 30 January. Several days previously, she had
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Epidemiological investigation. The charts and microbiological data of the organ donor and the four recipients of organs and tissue (liver, kidneys, and 2 corneas) were reviewed. The donor MRSA isolate and MRSA isolates from the kidney and liver recipients’ blood and from the donor corneal ring were compared at a phenotypic level (antibiograms) as well as at a genotypic level to determine genetic relatedness. In addition, a strain (Halifax A) that caused an MRSA outbreak at the QEII the previous year, another Halifax MRSA strain (Halifax B), two MRSA strains (Aberdeen A and B) from another area of Nova Scotia, and two strains from the UAH (UAH A and B) were subjected to the same assays to assess relatedness. It had initially been questioned whether the donor had been resuscitated at the hospital where Aberdeen strains A and B had been isolated. This possibility was later excluded, and it was determined that there were no epidemiological links between the donor and that area of the province. To determine if medical staff–to-patient spread of MRSA had occurred at the UAH, six members of the surgical transplantation team were screened for MRSA carriage, as were seven patients sharing the same preoperative patient care unit. At the IWK, cultures of specimens from patients sharing the same intensive care unit as the donor and recipient were monitored for MRSA. Bacterial strains. MRSA isolates were collected from the kidney and liver transplant recipients’ blood cultures, a donor corneal ring culture, and the donor’s endotracheal tube aspirate. In addition, as stated in the previous section, two MRSA isolates circulating in Halifax, two from another area of Nova Scotia, and two from the UAH were collected for genotype comparisons with the donor and recipient strains. The isolates were demonstrated to be oxacillin-resistant with use of Mueller-Hinton agar with 4% NaCl and oxacillin (6 mg/L), as per National Committee for Clinical Laboratory Standards (NCCLS) guidelines [9]. This was also confirmed by amplification of the mecA gene with use of PCR as previously described [10]. The template for PCR was prepared as follows. Colonies growing on a blood agar plate were scraped off and resuspended in lysis buffer (100 mM of NaCl, 10 mM of Tris-HCl [pH, 8.3], l mM of EDTA [pH, 9.0], and 1% Triton X-100 [Sigma Chemical, St. Louis, MO]). The suspension was boiled for 10 minutes and then subjected to centrifugation at 16,000g for 10 minutes. Approximately 2 mL of the supernatant was used as template in a 50-mL PCR reaction [10]. The susceptibilities of the MRSA isolates to cefazolin, oxacillin, ciprofloxacin, clindamycin, erythromycin, trimethoprim-sulfamethoxazole, and vancomycin were determined according to NCCLS guidelines, with use of disk diffusion [9]. Molecular typing. The MRSA isolates were typed at the molecular level by intergenic spacer PCR (ITS-PCR) and restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). For ITS-PCR, the
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Figure 1. Intergenic spacer (ITS)–PCR profiles of methicillinresistant Staphylococcus aureus (MRSA) strains. Following PCR amplification, 5 mL of amplicon was applied to a 1.5% agarose gel for electrophoretic separation. Lane 1 contains the donor MRSA strain; lane 2, the donor corneal ring strain; lane 3, the liver recipient strain; lane 4, the kidney recipient strain; lane 5, Aberdeen A strain; lane 6, Aberdeen B strain; lane 7, Halifax A strain; lane 8, ATCC 33591 S. aureus; and lane 9, a 1-kb ladder. The donor and recipient MRSA isolates and the Aberdeen A isolates are identical in their ITS-PCR profiles, whereas the Aberdeen B and Halifax A MRSA isolates are not. S. aureus ATCC 33591 was used as a positive control.
to ciprofloxacin, clindamycin, erythromycin, trimethoprimsulfamethoxazole, and vancomycin. The remaining strains (Halifax A and B, Aberdeen A and B, and UAH A and B) were susceptible only to trimethoprim-sulfamethoxazole and vancomycin. The susceptibility data alone suggest that the donor and recipient MRSA strains were not related to any MRSA strains circulating in the hospitals in question. Molecular typing. ITS-PCR and RFLP analysis were used to type the isolates at a molecular level. Both typing methods identified the donor and recipient isolates as identical. ITSPCR did not reveal a difference between Aberdeen A and the outbreak strains. Aberdeen B was clearly different (figure 1). However, the RFLP data analysis indicated that the Aberdeen A strain and the donor/recipient strains were possibly related if the guidelines for interpreting RFLP patterns for PFGE, as published by Tenover et al. [14], are used (figure 2). There are five band differences between donor/recipient strains and the Aberdeen A strain by PFGE. This would suggest ITS-PCR is not as discriminatory a typing method for MRSA as PFGE. In addition, RFLP analysis clearly shows that the two Halifax strains and the two UAH strains are not related to the donor/recipient MRSA (figure 2).
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been bacteremic with methicillin-susceptible S. aureus, and on admission she was receiving cephalexin. Postoperatively she received cefotaxime (as it was now known that the donor had been bacteremic with K. pneumoniae prior to death) and vancomycin for her previous S. aureus infection. Immediately postoperatively, blood cultures were obtained. There was no documentation of why these cultures were performed. One of these three sets was positive for MRSA. Many subsequent blood cultures were negative, and no focus of infection other than the bloodstream was identified. She received cefotaxime for 8 days and vancomycin for 16 days. Follow-up cultures of specimens from the axilla, groin, nares, and perineum were all negative for MRSA. Organ recipient number 2. A 6-month-old boy was admitted to the UAH in November 1996 for treatment of biliary atresia. Nasal screening cultures of the patient were negative for MRSA on admission and in December 1996. On 30 January the patient underwent transplantation of the donor liver from Halifax. A blood culture performed at the end of the procedure was reported positive for MRSA 2 days postoperatively, and vancomycin therapy was initiated. Subsequently, the patient developed multiple abscesses involving pleural, pericardial, and intraabdominal sites, from which MRSA was grown. He required several drainage procedures and prolonged antimicrobial therapy. Cornea recipients. A 25-year-old man from New Brunswick underwent cornea transplantation on 3 February at the QEII and was discharged from the hospital the following day. A culture from the donor’s corneal ring was positive for MRSA. Surveillance cultures of specimens from this recipient (nares, axilla, groin, and rectal) were all negative. The second cornea was transplanted into a 39-year-old woman from Nova Scotia. The culture from this donor ring was negative for MRSA. Follow-up surveillance cultures were not done for this recipient. Both cornea recipients received subconjunctival injections of cefazolin and tobramycin intraoperatively and tobramycin eyedrops postoperatively. Neither had infectious complications related to the corneal transplant. Contact tracing. Neither of the hospitals involved in the solid organ transplants has endemic MRSA. The UAH handles, on average, ,20 sporadic cases per year, and the IWK, ,10 cases per year. There was an outbreak of MRSA the previous year at the QEII (where the cornea transplantations were performed), but ophthalmology patients were not involved, and this facility also did not have endemic MRSA. There were no MRSA carriers identified among patients or health care workers who were screened. Bacterial strains. All donor and recipient S. aureus isolates were mecA-positive, as determined by PCR directed toward the mecA gene. The isolates had a similar antimicrobial susceptibility pattern, different from that of the QEII outbreak strain of the previous year. All strains were resistant to cefazolin and oxacillin. The donor and recipient strains were susceptible
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Discussion This study illustrates several interesting features relating to infectious complications arising directly from transplantation. It conclusively documents donor-to-recipient transmission of a bacterial pathogen through kidney and liver transplantation, confirming that this represents a real although unquantified risk to recipients. Such transmissions may occur more commonly than has been recognized. A recent report describes donor-to-recipient transmission of MRSA through heart transplantation [8]. It may be that these infections would not have been recognized as donor-related had more commonly seen pathogens been isolated. Since MRSA was not endemic to the three involved facilities (UAH, QEII, and IWK) during the time of the transplantation, its isolation prompted consideration that the transmissions were from a contaminated donor source. Thus, the donor as source should always be considered when bacterial infection occurs early in the transplant period. If donor-to-recipient transmission is more common than previously appreciated, strategies for its recognition and opportunities for prevention will have to be developed. Preventive strategies are important both from a patient care perspective and for infection control, especially when antimicrobial-resistant pathogens are at issue. However, it is not clear what the most cost-effective or feasible method of screening would be. Given the time constraints involved in organ transplantation, delays to await culture results are not possible. Screening on
clinical grounds would have missed this donor’s infection, as infection was not suspected premortem; nor was MRSA colonization suspected in the donor before organ retrieval. MRSA was and is not known to be endemic in the area where the donor lived. Furthermore, MRSA carriage in a healthy toddler is considered quite unusual. Screening for MRSA colonization would not, therefore, be routine in this situation. As other antimicrobial-resistant bacteria are identified, they too would have to be considered as organisms that could possibly be spread through transplantation. The benefits of routine culture to identify potential bacterial colonization of donor organs have not been demonstrated. This clearly represents an area where more data are required. Whether routine bacterial culture screening of all donors is justified in areas of low endemicity as a preventive measure remains to be determined. There was a delay in notifying the recipients’ institutions of the donor’s MRSA colonization. It is not clear that this information would have altered the outcome of these transmissions. However, in situations where recipients develop infectious complications and culture results are negative, this might prove to be very useful information upon which empirical antimicrobial therapy may be based. Microbiology laboratory staff members, infection control personnel, and transplant coordinators must work together to develop strategies to notify recipient facilities when donors are found to be infected or colonized with significant organisms. This outbreak is reflective of the spectrum of potential outcomes following donor-to-recipient transmission of bacterial pathogens. The cornea recipient had no clinical sequela despite not receiving specific antimicrobial therapy and did not become infected or colonized with MRSA. The kidney recipient fortuitously received “preemptive” therapy with vancomycin, beginning within hours of transplantation and before her positive blood culture results were available. She likewise had no morbidity related to receipt of a contaminated organ. The liver recipient developed disseminated staphylococcal infection complicating his transplantation. MRSA was not identified until 2 days postoperatively, and thus vancomycin administration was unavoidably delayed. The earlier and fortuitous administration of vancomycin in the kidney recipient may have prevented the serious infection experienced by the liver recipient. The literature is scant but would suggest that the outcome of S. aureus donor-to-recipient-transmitted infection is poor in cases of solid organ transplantation. Doig et al. reported death following transplant nephrectomy in one recipient and transplant nephrectomy in the second in which such infection occurred [7]. Coll et al. reported the death of a heart recipient due to fulminant MRSA myocarditis; in this case the donor had MRSA bacteremia [8]. Our data suggest that the course of these infections may be modified by early antimicrobial therapy, as was demonstrated in the kidney recipient.
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Figure 2. Restriction fragment length polymorphism (RFLP) analysis of MRSA strains by pulsed-field gel electrophoresis. Lane 1 contains molecular size markers as indicated; lane 2, the donor MRSA strain; lane 3, the donor corneal ring strain; lane 4, the liver recipient strain; lane 5, the kidney recipient strain; lane 6, Aberdeen A strain; lane 7, Aberdeen B strain; lane 8, Halifax A strain; lane 9, Halifax B strain; lane 10, UAH A strain; lane 11, UAH B strain; and lane 12, molecular size markers as indicated. Only the donor and recipient strains are identical in their RFLP profiles.
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References 1. Gottesdiener KM. Transplanted infections: donor-to-host transmission with the allograft. Ann Intern Med 1989;110:1001–16. 2. Kakaiya R, Miller WV, Gudino MD. Tissue transplant–transmitted infections. Transfusion 1991;31:277– 84. 3. Spees EK, Light JA, Oakes DD, Reinmuth B. Experiences with cadaver renal allograft contamination before transplantation. Br J Surg 1982;69: 482–5.
4. Tompkins LS. Current concepts: the use of molecular methods in infectious diseases. N Engl J Med 1992;327:1290 –9. 5. Bijnen AB, Weimer W, Dik P, Oberop H, Jeekel J. The hazard of transplanting contaminated kidneys. Transplant Proc 1984;16:27– 8. 6. Escapini H Jr, Olson RJ, Kaufman HE. Donor cornea contamination with McCarey-Kaufman medium preservation. Am J Ophthalmol 1979;88: 59 – 62. 7. Doig RL, Boyd PJR, Eykyn S. Staphylococcus aureus transmitted in transplanted kidneys. Lancet 1975;2:243–5. 8. Cole P, Montserrat I, Ballester M, et al. Epidemiological evidence of transmission of donor-related bacterial infection through a transplanted heart. J Heart Lung Transplant 1997;16:464 –7. 9. National Committee for Clinical Laboratory Standards. Approved standard M7-A3. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova, Pennsylvania: National Committee for Clinical Laboratory Standards, 1995. 10. Kobayashi N, Wu H, Kojima K, et al. Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction. Epidemiol Infect 1994;113:259 – 66. 11. Jensen MA, Webster JA, Strauss N. Rapid identification of bacteria on the basis of polymerase chain reaction–amplified ribosomal DNA spacer polymorphisms. Applied Environ Microbiol 1993;59:945–52. 12. Tyrrell GJ, Bethune RN, Willey B, Low DE. Species identification of enterococci via intergenic ribosomal PCR. J Clin Microbiol 1997;35: 1054 – 60. 13. Chang N, Chui L. A standardized protocol for the rapid preparation of bacterial DNA for pulsed-field electrophoresis. Diagn Microbiol Infect Dis 1998;31:275–9. 14. Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. 15. Kumari DNP, Keer V, Hawkey PM, et al. Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains. J Clin Microbiol 1997;35:881–5.
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This report suggests greater specificity of RFLP analysis by PFGE in comparison to ITS-PCR. ITS-PCR of the donor/ recipient MRSA strains resulted in an identical pattern to that of the Aberdeen A MRSA strain. This apparent genetic relatedness was not associated with an epidemiological link between the donor and the area where these isolates were obtained. However, RFLP analysis demonstrated that the donor/ recipient MRSA strain pattern was not identical to that of the Aberdeen A strain. Close examination of the PFGE gel (figure 2) shows that there are five band differences between the strains. Previous investigators have found ITS-PCR (referred to as RS-PCR in their work) to be not as discriminatory as PFGE [15]. However, ITS-PCR can aid in determining if an outbreak of MRSA is occurring in a particular situation, as all MRSA connected to that outbreak should have identical ITS-PCR profiles. In summary, this report highlights the potential for wide geographic spread of multiresistant bacterial pathogens through organ and tissue transplantation. As organs are increasingly transported across borders, this potential will grow and possibly introduce MRSA into areas of nonendemicity.
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