American Journal of Transplantation 2010; 10: 2161–2167 Wiley Periodicals Inc.
C 2010 The Authors C 2010 The American Society of Journal compilation Transplantation and the American Society of Transplant Surgeons
Case Report
doi: 10.1111/j.1600-6143.2010.03216.x
Fatal Apophysomyces elegans Infection Transmitted by Deceased Donor Renal Allografts B. D. Alexandera,b,∗ , W. A. Schella , A. M. Sistonc,d , C. Y. Raoe , W. A. Bowerf , S. A. Balajeeg , D. N. Howellb , Z. S. Mooreh , J. Noble-Wange , J. A. Rhynei , A. T. Fleischauerh , J. M. Maillardh , M. Kuehnertf , D. Vikramanj , B. H. Collinsj , C. E. Marroquinj and B. J. Parkg a Department of Medicine, b Department of Pathology, Duke University Medical Center, Durham, NC c South Carolina Department of Health and Environmental Control, Columbia, SC d Epidemic Intelligence Service, Office of Workforce and Career Development, e Division of Healthcare Quality Promotion, f Office of Blood, Organ, and Other Tissue Safety, g Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA h North Carolina Division of Public Health, Raleigh, NC i New Hanover County Health Department, Wilmington, NC j Department of Surgery, Duke University Medical Center, Durham, NC *Corresponding author: Barbara D. Alexander,
[email protected]
Two patients developed renal mucormycosis following transplantation of kidneys from the same donor, a near-drowning victim in a motor vehicle crash. Genotypically, indistinguishable strains of Apophysomyces elegans were recovered from both recipients. We investigated the source of the infection including review of medical records, environmental sampling at possible locations of contamination and query for additional cases at other centers. Histopathology of the explanted kidneys revealed extensive vascular invasion by aseptate, fungal hyphae with relative sparing of the renal capsules suggesting a vascular route of contamination. Disseminated infection in the donor could not be definitively established. A. elegans was not recovered from the same lots of reagents used for organ recovery or environmental samples and no other organ transplant-related cases were identified. This investigation suggests either isolated contamination of the organs during recovery or undiagnosed disseminated donor infection following a near-drowning event. Although no changes to current organ recovery or transplant procedures are recommended, public health officials and transplant physicians should consider the possibility of mucormycosis transmitted via organs in the future, particularly for near-drowning events. Attention to aseptic technique during organ recovery and processing is re-emphasized.
Key words: Fungal infection, moulds, mucormycosis, renal allograft, renal graft loss, transplant infectious diseases Received 14 April 2010, revised 26 May 2010 and accepted for publication 08 June 2010
Introduction A spectrum of infectious agents can be transmitted with the donated graft during solid organ transplantation but mold infections are rarely reported (1–3). Transmission of invasive mucormycosis (zygomycosis) with a renal graft has never been reported in the United States and only six cases exist in the world literature (4–8).
Apophysomyces elegans is an opportunistic mold, most commonly associated with skin and soft tissue infection following contamination of traumatic wounds or burns in otherwise immunocompetent hosts (9). It is responsible for only 1% of cases of mucormycosis in solid organ transplant (SOT) recipients (10). As with other members of the Mucoromycotina, A. elegans has broad, branching hyphae and only sparse septations. It is commonly found in soil and decaying vegetation. Two patients received kidneys from a near drowning victim in a motor vehicle crash (MVC). Both recipients developed invasive A. elegans infection of the transplanted grafts with subsequent dissemination including direct extension across tissue planes. Both recipients required graft nephrectomy and one patient died. To our knowledge, this is the first report of renal mucormycosis transmitted with the donated organ in the United States. The following is a summary of these cases and investigation into the source of the infections.
Materials and Methods A traceback investigation was performed to determine possible sources of A. elegans common between the two cases. Approximately, 3 months after the MVC, medical personnel were interviewed and environmental sampling was conducted at possible locations of contamination. The United Network for Organ Sharing (UNOS) and manufacturers of organ transplantation equipment and solutions were contacted to determine if additional cases of A. elegans had been reported.
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Alexander et al. Table 1: Primers employed for inter simple sequence repeat (ISSR) typing Primer ISSR 1 (BDB(ACA)5, trinucleotide) ISSR 8 (DDB(CAG)5, trinucleotide) ISSR 12 (DDB(AACG)4, tetranucleotide) ISSR 15 (DDB(CT)8, dinucleotide)
Morphological evaluation and DNA sequencing were used to identify molds recovered to the genus and species level. Random Amplified Polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) subtyping distinguished strains. For genotyping, genomic DNA was extracted for analysis of the ITS1, ITS2 and the 28s regions of the rRNA gene as previously described (11). Four different primers were used for RAPD subtyping; primers employed for ISSR subtyping are listed in Table 1. Subtyping was performed in duplicate for five A. elegans isolates: two isolates from this study and three A. elegans isolates unrelated to this study.
Results Clinical course of donor A 17-year-old female involved in an MVC was found by emergency personnel in the over-turned vehicle with her head under water in a shallow retention ditch. She was intubated at the scene; mud was suctioned from her airway. Chest X-ray (CXR) revealed left upper lobe opacification. On hospital day (HD) 2, she developed fever to 39.1◦ C with a subsequent drop in white blood cell (WBC) count to 1600 (absolute neutrophil count between 768 and 1600) cells/mm3 of blood. Sputum grew Escherichia coli and Enterobacter amnigenus treated with cefipime. By HD 5, her WBC count had recovered and she was no longer hyperthermic but on HD 6, she developed a respiratory acidosis and hypotension. Given the overall poor prognosis, on HD 7 support was withdrawn and the organs were recovered by the donation after cardiac death protocol. Ultimately, Acinetobacter baumannii and Stenotrophomonas maltophilia, both resistant to cefipime, were recovered from endotracheal suction and blood, respectively.
Details of organ recovery Following declaration of death, the distal aorta was cannulated and VIASPAN solution (Barr Laboratories, Inc., Pomona, NY) was circulated until venous return was clear. The solution was left in the organ vasculature; each organ was then placed in a sterile plastic bag containing VIASPAN solution for further packaging and transport to the organ processing center where each kidney was flushed with Kidney Perfusion Solution-1 (KPS; Organ Recovery Systems Inc., Des Plaines, IL), placed in a separate sterile pump cassette filled with KPS-1, and placed onto a pump for continuous circulation of KPS-1 through the organ during transfer to the transplant center. 2162
Clinical course of recipient A Recipient A, a 61-year-old male, received induction R and solumedrol. The left kidney was imThymoglobulin planted into a left lower quadrant retroperitoneal pocket. After reperfusion, the transplanted kidney was pink with firm turgor. On posttransplant (PT) day 1, tacrolimus and prednisone were started. Initial low urine output and hyperkalemia necessitated hemodialysis, but resolved by PT day 5. Although he was afebrile and normoglycemic, his WBC count began to slowly rise, reaching 25 900 cells/mm3 by PT day 9. Renal vein thrombosis was diagnosed and the patient was urgently taken to the operating room where a necrotic kidney was encountered necessitating transplant nephrectomy. The renal artery was still patent and the retroperitoneal tissues appeared unremarkable. Transplant immunosuppressives were stopped; cumulatively, Recipient A had received the equivalent of 585 mg of prednisone during his PT care. Two days following explantation of the renal graft, the patient suffered a cardiopulmonary arrest. Computed tomography (CT) revealed multiple foci of air and fluid in the explanted kidney bed, lesions in the spleen and liver, a thrombosed left common femoral vein and bilateral pulmonary opacities. Aseptate, fungal hyphae, many of which were present within arterial lumens, including the main renal artery, were seen throughout the explanted renal graft with coagulative necrosis in >90% of the kidney. The organisms were difficult to visualize on hematoxylin and eosin stains, and exhibited only weak, variable staining with methenamine silver (Figure 1). Based on the extensive amount of intravascular fungus, widespread distribution within the organ, and relatively noninflamed retroperitoneal pocket and incision at the time of explantation, a vascular route of organ exposure was suspected. Recipient A was immediately started on intravenous amphotericin B lipid complex (ABLC) despite which his condition continued to deteriorate prompting re-exploration of the transplant bed. Despite multiple debridements and aggressive medical care including the addition of micafungin, the patient’s clinical condition deteriorated with refractory hypotension and metabolic acidosis and the patient died on PT day 12. Retroperitoneal pocket and abdominal tissues taken during the final debridement grew A. elegans. Autopsy revealed disseminated, angioinvasive mold involving lungs, heart, spleen, liver and intestines.
Clinical course of recipient B Recipient B, a 31-year-old female underwent transplantation at the same center by the same surgical team approximately 5.5 h after the first, but in a different operating R and solumedrol were adroom. Induction thymoglobulin ministered. The right kidney was implanted into a right lower quadrant (RLQ) retroperitoneal pocket. Following American Journal of Transplantation 2010; 10: 2161–2167
Renal Transplant Transmitted Mucormycosis
vein. ABLC and micafungin were started empirically and she was urgently taken for exploratory surgery. The transplanted kidney appeared somewhat abnormal but overall grossly viable with a patent renal artery and vein. Tissues and fascia surrounding the graft appeared normal. However, because of the clinical situation, transplant nephrectomy was performed and transplant immunosuppressive medications were stopped (cumulative prednisone equivalent dose 675 mg). Intraoperative cultures of the explanted graft and deep retroperitoneal tissues grew A. elegans. A second look retroperitoneal exploration was performed the following day. An extensive amount of dead muscle was encountered. The peritoneal layer appeared dusky and was excised thereby exposing necrotic cecum, appendix and ascending colon, all of which were resected. Her subsequent course included two additional exploratory surgeries with amphotericin B irrigation and was complicated by bacterial intrabdominal abscesses. Micafungin and ABLC were continued for 32 days after which micafungin was stopped and ABLC was continued. The patient was discharged after an 84-day hospitalization, eventually completing 140 total days (∼40 g) of amphotericin B-based therapy without evidence of recurrent infection 6 months after stopping antifungal therapy.
Figure 1: Recipient A, histopathology of explanted renal graft showing coagulative necrosis and broad, faintly staining, aseptate, fungal hyphae present within arterial lumens on (A) hematoxylin and eosin and (B) methenamine silver stains.
reperfusion, the kidney was pink and regained good turgor. Prednisone and tacrolimus were started on PT day 1. By PT day 4, she was producing approximately 100 mL of urine/h. She was discharged on PT day 5. On PT day 10, based on data accumulating from the care of Recipient A, Recipient B was asked to return to the transplant center for evaluation. She reported decline in urine output and fever onset over the preceding 24 h. Her vital signs were stable, she was normoglycemic, and the RLQ transplant incision was intact with minimal erythema and no drainage, but her WBC count was 38 700 mm3 and she was anuric. CT showed peritransplant fat stranding but no fluid collection and no pulmonary lesions. Renal ultrasound documented blood flow in the main renal artery and American Journal of Transplantation 2010; 10: 2161–2167
Mycology and environmental results The fungi recovered from both recipients grew as sterile zygomycetous molds, ultimately sporulating in water culture containing yeast extract. Based on phenotypic characteristics (Figure 2), they were identified as A. elegans. Comparative sequence based analysis of the ITS and 28s RNA regions confirmed the identity of these isolates as A. elegans. Both RAPD and ISSR subtyping methods revealed the isolates to be genotypically indistinguishable but distinct from isolates not related to this study. Laboratory records from the recovery hospital and transplant center confirmed that A. elegans had not been recovered from any clinical specimen during the preceding year.
Public health actions After identification of the potential organ-related infection in Recipient A, the transplant center notified the organ procurement organization (OPO), which in turn notified UNOS of the probable transplant transmitted infection. The OPO confirmed that no donor cultures were growing fungus, including endotracheal suction collected on HD2, HD5 and HD6 as well as blood and urine collected on HD2 and HD6. No other organs had been placed from the donor; all tissues from the donor were withdrawn from tissue banks. Transport cassettes and KPS solution from the same lots used for organ procurement were withdrawn from use. Pumps used for the right and left kidney transports had been used during the interval to transport four and one other kidney, respectively; both pumps were subsequently quarantined. 2163
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Epidemiological investigation Traceback investigation yielded numerous potential areas where common exposure to A. elegans could have resulted in contamination (Figure 3). In total, 55 environmental specimens were analyzed from the various sites identified. Zygomycetous molds were isolated from two air and one mud sample at the MVC site; however, none were identified as A. elegans (Table 2). Culture of unopened transport cassettes and KPS solution from the same lots used for procurement of the kidneys did not yield fungal growth. VIASPAN solution from the lots in use at the time of recovery was not available for testing. The clinical course of patients who received pumped kidneys at the same transplant center within 3 months of the index transplant using KPS solution or transport cassettes from the same lots was reviewed but no additional cases were identified. No additional reports of transplantation or device-related fungal infections were reported from UNOS or from any medication or equipment manufacturer. The clinical status of recipients of organs from another donor, also a near-drowning victim and occupant in the same MVC, was ascertained. This donor received his postcrash care in the same trauma center as the index donor; his organs were recovered 12 h following recovery of organs from the incident donor but in a different operating room. Right kidney, left kidney/pancreas and liver from the second donor were transplanted into recipients at other transplant centers. None of these recipients developed invasive mucormycosis.
Figure 2: Apophysomyces elegans , microscopic morphology including sporangiophores having funnel-shaped apophyses (solid arrow), pyriform sporangia (dashed arrow) and rhizoids arising opposite the sporangiophores (not shown).
Discussion Mucormycosis is an opportunistic infection, primarily associated with diabetic ketoacidosis, deferoxamine
Figure 3: Traceback investigation to determine common sources of possible contamination of the donor organs, relative to the date of the initial motor vehicle crash.
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Renal Transplant Transmitted Mucormycosis Table 2: Environmental sampling1 results
Location/equipment sampled
Number of samples collected/ tested
Motor vehicle crash site
15
Motor vehicle interior/exterior
15
Recovery hospital
15
Organ processing center
10
Reagent/equipment
1 1
Specimen type
Zygomycetous mold recovered from culture
Air Soil/mud Water Air Air/soil Air Surface Air Surface KPS solution Transport cassettes
Yes, not A. elegans2 Yes, not A. elegans2 No No No No No No No No No
1 Air
samples were collected directly onto Sabouraud Dextrose Agar with benomyl (SDA) using an SAS Super 90 multiple hole impactor (International PBI, Milano Italy). Surface specimens were collected using a sterile sponge premoistened with neutralizing buffer (3MTM Sponge-Stick, St. Paul, MN) and soil/mud and water samples were collected in sterile conical tubes then inoculated onto SDA and incubated at 42◦ C. 2 Apophysomyces elegans.
therapy, neutropenia, hematologic malignancies, high-risk newborns and trauma (12). It is a rare complication of solid organ transplantation. A Centers for Disease Control and Prevention-sponsored study documented that mucormycosis was responsible for only 2% of all invasive fungal infections (IFIs) reported among a cohort of ∼17 000 SOT recipients, including 2% of IFIs reported among kidney transplant recipients (13). Further, median time to onset for invasive mucormycosis was 312 days and it most commonly presented as pulmonary, sinus or cutaneous disease (13).
ology of invasive mucormycosis, the most likely scenario is organ contamination via an intravascular source, possibly during organ recovery and processing, but more likely from an undiagnosed infection in the donor that disseminated to the kidneys prior to organ recovery. Infection resulting from contamination of the surgical wound or external surface of the kidney is considered unlikely as the infection would have characteristically presented as a rapidly spreading cellulitis radiating from the incision (10) with evidence of a more advanced infection in tissues surrounding the graft.
Two recipients of kidneys from the same donor, a neardrowning victim in an MVC, developed invasive A. elegans infection of the transplanted grafts with subsequent dissemination. The presentation of infection was similar in both. Both developed initial symptoms on postoperative day 9, neither had visual evidence of cutaneous mucormycosis of the surgical incision or the graft pocket at the time of graft nephrectomy, and the patterns of tissue involvement suggested a mixture of vascular spread and direct extension across tissue planes. The A. elegans isolates recovered from the recipients had genotypically indistinguishable fingerprinting patterns. Although the genotypic diversity of A. elegans is unknown, the genotypes of several A. elegans unrelated to this study were distinct from the isolates recovered in this study. Taken together, this finding indicates that both recipients were infected by the same A. elegans isolate.
Intravascular contamination of organs with mold during the preservation phase of renal grafts has been previously described with Aspergillus (1). In the current cases, equipment and reagents from the same lots used to preserve and transport the organs were tested and were sterile. Perhaps even more importantly, no infections occurred in recipients transplanted at the same center, recipients of organs recovered from another donor on the same day and in the same hospital as the index donor, and recipients of organs recovered and transplanted at other centers using reagents and equipment from the same lots. These factors suggest that a product contamination is unlikely.
The investigation did not pinpoint a definitive source for the A. elegans infections. However, other zygomycetous molds were recovered from the MVC site, indicating the environment at the site supports the growth of zygomycetous molds. The yield from the environmental sampling may have been limited due to the 3 months that elapsed between the MVC and the investigation. Based on the clinical presentations of the recipients and known pathophysiAmerican Journal of Transplantation 2010; 10: 2161–2167
No definitive conclusions can be drawn regarding possible disseminated mucormycosis in the donor at the time of recovery. In this scenario, dissemination from the lung would have required fungal hyphae to travel via the blood for distal inoculation of both kidneys. Although the donor was a near fresh-water drowning victim with mud recovered from her airways, radiographic evidence consistent with pneumonia, and probable sepsis on the last day of life, this was easily explained by the bacterial infections that were not covered by her antibiotic regimen. Further, respiratory samples collected from the donor premortem did not grow a mold and her radiographic study at the time of clinical deterioration was read as stable compared to the 2165
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preceding several days. Disseminated mucormycosis arising from lungs would have been expected to be present with worsening pulmonary opacification and extension into adjacent tissues.
Funding note: This work is supported by NIH.
Awareness of the possible infection in Recipient B resulted in aggressive surgical and medical intervention, which was ultimately life-saving. Unlike many pathogenic fungi which react strongly with histochemical stains, members of the Mucoromycotina are often difficult to detect. Pathologists must maintain a high index of suspicion for IFIs, especially in recipients with clinical signs and symptoms compatible with an infectious process. Moreover, early antifungal therapy for mucormycosis is critical. A retrospective review of hematology patients with mucormycosis revealed significantly higher mortality with delayed amphotericin B (AMB) administration (14). Although most experts now consider a lipid formulation of AMB to be first line therapy for mucormycosis (15), posaconazole has demonstrated promising activity for this infection (16). However, the lack of an intravenous formulation, moderate variability in absorption (17) and preclinical data suggesting less efficacy compared to AMB (18) has limited the use of posaconazole as first-line therapy for mucormycosis. The decision to use posaconazole as sequential therapy for patients who have responded to an initial course of lipid AMB must be individualized based on the infecting pathogen and pharmacokinetics in the patient. In our patient, we were not able to demonstrate acceptable systemic posaconazole blood concentrations despite aggressive attempts to maximize absorption.
The findings and conclusions in this presentation/report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention. This information is distributed solely for the purpose of predissemination peer review under applicable information quality guidelines. It has not been formally disseminated by the Centers for Disease Control and Prevention. It does not represent and should not be construed to represent any agency determination or policy.
In summary, components of the care process for these two kidney transplant recipients were closely examined. Although no definitive source of the A. elegans was established, the organs appear to be the most likely source. Importantly, no other patients were infected and there did not appear to be a larger public health risk. No changes in the current organ procurement or transplant procedures can be recommended based on the results of this investigation, although attention to aseptic technique during organ procurement and processing is re-emphasized. Further, when any organ-transmitted infection is suspected, rapid communication among physicians, transplant centers, organ procurement organizations, UNOS and public health authorities is imperative (19). In cases of renally transmitted mucormycosis, initiation of AMB-based antifungal therapy, immediate removal of the infected graft, close monitoring over several days for progression across tissue planes and aggressive surgical debridement are key to survival (10,20).
Acknowledgments The authors thank Dr. D.A. Sutton, University of Texas Health Science Center, San Antonio, TX, for kindly providing three A. elegans isolates unrelated to this study for subtyping and comparison.
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Disclaimer
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. B.D.A. has received investigator initiated research funding from Pfizer and Astellas pharmaceutical companies.
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