Medical Mycology August 2004, 42, 373 /378

Case Report

Wound infection due to Absidia corymbifera and Candida albicans with fatal outcome
*, S. HERFF*, G. MARKLEIN*, H. ZHOU$, I. HEINZE%, G. S. DE HOOG, R. RU *, B. JOVANIC CHEL & R. HORRE K. P. SCHAAL* Institutes for *Medical Microbiology and Immunology, $Pathology, %Department of Anaesthesiology, University of Bonn, Bonn, Department of Bacteriology, University Hospital, Go ttingen, Germany and Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands

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A case of a mixed infection due to Candida albicans and the zygomycete Absidia corymbifera in a 38-year-old, previously healthy, Caucasian male is presented. The infection developed following serial rib fractures, and ruptures of kidney, liver and biliary tract as well as a pancreatic contusion resulting from a traffic accident. During intensive care treatment the patient underwent several surgical procedures but subsequently experienced multi-organ failure and sepsis. Some weeks later, fungal growth was observed macroscopically on the patients skin and wounds. From wound swabs C. albicans and A. corymbifera were grown. Histopathology of abdominal tissue yielded pseudohyphae and coenocytic hyphae. Although surgical debridement and antifungal treatment with amphotericin B and 5-flucytosine were started immediately, the patient died in therapy-refractory septic multi-organ failure. Keywords Absidia corymbifera , Candida albicans , zygomycosis

Introduction
During the past 10 years, fungal infections have gained considerable medical importance, particularly in patients with severe underlying diseases. While Candida species were previously the predominant causative agents of invasive mycoses, in recent years opportunistic moulds, such as Aspergillus species or other hyphomycetes, and zygomycetes, have increasingly been implicated in human infection. In contrast to the yeasts, several of these moulds show a high degree of resistance to most antifungal drugs [1]. The yeast Candida albicans occurs worldwide as a common colonizer of mucosal sites in humans and warm blooded animals. With severe underlying illness it may affect any organ by continuous or haematogenous

spread. Clinical symptoms are usually non-specific [2]. Absidia corymbifera is a member of the class Zygomycetes , order Mucorales . It can be isolated from soil, plants, and air. Rhinocerebral mycosis due to this mould is the most common clinical manifestation, but gastrointestinal, pulmonary, cutaneous, and disseminated infections have also been reported [2]. These infections are characterized by vascular invasion, thrombosis, and tissue necrosis [3 /6]. The disease is usually fulminant and has a high mortality rate [7]. Complete debridement of the afflicted tissue forms the basis of an effective treatment of zygomycosis [8 /10].

Case history
A 38-year-old, previously healthy, Caucasian man suffered from multiple traumata after a bike accident. He was hospitalized in an intensive care unit of a peripheral hospital because of scapula and serial rib fractures, right-sided kidney, liver and biliary tract ruptures, and pancreatic contusion as a consequence
DOI: 10.1080/1369378032000141426

Received 1 June 2003; Accepted 4 August 2003 Correspondence: Regine Horre , Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger Allee 3, D-53175 Bonn, Germany. Tel: '/49 228 207 3267; E-mail: horre@bfarm.de

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of blunt abdominal and thoracic injury. No wounds could be observed on the patients head, neck or extremities. The patient rapidly developed haematothorax and was subjected to abdominal and thoracic surgery, including right-sided nephrectomy. One week later, he was transported to the Department of Anaesthesiology, University of Bonn, Germany, because of suspected abdominal bleeding, signs of multiorgan failure and sepsis. The patient was intubated, put on catecholamines and treated for pain. Intravenous antibiotic therapy was started with ciprofloxacin (0.4 g/ day) and piperacillin/combactam (4.0/1.0 g/day). Bleeding of the liver ceased, but 8 days after accident, the patient developed an intra-abdominal compartment syndrome and a leakage of his biliary tract. Repeated abdominal surgery followed, whereby the abdomen had to be kept open because of elevated intra-abdominal pressure due to retroperitoneal haematoma and swelling of the intra-abdominal organs. Therefore, daily surgical wound revision followed, after which the wound was covered with sterile organic foil (Sterile Vi-Drape Isolation Bag; Medical Concepts Development, Woodbury, USA) and rinsed daily with sterile physiological NaCl. From abdominal wound swabs as well as from tracheal secretions Escherichia coli and an Enterococcus species were isolated first, but 10 days after the accident, C. albicans was cultured from a tracheal secretion; 2 days later (12 days after the

accident), C. albicans and A. corymbifera were grown from an abdominal swab. Amphotericin B in liquid glucose solution (40 mg/500 ml) was then used for rinsing twice daily. On day 16 after the accident, A. corymbifera was also observed from a drainage catheter and fungal growth could be seen macroscopically on the patients abdominal wound and on the surface of abdominal organs, which showed signs of necrosis (Figs. 1 and 2). The situation was further complicated by the development of pulmonary infiltrates. Tracheal secretions now turned sanguineous and purulent. Although an intravenous antimycotic treatment with amphotericin B (initially 40 mg/day; then 80 mg/day) and flucytosine (2 g/day) was started 14 days after the accident in addition to daily surgical debridement, the patient died in therapy-refractory multi-organ failure 4 days later, 18 days after the accident. Several tissue specimens were subjected to histopathological and microbiological examination. Clinicochemical infection markers during disease showed elevated leukocyte counts (day 1, 8.2; day 17, 29.5 mm (3 [normal, 4.3 /10.5 mm (3]) and C-reactive protein values (day 1, B/1.0 mg/ml; day 7, 17.4 mg/ml; day 17, 21.2 mg/ml [normal, B/10.0 mg/ml]).

Histopathology
Microscopy of skin, soft tissue and muscles from the patients abdomen stained with Gomoris methenamin-

Fig. 1 The patients abdominal site in a case of wound infection due to Absidia corymbifera and Candida albicans .

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Fig. 2 Macroscopically visible fungal growth at the margin of the patients wound.

silver stain (GMS) showed invasive pseudohyphae suggesting a yeast-like fungus, in addition to broad, irregularly branched, non-septate hyphae, characteristic of zygomycetes (Fig. 3). In samples from the surface of the wound sporangia could be seen (Fig. 4).

Microbiology
Fungal isolation and identification
During the patients intensive care treatment, tracheal secretions, wound fluids, tissue specimens and blood-

Fig. 3 Tissue section showing pseudohyphae from the yeast Candida albicans in addition to coenocytic hyphae from the mould Absidia corymbifera (GMS )/100).

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Fig. 4 Tissue sections from the surface of the wound showing sporangia of Absidia corymbifera (GMS )/100).

cultures (Becton Dickinson, Germany) were sent for microbiological examination. For fungal culture CandiSelect agar (CSA; BioRad, Germany) (2 days at 378C) and Sabourauds glucose agar (SGA) (10 days at 308C) were used. Fungal growth was observed on SGA and CSA after incubation for 2 /3 days. No growth was observed from the blood cultures, but abdominal wound secretions yielded C. albicans as well as A. corymbifera . The yeast grew in typical colonies on SGA, with blue colour on CSA, which is indicative of C. albicans . With the API 32C Identification System (Api Biome rieux, France) the identification as C. albicans was confirmed. The mould was woolly and whitish-grey in colour; the reverse of the culture was whitish. It was identified by morphological criteria and thermotolerance at 508C, as described by De Hoog et al . [2]. A. corymbifera was deposited in the culture collection of the Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, as CBS 112528.

Discussion
Infections due to C. albicans currently are common diseases in intensive care patients. Probable risk factors of the patient described in this article may have been the antibacterial therapy and the prolonged abdominal surgery as well as the leakage of gastrointestinal contents. The aetiological agent mostly is one of the patients indigenous yeasts colonizing the respiratory and gastrointestinal tracts.

The situation in infections due to zygomycetes is different. In older literature [6,11,12] most described cases concerned colonisation of nasal sinuses, eventually leading to direct or vascular invasion of the brain. Major risk factors for such rhinocerebral mycoses are ketoacidosis as a consequence of diabetes mellitus or alcohol abuse and deferoxamine therapy for iron overload [11,12]. More recently, zygomycosis is increasingly seen in haematological malignancies [5,13]. Most of the infections are pulmonary or disseminated [6]. Localized infections after traumatic inoculation of the fungi have also been observed [14]. Clinical manifestation depends on the route of infection [3]. In the case described here, nothing is known about the previous fungal colonization of the patients mucosal surfaces and there was no indication of diabetes mellitus, alcohol abuse, or traumatic inoculation of the zygomycete. The patient had no damage of the skin; all traumatic lesions were located subcutaneously. Inoculation of A. corymbifera therefore might have occurred during or after surgery, possibly when the abdomen was left open after the compartment syndrome. Immunocompromized patients undergoing major surgery are at an increased risk, as infectious sporangiospores may spread nosocomially by various means [4,15,16]. Co-infections with other microorganisms may also be a predisposing factor for infection with zygomycetes [14,16 /18]. The major zygomycetes causing infections in humans belong to the genera Rhizopus , Rhizomucor, Mucor,
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and Absidia , but a variety of members of other genera have occasionally been reported [4]. It has been assumed that the first described human case of zygomycosis, from the 1800s, was due to A. corymbifera [4]. This fungus grows more rapidly at 378C than at 258C and tolerates temperatures up to 528C, which distinguishes it from other Absidia species [2,19]. Thermotolerance may be an important virulence factor for human infection. Cutaneous infections with A. corymbifera can be visible as grey-black plaques that rapidly increase in size over a 24-h period [14]. In the patient described here, fungal growth was visible macroscopically, mainly at the margin of the patients wounds, where local necrosis and acidosis may have favoured fungal development. Arterial invasion and thrombosis is a common condition in zygomycosis and can result in ischaemia and gangrene [20]. Conditions favourable to germination of A. corymbifera include low pH, high glucose content [21], increased iron [22] and decreased phagocytic defence [23]. The angioinvasive property of these fungi is another virulence factor that may be responsible for high lethality [7]. In our patient, the infection can only be assumed as hospital acquired. Cutaneous zygomycosis is a rare but serious infection in trauma patients with wounds contaminated by environmental debris or soil. In this case, the patient was traumatised, but fungal inoculation during the accident was unlikely, because there was internal damage only. For the definite diagnosis of zygomycosis, the detection of the non-septate hyphae in tissue sections and confirmation by culture is necessary [24]. Neither serological tests nor PCR-based specific tools are commercially available. In this patient, the first isolate of A. corymbifera was judged as fungal contamination. Daily surgery and twice daily rinsing with amphotericin-B-containing solution were performed until further microbiological probes and histopathology confirmed the infection due to A. corymbifera . Four days later, the patient died due to multiorgan failure; a change of therapeutic regimen was being discussed. Treatment of infections due to A. corymbifera is difficult; in -vitro test results often do not correlate with in -vivo sufficiency. In -vitro synergy of amphotericin B with rifampicin has been shown [25,26] but proved to be ineffective in a 3-year-old boy with haematological neoplasia who suffered from cerebral zygomycosis [27]. Another patient with a localized infection of his foot following trauma was cured with local and systemic ketoconazole associated with hyperbaric oxygen therapy [28]. Invasive cutaneous A. corymbifera infection was successfully treated in an allogeneic bone marrow
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transplant patient with liposomal amphotericin B for 6 weeks and surgical debridement [9]. One patient with catheter-related cutaneous infection was cured with liposomal amphotericin B and granulocyte growthstimulating factor after removal of the catheter and tissue debridement [10], and healing has been reported in one case of a kidney infection after kidney transplantation with transplantectomy alone [15]. All of these reports describe only single cases; the mortality rate in zygomycosis is still very high. For successful treatment it seems to be most important that the patient recovers immunologically and that the necrotic tissue is removed. In addition, drug administration should be performed as early as possible. Although rare reports suggest that fluconazole may be effective [28 / 30], in -vitro tests usually show resistance of Mucorales to azole compounds. However, the new antifungal compound posaconazole shows a high in -vitro activity against zygomycetes compared with the other azoles tested [1] and has been used successfully in the treatment of Mucor infection in an immunosuppressed mouse model [31]. This might indicate that posaconazole may be sufficient in the treatment of zygomycete infections also. Furthermore, the first single cases of the effectiveness of echinocandins (FK463) in the eradication of invasive mucormycosis have been reported [32]. Today, optimal treatment consists of aggressive surgical debridement additionally to intravenous administration of amphotericin B [8], especially lipid formulations, if possible [33].

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