Zygomycosis is an infection caused by fungi classified

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1 Original Articles Zygomycosis Due to Apophysomyces elegans Report of 2 Cases and Review of the Literature Masatomo Kimura, MD, PhD; Michael B. Smith, MD; Michael R. McGinnis, PhD Objective. The zygomycete Apophysomyces elegans is an unusual human pathogen that is being reported with increasing frequency as a cause of infection in previously healthy patients following trauma or after invasive procedures. We report 2 cases of infection caused by this emerging fungal pathogen. Methods. Histologic sections of tissue removed from the infected patients and the isolates in culture were examined. Other infections caused by A elegans that have been reported in the literature were reviewed. Results. Both patients developed infection due to A elegans after sustaining trauma that required tissue debridement because of tissue necrosis. Histologic examination showed broad, sparsely septate, thin-walled hyphae and angioinvasion with thrombosis. Extensive coagulation Zygomycosis is an infection caused by fungi classified within the class Zygomycetes. These fungi can produce serious and rapidly fatal infections, especially in immunocompromised patients. 1 The primary genera known to cause such infections are Absidia, Mucor, Rhizomucor, and Rhizopus. Apophysomyces elegans has been reported as an emerging pathogen that can cause primary cutaneous zygomycosis in healthy patients. 1 Only 14 cases caused by this agent have been reported in the English literature We present 2 additional cases of zygomycosis due to A elegans and review the literature. REPORT OF CASES Case 1 On November 20, 1994, a previously healthy 6-year-old boy fell off a truck and was pinned on his back underneath the tire of a trailer; the boy was then dragged on the road for a short distance. He suffered multiple soft tissue loss, primarily on his left back, as well as an unstable pelvic fracture. He was seen at a local hospital, underwent multiple debridement, and was started on multiple antibiotics. He became septic and was transferred Accepted for publication January 18, From the Second Department of Pathology, Kinki University School of Medicine, Osaka-Sayama, Japan (Dr Kimura), and the Division of Microbiology (Dr Smith) and Medical Mycology Research Center, Department of Pathology (Dr McGinnis), The University of Texas Medical Branch, Galveston. Reprints: Michael R. McGinnis, PhD, Department of Pathology, University of Texas Medical Branch, Keiller Bldg 1.116, 301 University Blvd, Galveston, TX necrosis of surrounding tissue was seen. A rapidly growing mold with sporangiophores having funnel-shaped apophyses and pyriform sporangia, characteristic of A elegans, was isolated from each case. Conclusion. Apophysomyces elegans is an opportunistic pathogen that can cause infection in previously healthy patients who suffer an injury to the cutaneous barrier, such as trauma or burns. Infection with this zygomycete should be considered when there is progressive necrosis of a wound in a previously healthy patient. Successful treatment requires tissue debridement and amphotericin B. Histologic examination for early diagnosis and frozen section evaluation of surgical margins are required for optimal therapy. (Arch Pathol Lab Med. 1999;123: ) to a second hospital on December 7. At the time of admission, he had a large soft tissue defect on the lower back, extending to the left flank with exposed pelvic bone; spinous processes L3, L4, and L5; left kidney; and viscera. Laboratory studies disclosed the following values: glucose, 6.9 mmol/l; albumin, 2 g/l; creatinine, 38 mol/l; white blood cell count, /L; and hemoglobin, 105 g/l. He continued to receive intravenous antibiotics. On December 8, the patient underwent jet lavage and debridement with a homograft being placed over the large posterior defect. Histologic examination of debrided tissue showed invasion of viable skin by fungal hyphae consistent with a zygomycete. The patient was started on amphotericin B. Subsequent cultures of the wound grew A elegans, Pseudomonas aeruginosa, and Klebsiella species. On December 10, the patient underwent a nephrectomy for a nonviable left kidney, and 2 days later, en-block resection of the spleen and tail of pancreas, removal of the left colon and a part of jejunum, and debridement of the left diaphragm. Histopathologically, infection caused by zygomycete hyphae was seen in the left kidney, pancreas, mesentery, soft tissue, and muscles. Hyperbaric oxygen therapy was initiated, and he was started on itraconazole in addition to amphotericin B. He was partially autografted on December 19 after improvement of his wounds; however, a radiograph showed pneumonia. On December 29, the patient became bradycardic and hypotensive and continued to worsen. He died and was autopsied on January 12, Case 2 A 55-year-old man with alcoholic liver cirrhosis fell on a crab trap and injured his right knee 2 weeks prior to admission. He scraped his knee and put dirt from an anthill onto the wound to stop the bleeding. He developed a progressive swelling, pain, and redness spreading down the anterior surface of the lower leg 386 Arch Pathol Lab Med Vol 123, May 1999 Apophysomyces elegans Kimura et al

2 to the ankle over several days. On June 14, 1997, he was admitted with a right lower leg cellulitis. There was necrotic eschar over the knee. Laboratory studies disclosed the following values: hemoglobin, 139 g/l; total white blood cell count, /L; and blood glucose, 4.6 mmol/l. The patient was started on antibiotics, but progression of the cellulitis continued. Computed tomographic scan showed diffuse soft tissue swelling on June 19. Direct smear of the wound surface disclosed fungal hyphae, cultures of which subsequently yielded Fusarium species, A elegans, Enterococcus species, and Staphylococcus species. The patient was started on amphotericin B on June 20. The knee eschar was debrided on June 21, followed by wider debridement of surrounding tissue on June 23. Histopathologic examination of the debrided tissue showed angioinvasion by hyphae that were compatible with a zygomycete. Treatment with hyperbaric oxygen was initiated on June 24. Because of a rise in the patient s serum creatinine level, antifungal therapy was changed to liposomal amphotericin B on June 26, with apparent improvement. A third debridement was performed because there was still some necrotic tissue along the wound bed, with the subsequent histopathologic result of no fungal elements being seen. The knee wound began to granulate, and the patient was discharged on July 10. Allograft and then split-thickness skin grafting were performed by mid- August. No recurrence of his leg infection was detected after 8 months of follow-up. PATHOLOGIC FINDINGS Case 1 Debrided cutaneous, subcutaneous, and underlying muscle tissue showed liquefactive and coagulative necrosis with little inflammatory cell infiltration. Irregularly broad(3to15 m in diameter), sparsely septate, thinwalled hyphae with right angle branching, consistent with a zygomycete, were found invading the necrotic tissue. A focal neutrophilic infiltration was seen at the site of hyphal invasion in the viable tissue. Hyphae were seen in the vascular lumen and vessel wall. The left flank wound border showed necrosis of muscle with marked fungal colonization on the surface. The resected kidney showed infarction. Most of the blood vessels were thrombosed and contained fungal elements similar to those seen in the debrided tissue (Figure 1). Extensive areas of mesentery and focal areas of debrided left diaphragm were necrotic and showed fungal invasion. The spleen showed infarction without apparent fungal infection. A focal neutrophilic infiltration with fungal hyphal invasion was seen in the tail of the pancreas. Autopsy revealed diffuse massive interstitial pneumonitis as the possible cause of death. Other findings were diffuse severe fatty metamorphosis, multifocal cytomegalovirus infection, and disseminated intravascular coagulation. Figure 1. Case 1. Mycotic thrombus found in the resected left kidney. An artery of the renal medulla is thrombosed, and numerous ribbonlike hyphae are seen in the vascular lumen (Movat s pentachrome stain, original magnification 100). Figure 2. Case 2. Mat of Fusarium hyphae with macroconidia and microconidia at surface of wound (hematoxylin-eosin, original magnification 100). Figure 3. Case 2. Hyphae of Apophysomyces in viable tissue below surface mat of Fusarium hyphae (hematoxylin-eosin, original magnification 100). Case 2 Microscopic examination of the first debrided tissue revealed mixed colonization by narrow septate hyphae and phialides with microconidia and macroconidia, the macroconidia being curved with foot cells consistent with Fusarium oxysporum (Figure 2), and secondly, irregularly broad, sparsely septate hyphae (Figure 3) similar to those seen in case 1, with bacterial colonies on the surface of the tissue. Most areas of the tissue showed coagulative necrosis with focal suppurative inflammation. The septate narrow hyphae invaded only the superficial necrotic tissue. The zygomycetous hyphae invaded subcutaneous adipose tissue and showed angioinvasion with thrombosis. The second debridement showed invasion of zygomycetous hyphae into necrotic adipose tissue with mycotic thrombosis. The third debridement did not reveal fungal organisms in the tissue. MYCOLOGIC CULTURE RESULTS Debrided tissues from both cases were inoculated onto blood agar, inhibitory mold agar, and malt extract agar and resulted in the growth of a rapidly spreading, white Arch Pathol Lab Med Vol 123, May 1999 Apophysomyces elegans Kimura et al 387

3 Reported Cases of Infection Due to Apophysomyces elegans* Patient No. Source,y Age, y/sex Location Underlying Condition Site of Involvement Circumstances of Inoculation 1 Wieden et al, /F Arizona Diabetes mellitus Left leg Scraping of leg following a fall 2 Lawrence et al, /M Texas None Left kidney, urinary Unknown bladder 3 Cooter et al, /M North Australia None Right ankle Rolled on ground to extinguish flames 4 Huffnagle et al, /M Texas None Right arm and leg Fell (55 ft) onto grass 5 Weinberg et al, /M Florida None Right shoulder Bite or sting 6 Lakshmi et al, /M South India None Abdominal wall to left thigh, inguinal canal, left testis 7 McGinnis et al, /M Texas None Entire back, lungs, kidneys, intestine, abdominal aorta, vertebrae Inguinal herniorrhaphy Motorcycle accident 8 Okhuysen et al, /M Texas None Right flank, left kidney Motor vehicle accident 9 Eaton et al, /M Florida None Mid chest Struck by the cut end of a tree limb 10 Meis et al, /M Caribbean None Left arm, shoulder Unknown 11 Radner et al, /M Mexico None Palate, left eye, Fell into a ditch paranasal sinuses 12 Naguib et al, /M Oklahoma Kidney transplant Right forearm Fell off bicycle onto a gravel road 13 Chakrabarti et al, /F India N/A Left arm Intramuscular injection 14 Mathews et al, /F South India None Anterior abdominal wall 15 Case 1 6/M Mexico None Pelvis, back, left kidney, pancreas, mesentery, diaphragm 16 Case 2 55/M Texas Liver cirrhosis, alcohol abuse * N/A indicates data not available; AmB, amphotericin B. Right knee Caesarian section Motor vehicle accident Fell on crab trap floccose mold that was sterile. The organism was subcultured to potato dextrose agar, and after 3 days of incubation at 30 C, a block of the medium containing the fungus was placed in a Petri plate containing 25 ml of sterile distilled water supplemented with 0.2 ml of 10% filtered sterilized yeast extract solution, which was then incubated at 37 C. 16,17 After 7 days of incubation, tease mounts of the growth contained sporangiophores with campanulateshaped apophyses, and pyriform sporangia. The dark, thick-walled sporangiophores had an exceptionally darkly pigmented, thickened portion just below the sporangium. Morphologically, the fungi were identified as A elegans. 18 The isolates are maintained in our culture collection as UTMB 3646 (case 1) and 4761 (case 2). In addition, Fusarium was recovered. Unfortunately, the isolate was not kept following the patient s discharge. COMMENT Apophysomyces elegans is a rare human pathogen that is classified within the family Mucoraceae of the class Zygomycetes, order Mucorales. 1 This species was first isolated in 1979 from soil samples collected in a mango orchard in northern India. 18 Three years later, A elegans was isolated from bronchial washings of a patient in the United States. 16 The first human infection caused by this fungus was described in Since then, 13 additional cases 2 15 have been reported in the English literature. A summary of these cases is shown in the Table. Two cases by Winn et al 19 were not included in the Table because of insufficient clinical data necessary to adequately evaluate them. Cases by Newton et al 20 and by Chugh et al 21 were also excluded, because it was not clear if the fungal identification was correct. Most of the reported cases have come from regions with warm climates, such as the southern portions of the United States, Mexico, North Australia, and South India. Infections caused by zygomycetes in the order Mucorales usually occur in immunocompromised patients who have leukemia, lymphoma, or diabetes mellitus or in those who have undergone organ transplantation. 1 These patients develop rhinocerebral or pulmonary infection, subsequently followed by dissemination. In contrast, A elegans causes cutaneous and soft tissue infection following trauma, such as burns or invasive procedures, in previously healthy patients Of the 16 patients described in the literature, including our 2 cases, only 2 patients had a pre- 388 Arch Pathol Lab Med Vol 123, May 1999 Apophysomyces elegans Kimura et al

4 Histopathology Tissue Invasion Angioinvasion Management Extended AmB Surgical Outcome Yes Yes Yes Amputation of left leg N/A Yes Yes Yes Nephrectomy Cured Yes No Yes Debridement, right below-knee amputation Cured Yes N/A Yes Amputation of right arm and leg Died Yes Yes Yes Debridement, scapulectomy Cured Yes N/A Yes Debridement, left testectomy Died Yes Yes Yes Debridement Died Yes N/A Yes Debridement Cured Yes N/A Yes Debridement, sternectomy Cured Yes No Yes Debridement, forequarter amputation Cured Yes Yes Yes Exenteration of left orbit, bilateral maxillectomies, palatectomy, debridement of paranasal sinuses Cured Yes N/A Yes Debridement Cured N/A N/A N/A Debridement Cured N/A N/A Yes Debridement Cured Yes Yes Yes Debridement of skin and diaphragm, left nephrectomy splenectomy, distal pancreatectomy, hemicolectomy Died Yes Yes Yes Debridement Cured disposing condition namely, diabetes mellitus 2 and kidney transplantation with cyclosporine and steroid therapy. 12 In most cases, trauma or burns lead to disruption of the skin s function as a mechanical barrier. Suppression of the immune response by trauma and burns 22 can be the factor that enables the infection to occur. Furthermore, the deteriorated general condition may be accompanied by acidosis, which is a favorable condition for the proliferation of zygomycetes. Damage to skin with subsequent soil contamination are common features in patients with A elegans infection. Apophysomyces elegans is known to be a thermophile that can be isolated from soil. 4 The introduction of contaminated soil at the site of trauma may serve as the primary means of inoculation of this organism. Apophysomyces elegans tends to invade and grow within the vascular lumen like other species of the zygomycetes. Vascular invasion frequently causes thrombosis leading to ischemic tissue necrosis. Histopathologic examination of most cases infected with A elegans revealed hyphae in the thrombi. The rapid progress of necrosis, which usually occurs within days after inoculation, may be explained by the rapid growth within vessels. The possibility of A elegans infection must be considered in the differential diagnosis when a patient is seen with progressive necrosis of a wound, especially when there is an ineffective response to antibacterial chemotherapy in an otherwise healthy patient. Successful treatment requires an early diagnosis and removal of infected tissues in conjunction with the administration of amphotericin B The key to diagnosis is a high suspicion and early tissue biopsy examination and culture. The organism may be found in tissue sections invading blood vessels and viable tissue. Histologic examination is also necessary in deciding the extent of debridement that will be necessary. Surgical margins should be evaluated until they become free of fungus. Surgical margins in some cases were effectively evaluated by serial frozen sections. 6,15 The morphology of A elegans in tissue is similar to that of other species of zygomycetes. Only hyphae of this fungus have been found in tissue, whereas other fungal structures, such as sporangia and chlamydoconidia, have never been described in tissue. In our case 2, both Fusarium and Apophysomyces colonized the surface of necrotic tissue of the wound, but only Apophysomyces invaded into viable tissue. Fusarium only colonized the wound and was not an etiologic agent of the patient s disease. Why Fusarium did not invade deeply into Arch Pathol Lab Med Vol 123, May 1999 Apophysomyces elegans Kimura et al 389

5 viable tissue is unclear. It is likely that aggressive surgical management with amphotericin B chemotherapy prevented the Fusarium from invading viable tissue. The morphology of A elegans in culture is similar to that of Absidia species. However, only A elegans has dark brown campanulate or funnel-shaped apophyses and an exceptionally darkly pigmented, thick-walled zone of the sporangiophore just below the apophyses. 8,17,18 In addition, A elegans has a foot cell that resembles those produced by Aspergillus species. Apophysomyces elegans resembles Saksenaea vasiformis in gross colony morphology; however, the morphology of sporangia in these 2 fungi is significantly different. 8,17 References 1. Kwon-Chung KJ, Bennett JE. Mucormycosis. In: Medical Mycology. Philadelphia, Pa: Lea & Febiger; 1992: Wieden MA, Steinbronn KK, Padhye AA, Ajello L, Chandler FW. Zygomycosis caused by Apophysomyces elegans. J Clin Microbiol. 1985;22: Lawrence RM, Snodgrass WT, Reichel GW, Padhye AA, Ajello L, Chandler FW. Systemic zygomycosis caused by Apophysomyces elegans. J Med Vet Mycol. 1986;24: Cooter RD, Lim IS, Ellis DH, Leitch IO. Burn wound zygomycosis caused by Apophysomyces elegans. J Clin Microbiol. 1990;28: Huffnagle KE, Southern PM Jr, Byrd LT, Gander RM. Apophysomyces elegans as an agent of zygomycosis in a patient following trauma. J Med Vet Mycol. 1992;30: Weinberg WG, Wade BH, Cierny G III, Stacy D, Rinaldi MG. Invasive infection due to Apophysomyces elegans in immunocompetent hosts. Clin Infect Dis. 1993;17: Lakshmi V, Rani TS, Sharma S, et al. Zygomycotic necrotizing fasciitis caused by Apophysomyces elegans. J Clin Microbiol. 1993;31: McGinnis MR, Midez J, Pasarell L, Haque A. Necrotizing fasciitis caused by Apophysomyces elegans. J Mycol Méd. 1993;3: Okhuysen PC, Rex JH, Kapusta M, Fife C. Successful treatment of extensive posttraumatic soft-tissue and renal infections due to Apophysomyces elegans. Clin Infect Dis. 1994;19: Eaton ME, Padhye AA, Schwartz DA, Steinberg JP. Osteomyelitis of the sternum caused by Apophysomyces elegans. J Clin Microbiol. 1994;32: Meis JFGM, Kullberg BJ, Pruszczynski M, Veth RPH. Severe osteomyelitis due to the zygomycete Apophysomyces elegans. J Clin Microbiol. 1994;32: Radner AB, Witt MD, Edwards JE Jr. Acute invasive rhinocerebral zygomycosis in an otherwise healthy patient: case report and review. Clin Infect Dis. 1995;20: Naguib MT, Huycke MM, Pederson JA, Pennington LR, Burton ME, Greenfield RA. Apophysomyces elegans infection in a renal transplant recipient. Am J Kidney Dis. 1995;26: Chakrabarti A, Kumar P, Padhye AA, et al. Primary cutaneous zygomycosis due to Saksenaea vasiformis and Apophysomyces elegans. Clin Infect Dis. 1997;24: Mathews MS, Raman A, Nair A. Nosocomial zygomycotic post-surgical necrotizing fasciitis in a healthy adult caused by Apophysomyces elegans in South India. J Med Vet Mycol. 1997;35: Ellis JJ, Ajello L. An unusual source for Apophysomyces elegans and a method for stimulating sporulation of Saksenaea vasiformis. Mycologia. 1982;74: Padhye AA, Ajello L. Simple method of inducing sporulation by Apophysomyces elegans and Saksenaea vasiformis. J Clin Microbiol. 1988;26: Misra PC, Srivastava KJ, Lata K. Apophysomyces, a new genus of the Mucorales. Mycotaxon. 1979;8: Winn RE, Ramsey P, Adams ED. Traumatic mucormycosis due to Apophysomyces elegans, a new genus of the Mucorales [abstract]. Clin Res. 1982;30: 382A. 20. Newton WD, Cramer FS, Norwood SH. Necrotizing fasciitis from invasive phycomycetes. Crit Care Med. 1987;15: Chugh KS, Sakhuja V, Gupta KL, et al. Renal mucormycosis: computerized tomographic findings and their diagnostic significance. Am J Kidney Dis. 1993;22: Baker CC. Immune mechanisms and host resistance in the trauma patient. Yale J Biol Med. 1986;59: Arch Pathol Lab Med Vol 123, May 1999 Apophysomyces elegans Kimura et al