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Journal of Clinical Microbiology, September 2008, p. 3169-3172, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.00052-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Molecular Diagnosis of Saksenaea vasiformis Cutaneous Infection after Scorpion Sting in an Immunocompetent Adolescent{triangledown}

Pauline Lechevalier,1,2 Dea Garcia Hermoso,3 Agnes Carol,2 Stephane Bonacorsi,2 Latifa Ferkdadji,4 Franck Fitoussi,5 Olivier Lortholary,3,6 Antoine Bourrillon,1 Albert Faye,1 Eric Dannaoui,3,7 and Francois Angoulvant1*

AP-HP, Hôpital Robert Debré, Service de Pédiatrie Générale, Université Paris Diderot-Paris VII, Paris, France,1 AP-HP, Hôpital Robert Debré, Laboratoire de Microbiologie, Université Paris Diderot-Paris VII, Paris, France,2 Centre National de Référence Mycologie et Antifongiques, Unité de Mycologie Moléculaire, CNRS URA3012, Institut Pasteur, Paris, France,3 AP-HP, Hôpital Robert Debré, Service d'Anatomopathologie, Université Paris Diderot-Paris VII, Paris, France,4 AP-HP, Hôpital Robert Debré, Service de Chirurgie Orthopédique, Université Paris Diderot-Paris VII, Paris, France,5 AP-HP, Hôpital Necker-Enfants malades, Centre d'Infectiologie Necker-Pasteur, Université Paris Descartes, Paris, France,6 AP-HP, Hôpital Georges Pompidou, Unité de Parasitologie-Mycologie, Université Paris Descartes, Paris, France7

Received 10 January 2008/ Returned for modification 21 February 2008/ Accepted 3 July 2008


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ABSTRACT
 
We report the first case of cutaneous mucormycosis after a scorpion sting in Tunisia. Histopathology showed broad aseptate hyphae suggestive of a Zygomycete. Saksenaea vasiformis was identified by PCR amplification and sequencing of the fungal DNA on a cutaneous biopsy. Successful treatment was obtained by surgery and liposomal amphotericin B.


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CASE REPORT
 
A previously healthy 14-year-old patient was admitted to Robert Debré hospital (Paris) with a left calf lesion following a scorpion sting in Tunisia 10 days before. At that time, he had been hospitalized for 4 days in an intensive care unit for acute renal failure and pulmonary edema induced by the scorpion venom. When he came back to France, the lesion due to the scorpion sting failed to heal.

On the day of admission, the patient had a limping gait. Physical examination of the left calf revealed a painful, indurated, erythematous, 5-cm lesion. Ultrasonic tomography revealed a diffuse, soft tissue swelling. The white blood cell count was 14.1 x 109/liter with 71% neutrophils, the platelet count was 395 x 109/liter, and the C-reactive protein level was less than 10 mg/liter. The patient was started on antibacterial therapy with cefotaxime and fosfomycin according to our local guidelines for antibiotic treatment (9).

On day 5, the lesion grew to a 10-cm area of swelling and induration, with a central area of black blisters. No sample was taken at this stage. The patient had a fever (38.7°C) and experienced severe pain. The white blood cell count was 18.9 x 109/liter with 80% neutrophils, and the C-reactive protein level was 52 mg/liter. Three blood cultures performed between day 1 and day 5 were sterile. A second ultrasonic tomography showed an extension within subcutaneous tissues, with collection of fluids. Because of the increasing area of the wound, vancomycin and metronidazole were added to the treatment regimen.

On day 7, the patient required local debridement to remove devitalized tissues, including muscles and fascia. A 7-cm incision was made to evacuate the necrotizing tissue. A piece of debrided tissue was submitted for microbiological and histopathological examination. On day 12, histological analysis showed many areas of necrosis and nonspecific inflammation. Within the necrotic areas, branched, broad, nonseptate fungal hyphae suggestive of Zygomycetes were found. The material was inoculated onto routine bacteriological media and Sabouraud dextrose agar. On the Sabouraud dextrose agar, there were few colonies of filamentous fungi, with no evidence of sporulation, and the loss of viability of the isolate, possibly due to growth conditions, precluded subcultures on specific media (11), impaired any further mycological identification. All other cultures on solid media were negative for bacteria. The cutaneous biopsy, kept frozen at –80°C, was then sent to the National Reference Center for Mycoses and Antifungals, Pasteur Institute, for molecular studies. Tissues were ground, and DNA extraction was performed as previously described (16). Direct examination of the homogenate used for PCR amplification was performed by fluorescence microscopy after it was stained with calcofluor white and showed broad, nonseptate hyphae typical of a Zygomycetes (Fig. 1). The universal fungal primer pairs ITS1/ITS4 and NL1/NL4 (20) were used to amplify the whole ITS1-5.8S-ITS2 region and a part of the 28S region, respectively. PCR products were sequenced, and both strands and sequences were compared with available ribosomal DNA sequences in GenBank, using nucleotide-nucleotide BLAST with the default settings. Additionally, available sequences of S. vasiformis and Apophysomyces elegans, a closely related species, were subjected to multiple alignment, using ClustalW version 1.4 software, and neighbor-joining phylogenetic trees were constructed. For the internal transcribed spacer (ITS) region, the best matches (90% identity over 541 bp and 84% identity over 681 bp) were obtained with sequences of S. vasiformis isolate MCCL 111004 (GenBank accession number AY211275) and S. vasiformis isolate CNRMA 05.1337 (GenBank accession number EU182902), respectively. For the 28S region, the identity percentages were 94% (over 681 bp) to 98% (over 381 bp) with two isolates of S. vasiformis (GenBank accession numbers AF113483 and AY234872) and were 90% (over 690 bp) to 91% (over 689 bp) with two isolates of A. elegans (GenBank accession numbers AF113449 and AF113450). Neighbor-joining trees based on two different targets (Fig. 2) clearly showed clustering of the sequence obtained from the present patient (CNRMA 07.577) with those of S. vasiformis. Based on these data, the fungus was identified as S. vasiformis. Multiple alignments of the complete 5.8S and ITS2 sequences of CNRMA 07.577 with those of two S. vasiformis isolates (GenBank accession numbers EU182902 and AY211275) are shown in Fig. 3. Two independent DNA extractions and amplifications from two tissue samples from the biopsy were performed with the same results.


Figure 1
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  		FIG. 1. Direct examination of the biopsy homogenate used for PCR amplification. The sample was stained with calcofluor white and showed large aseptate hyphae typical of a Zygomycete. The bar represents 20 µm.


Figure 2
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  		FIG. 2. Neighbor-joining phylogenetic trees for isolates of S. vasiformis and A. elegans, based on the partial nucleotide sequences of the ITS (A) and 28S (B) regions. The GenBank accession numbers are shown. Rhizomucor pusillus was used as an out-group.


Figure 3
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  		FIG. 3. Multiple alignments of the complete 5.8S and ITS2 sequences of CNRMA 07.577 with those of two S. vasiformis isolates (GenBank accession numbers EU182902 and AY211275).

On day 12, amphotericin B therapy (0.5 mg/kg/day) was begun, and all other antibiotics were discontinued. On day 17, amphotericin B therapy was replaced by liposomal amphotericin B at 10 mg/kg/day. On day 24, because the patient's serum creatinine level had increased from 58 to 152 µg/liter, the regimen of liposomal amphotericin B was reduced to 3 mg/kg/day. The patient completed a 20-day course of amphotericin B therapy (cumulative dose, 3,700 mg).

Clinical and radiological assessments, including computed tomography of the sinus and chest, revealed no evidence of other localization of infection. Immunoglobulin serum level, complement fraction, fasting plasma glucose, and lymphocyte subpopulation tests performed on day 15 were normal. These tests were performed to rule out immunosuppression and, more specifically, diabetes mellitus, which is frequently associated with mucormycosis.

A control cutaneous biopsy of the wound bed was performed, with the subsequent histopathologic result of no fungal element being seen. A split-thickness skin graft (12 cm by 7 cm) from the right thigh was used to cover the wound on day 31 after admission. On day 36, the patient was discharged to go home in good general condition. After 3 years of monitoring, the skin graft is still intact.

Mucormycoses are infections caused by Zygomycetes and mostly occur in immunocompromised hosts The organisms most commonly implicated in human infections belong to the genus Rhizopus (14) and are mostly responsible for rhinocerebral (39%) and cutaneous (19%) infections, with fatality rates of 67% and 16%, respectively (15). Different genera, including Apophysomyces, Rhizopus, Rhizomucor, Absidia, Mucor, and Saksenaea, have been reported to be responsible for cutaneous mucormycoses (2, 7). Cutaneous mucormycosis is less frequently associated with systemic illness than other forms, but a local risk factor constituting a break in the skin's integrity has been identified in 86% of patients (2). Adam et al. reviewed 25 cases of cutaneous mucormycosis and identified some local risk factor, such as surgery (17%), burns (16%), motor vehicle-related trauma (12%), the use of needles (13%), knife wounds (3%), insect or spider bites (3%), and other types of trauma (23%) (2). Cutaneous mucormycoses have also been reported after natural disasters such as volcanic eruptions (13) and tsunamis (4). To our knowledge, this is the first case of mucormycosis after a scorpion sting. Scorpions live most of the time inside underground holes or under rocks, which could explain why their stings are colonized by Zygomycetes. Note that insect or spider bites are responsible for 3% of primary cutaneous mucormycoses. Other animal species may harbor Zygomycetes, and it is possible that bites from such animal species might inoculate Zygomycetes, leading to primary cutaneous mucormycosis cases (2, 5, 10). In the case of a scorpion sting, a primary inoculation could occur, causing cutaneous mucormycosis.

Up to now, 35 cases of S. vasiformis infection have been reported (1, 3, 6, 12, 17, 18). Twenty of the 28 patients reported in the review of Vega et al. were immunocompetent (18). There were 22 cutaneous infections, 3 cases of sinusitis, and 3 disseminated infections (18). Among the 22 cases of cutaneous infection, one occurred after insect bites (5) and one after a spider bite (10). Most cases of human disease occur in warmer, tropical and subtropical climates and have been found in the United States, Australia, New Zealand, Colombia, Ecuador, French Guiana, Israel, Thailand, Spain, India, and Iraq (6, 12, 17, 18). The case in this report is the 36th reported case of S. vasiformis infection and the first reported case contracted in Tunisia. Only a few cases of cutaneous infections have been described, which could be explained by the fact that S. vasiformis, as well as A. elegans, a phylogenetically related species (19), fail to sporulate on routine mycological media (14). Specific culture media have to be used to induce sporulation and to allow identification to the species level (11). Nevertheless, when cultures are negative or when the strain is lost or nonviable, as in the present case, the only possibility of identification relies on molecular analysis performed directly on the infected tissues. Several DNA targets and methodologies have been used to identify Zygomycetes directly from frozen or paraffin-embedded tissues (8). In the present case, we performed molecular identification on a frozen biopsy by a technique that has been validated in an experimental model of zygomycosis (16). This is the first case of a molecular diagnosis of a S. vasiformis infection from tissue. Molecular analyses of two different DNA targets were performed. Alignment of the sequences with S. vasiformis sequences retrieved from GenBank and construction of phylogenetic trees (with sequences of S. vasiformis and A. elegans, the most closely related species) allowed us to identify the fungus with confidence as the species S. vasiformis. It is interesting that there are relatively large genetic distances between isolates of S. vasiformis, as previously noted (6). A more comprehensive phylogenetic study of this rare genus would be of interest. Our patient was treated with the combination of extensive surgical debridement and antifungal chemotherapy (amphotericin B), which is the treatment associated with the best outcome for mucormycosis (15).

Nucleotide sequence accession numbers. Sequences of the ITS and 28S regions obtained from the biopsy were deposited in the GenBank database under accession numbers EU644757 and EU644756, respectively.


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ACKNOWLEDGMENTS
 
We thank Damien Hoinard for excellent technical assistance and Adela Enache-Angoulvant for her help in the preparation of the manuscript.

None of the authors has any conflict of interest.


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Pédiatrie générale, Hôpital Robert Debré, AP-HP, 48 Boulevard Sérurier, 75019 Paris, France. Phone: 33 1 40 03 53 61. Fax: 33 1 40 03 47 45. E-mail: francois.angoulvant{at}rdb.aphp.fr Back

{triangledown} Published ahead of print on 16 July 2008. Back


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Journal of Clinical Microbiology, September 2008, p. 3169-3172, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.00052-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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