Diagnosis of Cutaneous Mucormycosis Due to Rhizopus microsporus by an Innovative PCR-Restriction Fragment-Length Polymorphism Method

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SirMucormycosis is an opportunistic infection caused by saprophytic fungi belonging to the class Zygomycetes and the order Mucorales. These molds live in soil, air, decaying matter, and substrates such as fruits, cereals, and breads, and infections have been reported in rhinocerebral, pulmonary, gastrointestinal, cutaneous, and disseminated manifestations [1, 2]. Host risk factors include diabetes, leukemia, bone marrow transplantation, immunosuppressive chemotherapy, broad-spectrum antibiotic use, and breakdown of the cutaneous barrier [2–4]. Recently, the number of such breakthrough infections has significantly increased, with associated mortality rates of 70%100% [5]. We report a study that suggests the use of polymerase chain reaction (PCR) and restriction fragmentlength polymorphism analysis is a promising tool for an early diagnosis and targeted treatment of mucormycosis. A 63-year-old woman presented to the medical intensive care unit with septic shock in the context of aggressive B lymphoma, diagnosed a month before. She was sedated, underwent intubation and mechanical ventilation, and she received vasoactive support. Antineoplastic chemotherapy against lymphoma was rapidly initiated. Microbiologic findings revealed Pseudomonas aeruginosa, rare Candida parapsilosis, and cytomegalovirus, and there was a significant inflammatory syndrome (the C-reactive protein level was 80 mg/L, and the procalcitonin level was 7.92 ng/mL). Ten days after admission to the intensive care unit, the patient developed rapidly expanding lesions on her back that evolved toward a necrotic aspect. Because of the patient's profound immunosuppression, liposomal amphotericin B (10 mg/kg) was given intravenously. Findings of a mycological examination of a first incisional biopsy specimen were consistent with mucormycosis. A wide local surgical incision of the necrotic lesions was then performed. Numerous large and aseptate fungal hyphae were identified in epidermal and dermal structures by histological staining. Subsequent cultures of the surgically excised tissue yielded an aerobic mycelium that grew rapidly on Sabouraud media. This mold was microscopically classified as a Rhizopus species on the basis of the presence of stolons, brown pigmented rhizoids, sporangiophores, and globose sporangiaboth apophysate and columellate (figure 1). Figure 1. Microscopic features of the Rhizopus species isolated from culture (lactophenol blue preparation; original magnification, ×20). Sporangiophores are long and nondichotomous, usually terminating in large globose sporangia. Rhizoids originate at the base of sporangiophores. Stolons extend laterally from the node. The diagnosis of primary cutaneous mucormycosis seemed likely because respiratory and blood samples tested negative for Zygomycetes. However, despite an adequate antifungal treatment and use of vasopressive amines, the patient experienced multisystem organ failure, and died after 30 days in the intensive care unit. Using PCR and restriction fragmentlength polymorphism analysis, we retrospectively confirmed the identification of the Rhizopus genus and found the specific species to be Rhizopus microsporus. Primers targeted a 830-bp sequence in the 18S fungal ribosomal gene with exclusion of human DNA, and then digestion with restriction enzymes enabled a specific identification at genus and species level. The amplicon was then digested using the restriction enzymes PpuMI, AflII, BmgBI, and AclI, respectively specific for the genera Rhizomucor, Mucor, and Rhizopus, and for Absidia corymbifera. As expected from mycological examination findings, the digestion with BmgBI resulted in 2 fragments (600 bp and 230 bp in length). The PCR product was not digested with PpuMI, AflII, and AclI, because the target amplicon does not possess any restriction site for these enzymes. These results confirmed the presence of a Rhizopus species, and a second round of digestion with AseI revealed features specific for R. microsporus or R. azygosporus (figure 2). On the basis of epidemiological and clinical data, R. microsporus was found to be likely implicated in this infection [6, 7]. Figure 2. PCR amplification and restriction-enzyme digestion patterns for a clinical biopsy sample from the cutaneous lesion of a 63-year-old patient. Lane 1, 100-bp ladder; lane 2, nondigested PCR products; lane 3, specific BmgBI digestion of Rhizopus species, lane 4, specific AseI digestion for Rhizopus microsporus var. rhizopodiformis and R. azygosporus (the 70-bp fragment is not clearly distinguishable because of its small size); lane 5, PCR without DNA (negative control). The occurrence of mucormycosis in immunocompromised patients is increasing, and physicians who treat patients in intensive care units must be aware of these fatal emerging infections [8, 9]. Today, the diagnosis of mucormycosis relies on analyses of clinical samples by microscopy and culture. Unfortunately, the recovery of Zygomycetes from tissues can be problematic, and fungal detection is often hindered by the absence of fungal growth in culture [1]. Therefore, the diagnosis is often made after a too-long delay or even postmortem. The only effective treatment for these infections consists of high doses of amphotericin B, combined with surgical debridement and control of the underlying disease. Moreover, differences have been noted in the in vitro susceptibilities of genera and species in the class Zygomycetes to available antifungals [10]. Thus, amphotericin B seems less effective against Rhizopus than it is against the Absidia or Mucor genera. This heterogeneity may require an improved identification of Zygomycetes genera and species. The discrimination between Mucorales species belonging to a single genus usually must be done by a reference laboratory. Thus, frequently, when attempting to identify fungi, clinicians take an alternative approach and use panfungal primers for DNA sequencing [11]. Recently, Kontoyiannis et al. [12] reported a 21% discordance rate between results of the morphological and sequence-based methods used to determine the fungal genera. Therefore, we propose use of a molecular test that enables rapid identification of Mucorales at the genus and the species level. Further studies are now clearly warranted to prospectively assess the accuracy of the PCR and restriction fragmentlength polymorphism on clinical samples in comparison to classical methods. PCR and restriction fragmentlength polymorphism analysis could then become an easy-to-use diagnostic tool to help clinicians establish more-targeted therapeutic or preemptive strategies to use against this life-threatening infection at an earlier stage. We thank Dr. Quentin Vicens for his precious help in checking our English. Potential conflicts of interest. All authors: no conflicts.

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