Correct answer: 4

Cutaneous inoculation of fungi as a consequence of trauma is a well-described mode of acquiring invasive fungal infections. In immunocompetent hosts, mononuclear and polymorphonuclear phagocytes normally serve as a primary host defense against cutaneously inoculated spores. However, in the immunocompromised host, the spores are able to germinate, and the hyphae invade surrounding tissues.¹ Infarction and necrosis of host tissue ensue as the hyphae invade blood vessels. The differential for a patient presenting with a necrotic eschar accompanied by surrounding erythema and induration following trauma should include invasive cutaneous fungal infection, particularly for immunosuppressed patients and those with diabetes.² The diagnosis of mucormycosis infections requires a high degree of suspicion, awareness of host factors, and appropriate evaluation.

Answer 1 is incorrect. Swabs are inferior to tissue and fluid for recovery of fungi. Further, swabbing the base of the wound runs the risk of picking up wound colonizers and environmental contaminants rather than the actual pathogen causing disease in the deeper tissues.³

Answer 2 is incorrect. Viral infections such as herpes simplex and varicella zoster would more classically present with vesicular lesions. Further, reactivation of herpes viruses would be less likely to occur at the actual site of the trauma. However, bacterial infection is certainly on the differential in such cases.

Answer 3 is incorrect. And while advances in blood culture systems (e.g., specialized broth media, automated and continuous monitoring platforms, etc.) have improved recovery of some fungi including Candida from blood, blood cultures are typically not positive in cases of invasive aspergillosis and mucormycosis, even in the face of disseminated disease.

Visualization of fungal elements in tissue remains the cornerstone for diagnosing invasive mycoses: however, isolation and identification of the infecting organism are required for a precise diagnosis.

Discussion:

Correct Answer: 3 

Direct KOH examination of the tissue specimen shows broad, ribbon-like, hyphae that are approximately 15 µm in diameter with irregular branching and sparse septations. Calcofluor White is a fluorochrome that binds to chitin and β-glucans in fungal cell walls. When the specimen is examined under a fluorescence microscope with ultraviolet filters, the fungal elements fluoresce, making them easier to distinguish from background tissues and other artifacts such as cotton fibers.

Possible answer 1: While a report of ‘fungal elements’ is not incorrect, a more specific description provides critically important information to guide further management of the infection. Specifically, “broad, ribbon-like, sparsely septate fungal hyphae” suggests mucormycosis.⁴

Answer 2 is incorrect. A hyphomycete-type pathogen such as Aspergillus, Scedosporium, or Fusarium, wherein the hyphae tend to be smaller (3-5 µm in diameter) with regular septations present, was not seen.⁴

Answer 4 is incorrect. No budding yeast or pseudohyphae are seen (suggesting Candida), making this response incorrect.

Discussion:

Correct answer: 1

Guidelines for management of mucormycosis are based on consensus recommendations.^8,9 Amphotericin B and amphotericin B-based lipid formulations remain the agents of choice for empiric management of mucormycosis.^8,9 However, amphotericin B has notorious toxicities and requires IV administration. While posaconazole and isavuconazole are available orally with fewer toxicities, their in vitro activity against the Mucorales is not predictable.^10,11  While there are no established clinical breakpoints for the Mucorales for any of the antifungal drugs, standardized susceptibility testing to isavuconazole and posaconazole could provide insight regarding the level of activity for each drug against this patient’s specific pathogen, thereby guiding choice of step-down therapy.

Diagnosis of mucormycosis is typically made based on a combination of clinical, imaging, culture, and histopathologic findings.

Grinding tissue for culture risks disrupting the fragile and sparsely septate hyphae, resulting in no viable cells. Instead, tissue should be finely minced and placed directly on fungal media.⁴  When recovered, these fungi grow rapidly on laboratory media, producing abundant cottony colonies (Figure 3b). Identification is based on their sporangial structures.⁸ However, culture is only approximately 50% sensitive for their recovery, even in microscopically positive samples.  Thus, molecular-based assays for detection of Mucorales DNA in culture-negative specimens are emerging as a key means of identifying the pathogen, particularly when the organism does not grow in culture.⁸ 

Answers 2 and 3 are incorrect. In the current case, the fungal pathogen was recovered in culture, thus extraction of DNA and sequencing for identification would add unnecessary expense. Identification of Rhizopus to species level also does not offer further information to guide management.

Answer 4 is incorrect. The non-invasive blood-based beta-D-glucan and galactomannan tests are not helpful in the diagnosis of mucormycosis because Mucorales species lack significant quantities of these polysaccharides in their cell walls.⁹

There is growing interest in the utility of molecular testing to noninvasively detect Mucorales DNA, but such testing is not approved by the United States Food and Drug Administration at this time. One prospective trial found the sensitivity and specificity of a serum Mucorales quantitative PCR to be 85.2% and 89.8%, respectively, and the first positive PCR was seen a median of 4 days (interquartile range 0-9 days) before the first positive mycological or histologic specimen in their study.¹²  Another study retrospectively analyzed archived specimens from hematopoietic stem recipients with invasive pulmonary mold (primarily Aspergillus and Mucorales) infection for the presence of microbial cell-free (mcf) DNA in plasma (Karius Laboratory, Redwood City, CA). The median mcfDNA concentrations were higher in Mucorales compared with Aspergillus cases, and high mcfDNA concentrations at diagnosis (mcfDNA concentrations >4.0 log₁₀ molecules per microliter) were associated with 6-week mortality.^13,14 Left to be explained is the variation in fungal DNA concentrations for Aspergillus compared with Mucorales infections. Ultimately, further study is needed to understand the role of molecular testing for diagnosis of Mucorales infections as well as the implications of fungal DNA concentration and kinetics on therapeutic response.