The Brief Case: Yeast, chlamydospores, and hyphae—a case of disseminated mucormycosis

CASE

A 63-year-old male with refractory mantle cell lymphoma was admitted for chimeric antigen receptor T cell (CAR-T) therapy. His course was complicated by grade 4 cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and hemophagocytic lymphohistiocytosis requiring high degrees of immunosuppression. Clinical history was notable for an unresolved fracture to the maxillary anterior bridge and trauma of the intraoral soft tissues 6 months prior after biting the endotracheal tube. On day 12 of hospitalization, the patient required intubation after fever and acute respiratory distress. Hypoxia resolved but he remained intubated due to altered sensorium. During this time, CT imaging revealed an interval increase in the size of bilateral pulmonary nodules and axillary and mediastinal lymphadenopathy. The nodules were noted prior to receiving CAR-T therapy and attributed to underlying malignancy after a bronchoscopy was unrevealing for infection.

On day 11, a sacral wound with discoloration was noted, and by day 17, the patient had developed painful, bilateral knee bruises, even while on high-dose micafungin since day 7. Within a few days, he was transitioned to oral posaconazole after new violaceous, indurated purpuric lesions appeared on his lower extremities. The patient subsequently developed similar lesions on all extremities and the face and a gastrointestinal (GI) bleed that required artery embolization. Dermatology was then consulted for biopsy. Suspicion was highest for inflammatory drug reaction from CAR-T therapy or an atypical vasculitis. Over the next 24 hours, the lesions precipitously expanded. The rapidity of evolution raised concern for disseminated fungal infection, and combination therapy was started with high dose-IV liposomal amphotericin and IV voriconazole. On the same day, standard anaerobic and aerobic blood culture bottles (BacT/ALERT 3D) turned positive at 23 and 29 hours, respectively. Gram stains showed Gram-positive cocci in clusters, which were identified as Staphylococcus epidermidis.

Rapid floccose colony growth was observed on TSA with sheep blood after 1 day (Fig. 1A). Fungal elements were scarce in smears from both positive bottles and were not observed until reviewed later. Fungal morphology on Gram stain showed long, pauci-septate hyphal elements and yeast with multipolar budding (Fig. 1B). While not routine practice, we prepared calcofluor white fluorescently stained blood smears to confirm fungal morphologic features (Fig. 1C through D). Lactophenol aniline blue stain from colony growth on potato dextrose agar was used to visualize globose, terminal sporangia containing sporangiospores, rough sporangiophores, columella, broad pauci-septate hyphae, and chlamydospores. The mold was identified by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS) (VITEK MS v3.2, 99.9% confidence) as Mucor velutinosus although not part of the FDA-approved database. Sequencing of the internal transcribed spacer (ITS) region demonstrated high sequence similarity (both 99.5% match) between M. velutinosus (GenBank: OW987840.1) and Mucor ramosissimus (GenBank: LC390232.1). ITS sequencing alongside growth characteristics (37°C growth, rough sporangiophores) assigned identification of this isolate to M. velutinosus.

Fig 1.

Positive blood culture with M. velutinosus shows pleomorphic fungal elements. Positive blood subculture with rapid, cottony mold growth over bacterial colonies after 1 day of incubation at 37°C on TSA with sheep blood (A). Gram stain from positive blood culture bottles shows pauci-septate, ribbon-like hyphae characteristic of Mucor spp. at 1,000× (B). Calcofluor white was used to stain and image positive blood cultures and shows pleomorphic yeast and hyphal elements, including multipolar budding yeast (C) and 90° angle branching, terminal chlamydospores, and successive chlamydospores (D) at 400×.

The patient was diagnosed with disseminated mucormycosis after a skin biopsy from purpuric lesions showed abundant fungal organisms with vascular invasion and in conjunction with fungal recovery from blood cultures (Fig. 2A through D). Skin biopsy was not sent for fungal culture. The patient died within 48 hours of diagnosis.

Fig 2.

Histologic assessment of cutaneous lesions showing angioinvasion. Disseminated, purpuric cutaneous lesions on extremities (A). Representative histological section of the 63-year-old patient’s skin punch biopsy, showing separation of the epidermis as well as fungal elements (black arrows) present in the dermal stroma and small blood vessels. Hematoxylin and eosin (H&E), 10× magnification (B). Representative histologic sections from the patient’s skin showing invasion of fungal elements into the vessel walls and lumen near the resection margin (inked black). H&E, 10× magnification (C). Higher magnification shows broad, pauci-septate hyphae branching at 90° angles (black arrow), a morphology characteristic of Mucor spp. Note the presence of fungal elements approaching and invading the blood vessels as well as a peripheral nerve (blue arrow). H&E, 20× magnification (D).

DISCUSSION

Mucor spp. are in the Mucorales order and are characterized as having broad hyphae (6–15 µm wide) with few or no observable septations and a sac-like sporangium containing spores. Macroscopic colony morphology of various Mucorales is gray and rapidly fills the agar with cottony growth. Microscopic details including size and shape of sporangia and columella, characteristics of sporangiophores (branched, unbranched, rough, and smooth), presence or absence of an apophysis or rhizoids, and rhizoid location relative to sporangiophores can be utilized to differentiate Mucorales members to the genus level (1). Mucor spp. can grow well below or at 37°C (1–3).

Infections caused by fungi in the order Mucorales are described as mucormycosis (previously called zygomycosis) and develop by inhalation, ingestion, or percutaneous implantation of spores in a susceptible host. The disease typically presents as pulmonary, rhino-orbital-cerebral, or cutaneous, although disseminated and GI presentations can also occur (4). Mucormycosis in immunocompetent hosts is rare and is usually associated with trauma, manifesting as a cutaneous disease after inoculation of spores into the skin. More commonly, mucormycosis develops as an opportunistic infection in patients with predisposing comorbidities such as diabetes mellitus, neutropenia, corticosteroid therapy, hematological malignancies, transplantation, HIV/AIDS, severe burns, intravenous drug use, iron overload conditions, and malnutrition (4). The clinical presentation of mucormycosis is precipitous, where rapid disease progression leads to high mortality. Mucorales have a predilection for invading blood vessels (Fig. 2B through D) and subsequently cause thrombosis, infarction, and necrosis, which contribute to severe disease pathology and dissemination (1, 4). If mucormycosis is suspected when sending a tissue specimen, the laboratory should be notified for special processing. Mucormycetes are fragile and hyphae are easily destroyed by grinding or homogenizing tissues; tissue should be minced instead. For pathology stains, hematoxylin and eosin is adequate to show tissue invasion with broad, irregular hyphae with few septations and wide-angle branching.

Here, positive blood culture smears portrayed budding yeast, hyphal elements with minimal branching, terminal chlamydospores, and successive chains of chlamydospores, with other areas also showing long pauci-septate hyphal elements. Several Mucor spp. have been described to have dimorphic qualities, characterized as growing as a spherical multipolar budding yeast. However, Mucor spp. are not grouped together with thermally dimorphic fungi; thus, this aspect of their growth may be commonly overlooked. Multipolar budding yeast forms in Mucor spp. are attributed to specific growth conditions, including anaerobic or high-CO₂ environments, whereas hyphal growth is expected in aerobic conditions (5). It is important to keep in mind that Mucor may demonstrate many various morphologies when grown in oxygen-limited or elevated CO₂ conditions present in blood culture bottles, including hyphae, chlamydospores, and yeast-like cells.

Although Mucor spp. can cause serious disease, they are also common environmental contaminants and interpretation of their detection should be considered within the clinical context of the patient. Within the last 2 years, we have seen three patients with blood cultures (pediatric, aerobic, and anaerobic bottles) drawn at different locations, with yeast and mold forms of Mucor spp. in positive blood culture smears. This makes a single cause of environmental contamination unlikely. MALDI-TOF MS and ITS sequencing were used to identify two isolates as Mucor circinelloides, and this case as M. velutinosus. Reporting to the genus level is adequate for diagnosis and appropriate clinical treatment, and successful treatment is dependent on early diagnosis, treatment of the underlying condition, removal of the infectious focus, and/or concurrent antifungal treatment with amphotericin B (6). If complications arise with amphotericin B treatment, such as nephrotoxicity or electrolyte imbalance, isavuconazole and posaconazole are recommended second-line therapies for mucormycosis (6). The two patients with M. circinelloides had no evidence of clinical disease and therefore did not require treatment. In these cases, M. circinelloides was recovered alone from a positive blood culture bottle (BACTEC FX, Aerobic Plus) and co-isolated with Micrococcus luteus from a pediatric collection (BacT/ALERT 3D, PF Plus).

Despite the predilection for blood vessel invasion, Mucorales are rarely detected in the bloodstream (7). M. velutinosus was first described from a clinical sample in 2011 (2), with four case reports, one describing hematogenous dissemination causing cutaneous disease (8) and three cases of fungemia (3, 7, 9). M. circinelloides is also a rare cause of fungemia with only three published case reports (10–12). In one case, treatment was started after the patient also demonstrated cutaneous disease from which M. circinelloides was recovered; however, their clinical course worsened and the patient expired (12).

In this case, the 63-year-old patient was in a highly immunosuppressed state from his underlying refractory malignancy and complications from CAR-T therapy, which placed him at risk for disseminated fungal disease. Although the patient was on systemic antifungal therapy, he was not transitioned to liposomal amphotericin B until day 29 of his hospital admission. Importantly, echinocandins such as micafungin are not recommended as monotherapy for the treatment of mucormycosis (6). The disease onset of this patient remains unclear given his complicated clinical picture, but it could be subsequent to inhalation given pulmonary symptoms or implantation following maxillofacial trauma earlier in the year. Disseminated disease was evident with fungal organisms present in cutaneous lesions, and GI presentation cannot be ruled out given the GI bleed. The diagnosis was confirmed by pathology with extensive fungal angioinvasion and recovery of M. velutinosus from positive blood cultures. Despite treatment with systemic antifungals throughout his hospital course, the patient died shortly after the infectious diagnosis was made. An autopsy was declined by the family.

Here, we describe that growth in blood culture bottles may allow for the fungal dimorphic state of M. circinelloides and M. velutinosus to be observed. Increasing awareness of the growth characteristics of Mucor spp. is warranted. It is unclear if growth of Mucor alone would routinely allow for blood culture systems to flag bottles as positive or if growth with S. epidermidis and M. luteus allowed for incidental detection of Mucor in these cases.

Invasive mucormycosis caused by M. circinelloides and M. velutinosus have been subject to infrequent case reports over the decades. This may be due to the fact that many clinical laboratories do not identify Mucorales members to the species level. With the implementation of technologically advanced identification techniques, such as MALDI-TOF MS and sequencing, full identification and species-level reporting are becoming more common. MALDI-TOF MS for molds still has some weaknesses, including limited organism representation and spectra number, which may require repeat testing and can lead to low identification rates. There are two FDA-approved/cleared databases depending on the instrument in use, creating a constraint in the organisms represented. These limitations can be addressed with the use of expanded research-use only databases and the supplementation of mass spectral profiles into the library.

SELF-ASSESSMENT QUESTIONS

1. What is the most common site of mucormycosis in immunocompetent hosts?

   1. Bloodstream

   2. Cutaneous

   3. Pulmonary

   4. Rhino-orbito-cerebral

2. Which environmental condition induces yeast formation by Mucor species?

   1. Growth in an aerobic blood culture bottle at 35°C

   2. Growth in an anaerobic blood culture bottle at 35°C

   3. Growth on an aerobic agar plate at 25°C

   4. Answers a and b

3. Which of the following laboratory methodologies is most likely to provide a diagnosis of mucormycosis?

   1. Beta-D-glucan antigen testing

   2. Homogenized tissue plated for fungal culture

   3. Minced tissue plated for fungal culture

   4. Blood culture set drawn before antifungal treatment

ANSWERS TO SELF-ASSESSMENT QUESTIONS

• 1What is the most common site of mucormycosis in immunocompetent hosts?

  1. Bloodstream

  2. Cutaneous

  3. Pulmonary

  4. Rhino-orbito-cerebral

Answer: b. Cutaneous. The primary route of entry and infection with mold spores is through inhalation. In an immunocompetent host, the intact immune system clears these without establishing infection, unlike immunocompromised individuals who are at higher risk for rhino-cerebral or pulmonary mucormycosis. It is more common that an individual without an underlying immune disruption presents with cutaneous mucormycosis after traumatic inoculation with mold spores; this can occur after explosions, motor vehicle accidents, burns, natural disasters, etc. Notably, fungemia with Mucorales is quite rare. Although Mucorales have a predilection for blood vessel invasion, their recovery from blood is uncommon.

• 2Which environmental condition induces yeast formation by Mucor species?

  1. Growth in an aerobic blood culture bottle at 35°C

  2. Growth in an anaerobic blood culture bottle at 35°C

  3. Growth on an aerobic agar plate at 25°C

  4. Answers a and b

Answer: d. Answers a and b. The classic definition of dimorphic fungi is characterized by microorganisms in the form of yeast or spherules at higher temperatures (35°C–37°C) and filamentous mold at lower temperatures (25°C). The growth environments that allow for Mucor spp. to grow in the yeast phase are driven by anaerobic or high-CO₂ conditions, not temperature. As organisms proliferate in broth-based aerobic and anaerobic culture bottles, an increase in CO₂ occurs, supporting the growth of Mucor spp. yeast phase. For these three patients, we observed pleomorphic fungal elements representative of yeast and mold forms from the positive bottle Gram stains after incubation at 35°C and rapid growth of the mold form on subculture plates incubated at 25°C and 35°C.

• 3Which of the following laboratory methodologies is most likely to provide a diagnosis of mucormycosis?

  1. Beta-D-glucan antigen testing

  2. Homogenized tissue plated for fungal culture

  3. Minced tissue plated for fungal culture

  4. Blood culture set drawn before antifungal treatment

Answer: c. Minced tissue plated for fungal culture. Of the answers provided here, fungal cultures from tissue specimens processed by mincing tissue inflict less damage to hyphae as opposed to homogenization, thereby increasing the likelihood of retaining viable organism for recovery in culture. Mucormycetes do not produce Beta-D-glucan or galactomannan and therefore these fungal antigen tests cannot be used for the diagnosis of mucormycosis. And, although described in this case, recovery of Mucor spp. from positive blood cultures is infrequent. Blood cultures should not be relied upon for the attempted diagnosis of mucormycosis. Finally, experienced pathologic review of tissue sections can also be used for the diagnosis of mucormycosis, using morphologic characteristics, including broad, irregular hyphae with few septations, wide angle branching and common pathologic findings, such as angioinvasion.

TAKE-HOME POINTS.

• Mucormycosis can present with rhino-orbito-cerebral, pulmonary, cutaneous, and/or disseminated manifestations.

• Treatment of mucormycosis may prevent severe morbidity and mortality and can include treating underlying symptoms, removal of infected tissues, or amphotericin B treatment as first-line antifungal therapy.

• In general, Mucor spp. grow rapidly, filling culture plates within 1–2 days with cottony colonies, and are characterized by globose sporangia containing sporangiospores, columella, and broad ribbon-like pauci-septate hyphae.

• Some species of Mucor can exhibit multiple morphologies, including yeast, hyphae, and chlamydospores when grown in blood culture bottles, although they grow as a filamentous mold on subculture plates at 35°C.

• A diagnosis of mucormycosis can be made within the clinical context of patient presentation from positive microbiology culture(s) and/or histopathological evidence.