The Brief Case: Encephalitis in a West Texas Woman

CASE

A 59-year-old West Texas female who had a history of rheumatoid arthritis (RA), recently completed methotrexate and tocilizumab for RA management, and has type II diabetes mellitus and hypothyroidism presented to her local hospital with a chief complaint of severe headache in late summer. She was treated with antibiotics, clinically improved, and then discharged. In early fall, she presented with severe headache. A lumbar puncture (LP) revealed a white blood cell (WBC) count of 1,300 with neutrophil predominance and a protein level of 124 mg/dL. Following admission, vancomycin and meropenem were initiated and continued through year end. Repeat LPs revealed a downtrending WBC count, but a persistently elevated protein. All cerebrospinal fluid (CSF) cultures were negative. Brain magnetic resonance imaging (MRI) revealed an enhancement of unclear significance along the left frontal horn. Brain biopsy was recommended. The patient declined.

Early in the following year, the patient developed a severe headache with altered mental status. A brain MRI showed subependymal enhancement in the anterior aspect of the left lateral ventricle with obstructive hydrocephalus. The patient underwent external ventricular drain (EVD) placement and biopsy. Intraoperative cultures and histopathology were unrevealing. A week later, she was transferred to a tertiary care center. On admission (hospital day [HD]0), serum 1,3-β-d-glucan (BDG) was greater than 500 pg/mL, and CSF analysis showed glucose of 81 mg/dL, protein of 85 mg/dL, 7 nucleated cells (6/7 lymphocytes), and 8,000 red blood cells. CSF was also submitted for culture. The patient’s vitals were stable, but the neurological exam was variable. The patient was awake, nonverbal, and had spontaneous, purposeful movement on the left, but no movement on the right.

Given the patient’s previous broad coverage with antibiotics, continued laboratory abnormalities, and clinical decline, invasive fungal infection was high on the differential. Liposomal amphotericin B and fluconazole were empirically started on HD1. On HD2, mold was recovered from CSF cultures obtained on admission with the preliminary morphological identification suggesting Coccidioides sp. (Fig. 1A and B). Lactophenol cotton blue stain demonstrated rectangular arthroconidia with gaps between the arthroconidia, suggesting disjunctor cells. No pigment was visible. The isolate was subcultured and sent to a reference laboratory for definitive identification on HD4. By HD7, the mold developed pigment characteristic of dematiaceous molds (Fig. 1C and D). Fluconazole dosing was increased with the continuation of amphotericin B therapy. The clinical team ordered that a serum Coccidioides sp. antibody titer be sent to a reference laboratory, which resulted as negative on HD12.

FIG 1.

Comparative colony morphology and microscopic evaluation of Neoscytalidium sp. (A–D) and Coccidioides sp. (E–F). (A) Light, wooly growth of Neoscytalidium sp. on chocolate agar at 48 h incubation from CSF. (B) Lactophenol cotton blue (LCB) tape preparation (4000×) of the mycelial growth of Neoscytalidium at 48 h incubation, showing rectangular, nonpigmented arthroconidia with spaces suggestive of dysjunctor cells. (C) Dark, wooly mold on chocolate agar at 7 days incubation. (D) LCB demonstrating the arthroconidia with melanized, irregular, rectangular arthroconidia without appreciable spaces (4000×). (E) Coccidioides sp. demonstrating velvety mold growth on Sabouraud agar at 5 days incubation (representative image for comparison), (F) LCB demonstrating alternating, barrel-shaped arthroconidia that lack the pigmentation, diagnostic of Coccidioides sp. (4000×) (representative image for comparison).

Despite aggressive intervention, the patient’s condition deteriorated. A second EVD was placed on the contralateral side on HD11. A brain MRI on HD14 revealed extensive intraventricular hemorrhage in the left lateral ventricle, worsened left to right midline shift, and worsening uncal herniation. On HD15, the reference laboratory definitively identified the mold as Neoscytalidium sp. by morphology. Management was changed to voriconazole and amphotericin B. Debridement was undertaken on HD25. The patient’s prognosis was poor. She continued therapy without improvement until discharge to her local hospital 2 months later for hospice care, where she passed away the following month.

DISCUSSION

Neoscytalidium sp. are members of the Botryosphaeriaceae family of fungi. As phytopathogens, they are present in soil and associated with canker disease and rot of plants and fruit trees. Human infections are uncommon and predominantly superficial (e.g., onychomycosis, dermatomycosis), although Neoscytalidium sp. does not grow on dermatophyte isolation media containing cycloheximide, which possibly contributes to under-recognition (1). These ascomycetes are also infrequent causes of phaeohyphomycosis. Rare cases of disseminated Neoscytalidium infections among solid organ transplant patients and patients with immune dysfunction have been reported (2). While this case’s epidemiology is unknown, this patient’s RA was managed using immunosuppressive agents, likely predisposing her to opportunistic infection.

Morphological identification remains among the most widely utilized diagnostic approaches in clinical mycology. While generally consistent, it is important to remember that characteristic structures or pigment may not always be identifiable, particularly in immature fungal growths or upon exposure to antifungal agents. Mycelial morphology can change significantly over time, so the careful consideration of all reasonable organisms when evaluating macroscopic and microscopic fungal characteristics is crucial. Consultation of those with technical expertise in mycology can improve diagnostic accuracy and outcomes.

Arthroconidia are asexual spores formed by the fracture of hyphae at the point of septation. Clinically relevant fungi that predominantly produce arthroconidia include Coccidioides sp., Trichosporon sp., Arthrographis sp., Malbranchea sp., Geomyces sp., Geotrichum sp., and Neoscytalidium sp. The most frequently encountered pathogenic species (especially in the southwestern United States) is Coccidioides sp. In contrast, these additional species generally cause invasive infections only in severely immunocompromised hosts. Coccidioides sp. produce delicate, hyaline hyphae with thick-walled, barrel-shaped arthroconidia. Characteristic thin-walled disjunctor cells alternate between the arthroconidia and lyse to release the spores from the hyphae (Fig. 1E and F) (1, 3).

Trichosporon sp. are associated with hair infection (white piedra) and disseminated infections in immunocompromised populations. Trichosporon sp. are hyaline, mold-like yeasts which produce budding yeast cells, hyphae, and abundant, nonalternating, rectangular arthroconidia. Arthrographis sp. is associated with mycetoma, onychomycosis, and (infrequently) eye infections, with nonalternating, rectangular arthroconidia that branch from a conidiophore. Malbranchea sp. are generally considered contaminants with arthroconidia and disjunctor cells similar to Coccidioides sp. Fragmented Malbranchea sp. arthroconidia bear an annular frill, which is the attached remnant of the disjunctor cell. In contrast, Malbranchea sp. have flat, rectangular arthroconidia, not barrel-shaped, like Coccidioides sp. Geomyces sp. rarely cause human infection and do have alternating, barrel-shaped arthroconidia, but Geomyces sp. arthroconidia branch from a conidiophore, like Arthrographis sp. Geotrichum sp. are commensal organisms of the skin, hair, and nails that elaborate nonalternating, rectangular arthroconidia and either conidiophores or blastoconidia, depending on the species. (1, 3).

In this case, the clinicians taking care of the patient had a high suspicion for invasive fungal infection, as she had been on long-term, broad spectrum antibiotics but had persisting abnormalities in her CSF and on imaging. Her serum BDG test demonstrated >500 pg/mL, which is highly suggestive of invasive fungal infection. Numerous fungi, including Candida sp., Aspergillus sp., Histoplasma sp., Coccidioides sp., and dematiaceous molds elaborate variable amounts of 1,3-β-d-glucan that can be measured in serum or body fluids. It is important to note that, although there was a high pretest probability that this patient had an invasive fungal infection, the BDG test is not specific. CSF culture yielded significant mycelial growth on conventional media within 48 h. Despite an initial white, cottony appearance, extensive melanized pigment developed within the mycelium over a one-week period. Thus, the primary diagnostic challenge in this case was that the dematiaceous nature of the mold was not immediately appreciable. The patient was also from a region of the United States where Coccidioides sp. is endemic, and the rapid fungal growth on routine media raised further suspicions for this organism. Moreover, when microscopically evaluated, the lack of melanotic pigment and the presence of arthroconidia were further suggestive of coccidioidomycosis. The thick walled arthroconidia in chains can be confused with Coccidioides sp. when the mold is immature or if the culture presentation is atypical. Following additional incubation, as the mold matured, pigment within the hyphae did develop, and the arthroconidia did not exhibit the characteristic barrel shape or disjunctor cells, leading to an accurate identification.

Arthroconidia production among dematiaceous molds is uncommon. Of the aforementioned arthroconidia-producing fungi, Neoscytalidium sp. is the only dematiaceous species. Septate hyphae are often present without conidiophores with accompanying thick-walled, oval-to-rectangular, nonalternating, pigmented arthroconidia. Nonpigmented mutants of dematiaceous Neoscytalidium sp. can occur (Neoscytalidium hyalinum), further complicating morphological identification. Dematiaceous fungi are also an infrequent cause of CNS infections in immunosuppressed patients. CNS infections in immunosuppressed populations caused by Cladophialophora bantiana and Bipolaris sp. have been reported but are uncommon. Morphologically, these fungi produce variably thick hyphae with occasional to frequent septations but do not produce arthroconidia (4). Like Neoscytalidium sp., macroscopic observations of immature growths of these phaeohyphomycotic molds may not exhibit characteristic melanin pigment.

Molecular methods of identification, including matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gene sequencing, and DNA probes offer alternatives to morphological diagnosis. The two most common MALDI-TOF MS vendors’ libraries are not uniform with respect to fungal target entries. Bruker Daltonics, Inc. (Billerica, CA) markets a research-use-only filamentous mold database which includes entries for Neoscytalidium sp. but does not contain entries for Coccidioides sp. (5). Conversely, bioMérieux, Inc. (Durham, NC) markets an FDA-cleared database which includes Coccidioides sp. but does not contain entries for Neoscytalidium (6). Thus, laboratories choosing to utilize MALDI-TOF MS for filamentous mold identification should be aware of the limitations of each manufacturer’s databases. Additionally, despite MALDI-TOF MS becoming the gold standard for bacterial identification, fungal identification using the technology is not yet available in all clinical laboratories.

Sequencing methods, including ITS gene sequencing, are commonly used to differentiate and identify fungi at the genomic level. Despite clinical utility, these methods are not as ubiquitous as morphologic identification in routine settings. Sequencing methods take time, are not always cost-effective, and frequently require the technical expertise of a reference laboratory. For laboratories without access to on-site expertise regarding atypical fungal morphology, molecular confirmation may be needed to confirm diagnostic accuracy.

CNS infections are treated with surgical debridement and prolonged therapy with an azole anti-fungal therapy. Multiple studies have investigated the in vitro susceptibility of Neoscytalidium to common antifungals. Amphotericin B, voriconazole, and terbinafine appear to be the most potent antifungal agents and exhibit the lowest MICs (7). Despite aggressive antifungal therapy and debridement, the mortality rate of CNS infections with this organism is nearly universal (2). Of the reported cases of Neoscytalidium CNS infection, morality has been 100%, despite aggressive antifungal therapy and, in some cases, surgical debridement.

SELF-ASSESSMENT QUESTIONS

1. Which of the following fungi predominantly produce arthroconidia?

   1. Bipolaris sp.

   2. Cladosporium sp.

   3. Neoscytalidium sp.

   4. Alternaria sp.

2. Which of the following fungi exhibit characteristic disjunctor cells between arthroconidia?

   1. Coccidioides sp.

   2. Neoscytalidium sp.

   3. Geotrichum sp.

   4. Histoplasma sp.

3. Which of the following therapies might be used to treat phaeohyphomycosis?

   1. Voriconazole

   2. Micafungin

   3. Fluconazole

   4. Nystatin