Infective Meningitis Caused by Phialemonium curvatum

Abstract

Infections caused by rarely encountered fungal pathogens have increased in recent decades. Phialemonium species are widely distributed in the environment and are also involved in human infections, affecting both immunocompromised and immunocompetent patients. The present study describes a case of meningitis caused by Phialemonium curvatum.

CASE REPORT

A 42-year-old male patient was admitted to the hospital for continuous headache without nausea and fever, with a maximum temperature of 39°C during the past 30 days. He was a poultry farmer and had experienced head trauma at the age of 19. The patient was conscious and claimed acroanesthesia. He denied any systemic disease, history of immunosuppression, or recent travel. When the patient was first admitted to the hospital, his physical examination showed that he had stable vitals, the neurological assessments were unremarkable, and no neck stiffness was noted. A routine blood test revealed a white blood cell (WBC) count of 11.6 × 10⁹/liter (53.7% neutrophils and 36.3% lymphocytes). The first lumbar puncture was performed under local anesthesia, revealing 110 × 10⁶/liter WBCs, 0.3 g/liter protein, 3.1 mmol/liter glucose, and 117 mmol/liter chloride. The pressure of the cerebrospinal fluid (CSF) was 250 mmH₂O. CSF cytology demonstrated the presence of approximately 70% lymphocytes and 30% neutrophilic granulocytes. A computed tomography scan of the head and a chest radiograph showed no alterations. Cranial magnetic resonance imaging was normal. An echocardiogram revealed that the left ventricle had a normal size and function, and no vegetation was detected. In addition, a serum test for human immunodeficiency virus (HIV) was negative. Aricine staining and India ink staining of the CSF were also negative. He was initially diagnosed with viral encephalitis and tuberculous meningitis and received antiviral therapy (ganciclovir, 0.25 g twice a day) for 10 days, as well as a standard 15-day course of antituberculosis therapy (isoniazid, 0.9 g/24 h, rifampin, 0.6 g once a day [q.d.], and pyrazinamide, 0.25 g three times a day [t.i.d.]). However, the treatment had little effect, and the patient still complained of a heavy headache. Measurements of CSF and blood (1-3)–β-d-glucan were 151 pg/ml and 466 pg/ml, respectively (with a cutoff value of <100 pg/ml). The later culture test showed that the four blood cultures were negative. Two of the CSF cultures became positive after 4 days of incubation in blood culture bottles (BacT/Alert, aerobic bottle; bioMérieux) without antibiotics. Subculturing of the positive CSF cultures revealed mold growth after 72 h of incubation. On traditional Sabouraud agar, the colonies were white, reaching 10 to 15 mm in diameter after 10 days at 25°C. Colonies were cream colored, and small areas of the colonies became light yellow after 30 days (Fig. 1). Microscopically, vegetative hyphae and conidia were hyaline. Accumulation of hyaline conidia was observed at the apex, and most of them were allantoid (Fig. 2). Scanning electron microscopy (SEM) images revealed that the shape of the conidia was either allantoid or obovate (Fig. 3). DNA was extracted using a TIANamp yeast DNA kit (Tiangen, Beijing, China) 10 days after fungal growth was detected in the CSF cultures. Five microliters of the extracted DNA was used to amplify the 28 S rRNA and internal transcribed spacer (ITS) genes using the primers P1 (forward, 5′-GATAGCGAACAAGTAGAGTGA-3′) and P2 (reverse, 5′-GTCCGTGTTTCAAGACGGGC-3′) and primers ITS1 (forward, 5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (reverse, 5′-TCCTCCGCTTATTGATATGC-3′). PCR was performed with a Bio-Rad MyCycler thermal cycler (Bio-Rad, Hercules, CA) as follows: 30 cycles of 30 s at 95°C, 45 s at 55°C, and 45 s at 72°C, followed by a final extension of 10 min at 72°C (1). The sizes of the PCR products were estimated using a standard DNA ladder and sequenced with an automated ABI Prism 377 DNA sequencer (Applied Biosystems Corp., Foster City, CA). Then, the sequences were analyzed by nucleotide-nucleotide basic local alignment search tool (BLAST) searches and DNAMAN 5.2 (Lynnon Biosoft, Pointe-Claire, Quebec). We found that the two amplified DNA fragments were 514 and 252 bp (GenBank accession numbers KF318613 and KF318614, respectively), which matched the Phialemonium curvatum sequences (GenBank AB568605) with high (99%) similarity. Ultimately, the patient was diagnosed with P. curvatum meningitis. In vitro antifungal susceptibility was determined by the broth dilution method. The MICs of the strain against fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), amphotericin B (AMB), and caspofungin were 32, 1, 0.5, 2, and ≥16 μg/ml, respectively.

FIG 1.

Culture on Sabouraud agar showed light yellow after 30 days.

FIG 2.

Microscopically, vegetative hyphae were hyaline, and conidia were hyaline (×1,000 magnification).

FIG 3.

The shape of the conidia was either allantoid or obovate (×6,000 magnification).

The patient was treated with intravenous VRC (0.40 g/12 h on day 1, and 0.20 g/12 h thereafter) for 2 weeks and subsequently made good progress toward recovery. Indeed, the symptoms of headache and fever decreased during the course of VRC treatment. However, the patient refused to continue this treatment due to his economic conditions. Thus, we treated him with FLC (0.40 g/24 h on day 1, and 0.20 g/24 h thereafter) and AMB (0.02 mg/kg body weight/day increased to 0.6 mg/kg/day) for approximately 2 months. Examination of CSF revealed 10 × 10⁶/liter WBCs (77% lymphocytes, 8% monocytes, and 15% neutrophils), 0.58 g/liter protein, 4.00 mmol/liter glucose, and 112 mmol/liter chloride after 2 months of treatment. The CSF pressure was 150 mmH₂O. Cultures of blood and CSF were both negative. The levels of CSF and blood (1-3)–β-d-glucan were 15 and <5 pg/ml, respectively. The patient continued medication (0.20 g/day FLC) after discharge. After 1 year of follow-up, the patient could work but still complained of acroanesthesia.

Phaeohyphomycosis is an infection caused by a large number of dematiaceous fungi, which affect cutaneous and subcutaneous tissues, the ocular region, the frontal and maxillary sinuses, lungs, bones, and the heart (i.e., endocarditis). The majority of Phialemonium infections are invasive, and the most frequent infections include peritonitis endocarditis, osteomyelitis, and cutaneous infections of wounds that may occur after a burn (2–6). In this case, the only theoretical factor that might raise doubts about the patient's complete immunological integrity was his occupation, bringing him in frequent contact with chickens, and the head trauma at the age of 19. In reported Phialemonium infection cases, the filamentous fungus is isolated mainly from blood samples obtained from patients with primary fungemia (3–5). In the current case, the diagnosis of meningitis caused by P. curvatum was confirmed by CSF culturing, but the blood cultures were negative.

Initially, clinicians misdiagnosed him with common encephalitis because he presented nonspecific signs and symptoms and denied any prior steroid use. Phialemonium is often mistaken as a yeast on Gram stains of blood and CSF specimens, and similar phenomena have been observed in cases of infection caused by Fusarium and Acremonium (4, 7). In recent years, molecular diagnostic tools such as PCR have been used to analyze fungal pathogens, especially Aspergillus and Candida species (8). In clinical samples, these sensitive and specific methods utilize primer pairs that are complementary to the highly conserved 18S, 5.8S, and 28S regions of the fungal rRNA genes and ITS regions, which enable differentiation of many fungal species. Using specific primer pairs for quick molecular detection of Phialemonium is expected to be the most therapeutically effective. The measurement of plasma (1-3)–β-d-glucan can also provide early and reliable information on deep fungal infections. In this case, plasma (1-3)–β-d-glucan measurement was sensitive for the diagnosis of fungemia.

In the current case, the antifungal susceptibility results for the case isolate were similar to those previously reported in the literature (1, 5, 9), i.e., the strain was sensitive to VRC. Due to his economic condition, we initially treated him with VRC but then used FLC and AMB for longer-term therapy. His headache and fever improved; however, the patient still complained of acroanesthesia after 1 year of follow-up. We attribute his recovery to the use of antifungal drugs and the improvement of his living standard. Nevertheless, we do not know whether the use of FLC and AMB was effective in the present case.

To our knowledge, this is the first case of meningitis infection due to P. curvatum reported in China. Phialemonium infections may be misdiagnosed initially because of the nonspecific clinical manifestations and lack of accurate identification. Our study demonstrates that molecular biological techniques and morphological observation contribute to the proper diagnosis of rare pathogens. In addition, we also alert clinicians about the rarity of the case and the increasing occurrence of emerging fungi in immunocompromised patients.