Chronic Pulmonary Aspergillosis Caused by Aspergillus tubingensis Diagnosed by a Bronchoscopic Biopsy

Abstract

We herein report a case of chronic pulmonary aspergillosis (CPA) caused by Aspergillus tubingensis diagnosed by a bronchoscopic biopsy with negative serological and sputum culture findings. A 66-year-old man was referred for the assessment of a pulmonary cavity. Computed tomography showed a thick-walled cavity in the upper right pulmonary lobe. Serum β-D glucan, Aspergillus galactomannan, and Aspergillus antibody tests were negative. Aspergillus species were not detected in the sputum. Culture and pathological specimens were obtained from the mass by bronchoscopy. Microscopic examination findings were consistent with Aspergillus niger complex morphologically and identified as Aspergillus tubingensis through DNA sequencing. The patient was diagnosed with chronic pulmonary aspergillosis.

Introduction

Aspergillus is the most frequent pathogen that causes pulmonary mycosis in humans. Chronic pulmonary aspergillosis (CPA) is diagnosed definitively based on microbiological and/or histopathological findings in respiratory specimens, referring to clinical, radiological, and serological findings (1-3). Specifically, Aspergillus antibody tests have a high sensitivity for the diagnosis of CPA.

Aspergillus niger is a black aspergillus, and Aspergillus section Nigri are the second-most prevalent causative species of aspergillosis after A. fumigatus in Japan (4,5). Black Aspergillus species are difficult to differentiate by conventional examination methods based on macroscopic and microscopic morphological criteria and have often been identified as Aspergillus niger complex (6). DNA sequencing of calmodulin and β-tubulin genes is performed to identify the Aspergillus species, differentiating this section's cryptic species, such as A. niger sensu stricto, A. tubingensis, and A. welwitschiae (6-8).

We herein report a case of CPA associated with A. tubingensis diagnosed by bronchoscopy, in which a fungal specimen was obtained from the fungal mass in the cavitary wall.

Case Report

A 66-year-old man was referred to our hospital for a further assessment of a pulmonary cavity diagnosed a few years earlier. He reported chronic cough, sputum, and intermittent hemoptysis but no fever, chills, or dyspnea. There was no medical history, medication use, or family history of respiratory diseases. He had smoked 30 cigarettes daily for 30 years. The patient denied alcohol consumption, illicit drug use, occupational exposures, any recent travel, or exposure to tuberculosis.

The patient's body temperature was 36.2°C, blood pressure was 148/93 mmHg, heart rate was 76 beats/min, SpO₂ was 99% on ambient air, and body mass index was 16.33 kg/m². Chest auscultation revealed decreased bilateral breath sounds. All other physical examination findings (abdomen, skin, and lymph node stations) were normal. A blood examination revealed C-reactive protein levels of 4.50 mg/dL and a white blood cell (WBC) count of 7.5×10³/μL (70.9% neutrophils, 19.1% lymphocytes, and 2.4% eosinophils). His renal function, liver function, HbA1c, and human immunodeficiency virus (HIV) antigen antibody test results were within normal ranges. Serum β-D glucan, Aspergillus galactomannan, and FSK1 Aspergillus immunodiffusion system findings (Microgen, Surrey, UK) as well as Platelia Aspergillus IgG (Bio-Rad, Hercules, USA), Capilia MAC Ab ELISA (TAUNS, Izunokuni, Japan), and T-SPOT.TB results (Oxford Immunotec, Abingdon, UK) were also negative.

Chest computed tomography (CT) showed a thick-walled cavity in the upper right pulmonary lobe and emphysematous changes in both lungs (Fig. 1). Klebsiella pneumonia, Pseudomonas aeruginosa, and a morphologically unidentifiable fungus were detected in the sputum. Mycobacteria were not detected. The fungus was identified as Coprinellus radians by DNA sequencing of the Domain 1 (D1)/Domain 2 (D2) and internal transcribed spacer region (ITS) 1/ITS 2 genes (DDBJ accession no. LC612525).

Figure 1.

Chest radiography and computed tomography (CT) findings. (A) Chest radiography shows a cavity shadow in the upper right lung field (black arrow). (B) Chest CT reveals a thick-walled cavity in the upper right lung lobe (black arrow) and emphysematous changes in both lungs.

Bronchoscopy was performed for a definitive diagnosis, suspecting chronic pulmonary mycosis (CPM). Bronchoscopy reached the cavity from the conducting bronchus (B3a), and a yellow-brown mass was observed on the cavitary wall (Fig. 2). Culture and pathological specimens were obtained from the mass. A black colony grew from the fungal mass on potato dextrose agar (Fig. 3A). A microscopic examination revealed long, smooth hyphae and phialides with radial heads throughout the endoplasmic reticulum, consistent with A. niger complex morphologically. The A. niger complex was identified as A. tubingensis through DNA sequencing of β-tubulin and calmodulin genes (DDBJ accession no. LC612595 and LC612596, respectively). The minimum inhibitory concentrations of A. tubingensis were as follows: itraconazole (ITCZ) 2 μg/mL, voriconazole (VRCZ) 4 μg/mL, and amphotericin B 1 μg/mL. The minimum effective concentration of micafungin was ＜0.015 μg/mL. In addition, the pathological findings of the mass revealed dichotomously branching hyphae, consistent with Aspergillus species, which was surrounded by an eosinophilic material known as Splendore-Hoeppli phenomenon (Fig. 3B). Consequently, the patient was diagnosed with CPA caused by A. tubingensis.

Figure 2.

Yellow-brown mass observed via bronchoscopy on the cavitary wall.

Figure 3.

(A) A black surface colony grew, which was consistent with A. niger complex. (B) The fungal mass was composed of dichotomously branching hyphae, consistent with Aspergillus species, surrounded by an eosinophilic material known as Splendore-Hoeppli phenomenon.

The patient was treated with ITCZ for one month but experienced a deterioration in his condition, as evidenced by chest radiography and the C-reactive protein level (CRP); therefore, micafungin was added to intensify the therapy. However, the treatment with itraconazole and micafungin was discontinued because the patient developed a skin rash. The patient was treated for three months with VRCZ after the skin rash had improved. On day 140, the CRP level was found to be elevated again, and the thick-walled cavity and pericavitary infiltrate indicated a worsening of the condition. The VRCZ trough concentration was 5.15 μg/mL, and he had no adverse effects of VRCZ with a high blood VRCZ concentration. Liposomal amphotericin-B was administered for one month with improvement. VRCZ was selected for subsequent maintenance therapy because the patient had developed drug eruption in response to ITCZ. Currently, oral antifungal therapy is ongoing (Fig. 4).

Figure 4.

Clinical course of the present case. Treatment was initiated with itraconazole. On day 28, fluid levels in the cavity were exacerbated, and CRP levels were elevated. Micafungin was added to intensify treatment. On day 35, the patient developed a drug rash, necessitating the discontinuation of medication for four weeks until the rash improved. On day 56, voriconazole was administered as an alternative antifungal therapy. On day 140, CRP was elevated again, and thickening of the cavity wall and consolidation around the cavity were observed. Subsequently, liposomal amphotericin-B was administered. On day 168, the pulmonary infiltrate showed improvement, and the patient was managed with voriconazole as maintenance antifungal therapy.

Discussion

We first suspected that the patient had CPA, but sputum culture and serological tests for aspergillosis were negative. A. niger complex, identified as A. tubingensis was isolated from the fungal mass. The pathological findings of the mass revealed dichotomously branching hyphae, which was consistent with Aspergillus species.

Aspergillus antibody tests have high sensitivity with efficacy to diagnose CPA clinically. However, the sensitivity was evaluated based on CPA caused by A. fumigatus primarily. We reported that the sensitivity of the Aspergillus antibody tests in patients with non-fumigatus CPA was lower than that in patients with A. fumigatus-associated CPA (9,10). Another factor associated with false negative results in Aspergillus antibody tests was the host immunodeficiency, although this patient had no immunodeficiency.

Aspergillus tubingensis isolates had higher minimum inhibitory concentrations against azoles than those of other species of Aspergillus section Nigri (8). The clinical breakpoint of A. tubingensis is undefined. The epidemiological cut-off values (ECVs) for individual Aspergillus species have not been established (11). However, patients with A. tubingensis-caused pulmonary aspergillosis might have a clinical course as poor as that of azole-resistant A. fumigatus-associated CPA. In the present case, azoles were ineffective.

Coprinellus radians, a basidiomycete, was detected only once in the patient sputum, so we considered it contamination. There has been no report of human infection caused by C. radians. Basidiomycetes have emerged as an important human pathogen causing allergic diseases and invasive infections in recent years. However, CPM associated with basidiomycetes is rare (12,13). Basidiomycetes are environmental fungi that inhabit the natural environment; they are known to float in the air as spores or mycelia (14). In the present case, basidiomycetes adhering to the nasal airway and pharynx might have been detected as contaminants in the sputum.

In conclusion, this report emphasizes the importance of making a definitive diagnosis based on specimens obtained during bronchoscopy, particularly when CPA is suspected with no positive serological findings or sputum culture. In cases with negative Aspergillus antibody test findings in patients with CPA, A. tubingensis, a cryptic A. niger complex species with a low susceptibility to azoles, might be the causative agent of CPA.

The authors state that they have no Conflict of Interest (COI).