Misidentification of Neosartorya pseudofischeri as Aspergillus fumigatus in a Lung Transplant Patient

Abstract

We present a case of disseminated Neosartorya pseudofischeri infection in a bilateral lung transplant patient with cystic fibrosis. The organism was originally misidentified from respiratory specimens as Aspergillus fumigatus using colonial and microscopic morphology. DNA sequencing subsequently identified the organism correctly as N. pseudofischeri.

CASE REPORT

A 36-year-old female with cystic fibrosis underwent bilateral lung transplantation in October 2011. Prior to her transplant, she had frequent respiratory exacerbations and her airways were chronically colonized with Aspergillus fumigatus and Pseudomonas aeruginosa. Posttransplant, she was placed on an immunosuppressive regimen of tacrolimus, azathioprine, and prednisone together with antibiotic prophylaxis with trimethoprim-sulfamethoxazole, itraconazole, and valganciclovir. The patient's postoperative course was complicated by tracheobronchitis caused by Pseudomonas aeruginosa and multiple episodes of steroid-responsive acute cellular rejection (International Society for Heart and Lung Transplantation [ISHLT] grade A3), requiring frequent adjustment of her immunosuppressive regimen. In January 2012, she developed areas of focal consolidation in her right lung but bronchoalveolar lavage (BAL) fluid cultures were negative for bacterial and fungal pathogens. She was empirically switched from itraconazole to posaconazole and was given a course of intravenous (i.v.) ceftazidime and oral levofloxacin. In February 2012, the patient was diagnosed with a paratracheal abscess, which was surgically debrided. The intraoperative cultures were positive for multidrug-resistant P. aeruginosa, which was treated with i.v. colistin, ciprofloxacin, and inhaled tobramycin. Subsequent evaluation showed the persistence of the abscess, and the patient was placed on long-term therapy with inhaled tobramycin alternating with colistin, i.v. piperacillin-tazobactam, and ciprofloxacin in July 2012. Spirometry showed a continued decline in lung function. Repeat bronchoscopy by BAL in September 2012 yielded multiple colonies of A. fumigatus, despite the patient being on oral posaconazole prophylaxis.

The A. fumigatus isolate was identified using colonial and microscopic morphology. White or greenish-gray fluffy colonies with white edges were observed after 2 to 3 days of growth on Czapek Dox agar (CZA) and inhibitory mold agar (IMA). Using a lactophenol aniline blue-based dye, a tape mount was prepared from each isolate, and columnar heads with phialides covering the upper two-thirds of the vesicle were observed consistent with A. fumigatus. Aspergillus antigen (Platelia Aspergillus enzyme immunoassay; Bio-Rad, Redmond, WA,) tests performed on serum were negative (index < 0.5). The Aspergillus antigen test performed on the BAL specimen was positive at an index of ≥3.75 in September 2012 but then reverted to negative in October 2012. Liposomal amphotericin B was initiated in addition to posaconazole. Due to worsening renal function, the dosage of amphotericin was lowered and caspofungin was added to her regimen.

Despite aggressive antifungal therapy, the patient's respiratory symptoms progressed over the next 10 months. A. fumigatus was identified from multiple respiratory specimens, including BAL fluid, sputum, and lung tissue, and even from a paraumbilical subcutaneous abscess by colony and microscopic morphology (Table 1). Beginning in June 2013, her level of Aspergillus antigen in serum and BAL fluid remained persistently positive at an index of ≥3.75. One fungal isolate grown in culture from lung tissue obtained via a computed-tomography (CT)-guided transthoracic biopsy procedure did not produce adequate microscopic structures for identification and appeared white on the IMA and CZA plates even after 16 days of incubation. The D2 segment of the 23S rRNA gene was therefore sequenced for identification, and the isolate had 100% sequence homology to a type strain of Neosartorya pseudofischeri. Based on these results, two available isolates from the umbilicus and sputum that were previously identified based on morphological features as A. fumigatus were sequenced using the 23S rRNA target and were also identified as N. pseudofischeri rather than A. fumigatus. Interestingly, a comparison of available in vitro susceptibility results showed significantly different MICs (Table 1).

TABLE 1.

Culture and susceptibility results of isolates identified as Aspergillus fumigatus or Neosartorya pseudofischeri from multiple sources

Collection date	Source	Colony color	Morphological identification	Sequencing identification	MIC (μg/ml)
					Posaconazole	Voriconazole	Caspofungin
7 September 2012	Bronchoalveolar lavage fluid	Green center with white edges	Aspergillus fumigatusa	NDc	0.25	0.5	0.5
14 December 2013	Bronchial washing	Two morphologies: (i) green center with white edges; (ii) white	Aspergillus fumigatusa	ND	2	16	0.5
4 January 2013	Bronchial washing	Green center with white edges	Aspergillus fumigatusa	ND	ND	ND	ND
17 January 2013	Sputum	White turning green	Aspergillus fumigatusa	ND	ND	ND	ND
21 January 2013	Bronchial washing	Three morphologies: (i) green center with white edges; (ii) blue-green with white edges; (iii) white	Aspergillus fumigatusa	ND	1	8	1
5 April 2013	Sputum	Green center with white edges	Aspergillus fumigatusa	ND	4	16	0.06
29 April 2013	Sputum	Green center with white edges	Aspergillus fumigatusa	ND	2	16	0.125
7 May 2013	Lung tissue	White	Not used	Neosartorya pseudofischeri	16	0.25	≤0.015
23 May 2013	Umbilicus	Green center with white edges	Aspergillus fumigatusb	Neosartorya pseudofischeri	ND	ND	ND
28 May 2013	Sputum	Green center with white edges	Aspergillus fumigatusa	ND	ND	ND	ND
2 July 2013	Sputum	Green center with white edges	Aspergillus fumigatusb	Neosartorya pseudofischeri	1	8	0.25
25 July 2013	Bronchoalveolar lavage fluid	Green center with white edges	Not used	Neosartorya pseudofischeri	>16	0.5	0.5
26 July 2013	Leg tissue	Green center with white edges	Not used	Neosartorya pseudofischeri	>16	0.5	0.5
26 July 2013	Leg tissue	Gray center with white edges	Not used	Neosartorya pseudofischeri	ND	ND	ND
2 August 2013	Bronchoalveolar lavage fluid	Green center with white edges	Not used	Neosartorya pseudofischeri	ND	ND	ND

^aIsolate not available for further investigation.

^bResults initially reported as A. fumigatus but revised upon further investigation.

^cND, not done.

In July 2013, while on posaconazole, liposomal amphotericin B, and caspofungin therapy, the patient developed skin lesions on her left leg which were culture positive for N. pseudofischeri. Since her respiratory status continued to inexorably decline, an echocardiogram was performed which revealed four large vegetations on the mitral chordae apparatus with a flail posterior leaflet. The patient was not considered a candidate for operative intervention. Blood cultures remained negative for fungi throughout the course of her illness. Despite aggressive antifungal therapy, the patient passed away in August 2013.

Autopsy revealed an infectious process that was grossly restricted to the heart, lungs, and kidneys, while evidence from excised skin lesions on the left lower leg was consistent with a recent history of treated N. pseudofischeri infection. On cardiac examination, multiple vegetations were identified on the tips of papillary muscles, cords, and the posterior leaflet of the mitral valve (Fig. 1A and B). These were associated with a prolapse of the mitral valve leaflets. Histopathological examination revealed the presence of valvular vegetations with fungal colonization on hematoxylin and eosin (H&E) and Grocott's methenamine silver (GMS) staining. Overall, these findings supported the antemortem diagnosis of infective endocarditis with mitral valve vegetations and mitral regurgitation based upon prior echocardiography.

FIG 1.

(A) Inferior view of a transverse section of the heart at the midventricular level showing a dilated right ventricle, indicated by a yellow asterisk (the left ventricle is indicated by a white asterisk). The arrow indicates the presence of white-colored fungal colonization of the papillary muscles. (B) Arrows indicate the tips of the papillary muscles (white), chordae tendineae (yellow), and mitral valve leaflets (red) with associated vegetations, which have a necrotic and hemorrhagic appearance. (C) Cut surface of the right lung demonstrates prominent hemorrhagic nodules, abscesses (upper arrow), and mucous plugging (lower arrow). (D) H&E staining of a pulmonary abscess at ×400 magnification, with accompanying acute necrotizing bronchopneumonia. (E) Grocott's methenamine silver stain showing septate filamentous fungi with acute-angled branching (magnification, ×600) from the pulmonary abscess. (Panels A and B courtesy of W. D. Edwards, Mayo Clinic; reproduced with permission.).

Pulmonary examination revealed a left-sided serous pleural effusion (200 ml) with marked pleural adhesions which were predominantly right sided. Gross lung examination revealed a prominent cavitary lesion (3.0 by 1.5 cm) in the posterosuperior aspect of the right lower lobe as well as hemorrhagic nodules, abscesses, mucous plugging, and prominent alveoli (Fig. 1C). Postmortem cultures of lung tissue and chest swabs of purulent exudates extending through the chest wall and subcutaneous tissue revealed growth of N. pseudofischeri. In addition, bilateral metastatic renal abscesses were identified (not shown).

Histopathological examination revealed acute necrotizing bronchopneumonia, focal abscess formation, and mycetomas in airways with airway dilatation. Abscesses were identified in cardiac, pulmonary (Fig. 1D), and renal tissues and showed septate hyphae with acute-angled branching, a morphological description that has been classically used to characterize colonization with Aspergillus species (Fig. 1E). Other relevant findings included scattered noncaseating granulomas in the cortex and deep gray structures of the brain and the brainstem and pancreatic exocrine atrophy as well as hepatosplenomegaly. However, no organisms were identified on GMS or Gram staining.

Death was attributed to complications of cystic fibrosis and lung transplantation, which included fungal infective endocarditis, acute necrotizing bronchopneumonia, lung abscesses, and disseminated N. pseudofischeri infection.

Neosartorya pseudofischeri is the teleomorphic (sexual) form of Aspergillus thermomutatus, typically an environmental organism, which has been identified as the causative agent in 5 case reports of human infection, including invasive pulmonary aspergillosis, osteomyelitis, peritonitis, and invasive otitis (1–5). Two separate groups have also identified N. pseudofischeri from the sputum of cystic fibrosis patients (2, 6). Colonization in these hosts may occur for extended periods of time, although the significance in terms of attributable disease is unclear. This is the first case report of disseminated N. pseudofischeri infection in a bilaterally lung-transplanted cystic fibrosis patient.

Many fungi produce similar hyphal structures, and it is likely that infections by N. pseudofischeri are underreported due to misidentification as A. fumigatus (7, 8). N. pseudofischeri may be misidentified since it produces colony and microscopic morphologies similar to those of A. fumigatus. In this case, one isolate was routed for molecular identification by sequencing because it sporulated poorly and produced white colonies that did not change color for 16 days (Fig. 2A). This presentation is typical for N. pseudofischeri (2, 3, 5, 6) (Fig. 2A and B). However, subsequent and prior isolates from nonrespiratory and respiratory sources sporulated well on IMA and CZA agar and produced fluffy white, white-gray, or white-green colonies with a pale-white reverse typical of A. fumigatus (Table 1).

FIG 2.

(A and B) Growth of Aspergillus fumigatus (A) and Neosartorya pseudofischeri (B) on Czapek's agar after 3 days. (C and D) Growth of Aspergillus fumigatus (C) and Neosartorya pseudofischeri (D) on inhibitory mold agar after 2 days. (E and F) Conidial heads of A. fumigatus (E) and N. pseudofischeri (F) from lactophenol aniline blue tape preparation after 2 days of growth on inhibitory mold agar.

All isolates also had fruiting structures similar to those of A. fumigatus, with phialides on the upper two-thirds of the vesicle (Fig. 2C and D). No additional structures were seen on either CZA or IMA media, although culture for 7 days on malt extract agar (MEA) revealed that some strains of N. pseudofischeri produce ascogonia and ascomata (4). Ascogonia are precursors to ascoma and appear as hyphal coils, while hyaline ascomata contain 8 ascospores with spiny valves (2, 6). These structures are unreliably produced and cannot always be used for identification, although lower temperatures may increase production (5). Aspergillus antigen tests are relatively nonspecific, and in this patient, cross-reaction with N. pseudofischeri in BAL fluid and serum specimens was useful for confirmation of disseminated infection (5).

In this case, the fungal isolate was correctly identified by sequencing of the D2 region in the 23S rRNA gene. Other techniques such as thermotolerance studies and multiplex PCR of the β-tubulin (β-tub) and Rodlet A (rodA) genes can also been used, since they can separate A. fumigatus from less-common species such as A. lentulus, A. novofumigatus, A. unilateralis, N. hiratsukae, N. pseudofischeri, and N. udagawae species by amplicon size (6, 9). To ensure that the organism was accurately identified, β-tub was also PCR amplified and then sequenced using previously published Bt2a and Bt2b primers (7, 10). NCBI BLAST searches of the sequence gave a 100% sequence identity to Neosartorya pseudofischeri.

The initial isolates from this patient's cultures that were originally identified morphologically as A. fumigatus were not available for retesting by sequencing. Furthermore, antifungal susceptibility patterns could not be used to gauge the probability that those isolates were actually N. pseudofischeri since the MICs of posaconazole and voriconazole for the isolates identified as N. pseudofischeri or A. fumigatus were highly divergent.

Results from several reports indicate that N. pseudofischeri tends to have low amphotericin B (≤1 μg/ml), itraconazole (≤2 μg/ml), and caspofungin (≤0.125) MICs, moderate voriconazole (≤8 μg/ml) MICs, and high flucytosine (>64 μg/ml) and fluconazole (>128 μg/ml) MICs (1, 2, 6, 11, 12). In this case, the echinocandin MICs were in the susceptible range for all isolates tested, which is consistent with the published literature. On the other hand, although voriconazole has been used to successfully treat N. pseudofischeri with a low MIC (1 μg/ml) (1), it was not chosen for this patient due to the high MICs from the early isolates (16 μg/ml). MIC values for posaconazole are noticeable absent in the literature, but due to the comparatively low MICs from the early isolates (Table 1), posaconazole in combination with liposomal amphotericin B and/or caspofungin was used for treatment.

This report reveals that microscopic morphology and currently known antibiotic susceptibility patterns cannot be reliably used to distinguish between N. pseudofischeri and A. fumigatus. At our institution, the white colony color typically associated with N. pseudofischeri is used as a flag for further identification by sequencing. However, this recommendation did not capture sequence-confirmed N. pseudofischeri cultures presenting with white-green colonies. Isolates that were not available for sequence confirmation were considered to be N. pseudofischeri, although the possibility of dual infection with A. fumigatus cannot be ruled out. Since some Neosartorya species are associated with food products and spoilage, accurate identification may begin to yield important information about routes of exposure for high-risk cystic fibrosis patients (13). Correct and early identification of N. pseudofischeri may also alert clinicians to consider more-aggressive therapy, such as multidrug antifungal regimens or surgical intervention, than would normally be done for most A. fumigatus infections.