patient and the third in the literature, following two cases

reported in children due to A. viridinutans [11].

Case report

A 56-year-old Caucasian male with diabetes mellitus type

II and rheumatoid arthritis, had been treated since 2003 with:

prednisolone, 17.5 mg/day; etarnecept, 50 mg/day; metho-

trexate, 2.5 mg 3 � /week; salazopirine, 1,500 mg/day; and

metformine, 1,700 mg/day. Due to the observation in March

2007 of a single pulmonary nodule in a routine chest X-ray,

a computed tomography (CT) was ordered which revealed

a nodular, 26 mm in diameter, hypodense, irregular, spicu-

lated lesion, suggestive of a neoplastic process, located in

the posterior segment of the upper lobe of the right lung.

The remaining upper lobe exhibited a micro-reticulo-nodular

pattern. Trans-thoracic needle aspiration biopsy and cyto-

logical examination of the aspirate revealed septate hyphae

suggestive of Aspergillus but the search for malignant cells

was negative. At the microbiology laboratory, septate

hyphae were found on direct exam (Fig. 1) but cultures of

the material, although incubated for 1 month, were nega-

tive. In the following weeks, the patient was afebrile, had

right-sided chest pain of moderate intensity, without inter-

ference with daily-life activities, and did not have other

associated complaints.

In April 2007 he started outpatient treatment with itra-

conazole PO 100 mg/day. One month later the lesion was

Received 11 September 2010 ; Received in fi nal revised from 8 January

2011 ; Accepted 18 January 2011

Correspondence: Maria Dolores Pinheiro, Laboratory of Microbiology,

Service of Clinical Pathology, Hospital de S. Jo ã o EPE, Alameda Prof.

Hern â ni Monteiro, 4200 319 Porto, Portugal. Tel: � 351 22 55 12 116;

telefax: � 351 22 55 12 261; E-mail: m.dolores.pinheiro@gmail.com

Case Reports

Aspergillus viridinutans: an agent of adult chronic

invasive aspergillosis

DANINA COELHO * , SUSANA SILVA * , LU Í S VALE-SILVA † , HELENA GOMES * , EUG É NIA PINTO † ,

ANT Ó NIO SARMENTO * & MARIA DOLORES PINHEIRO ‡

* Service of Infectious Diseases, Hospital de S. Jo ã o EPE, Porto, † Microbiology Service/CEQUIMED, Faculty of Pharmacy,

Universidade do Porto, and ‡ Laboratory of Microbiology, Service of Clinical Pathology, Hospital de S. Jo ã o EPE, Porto, Portugal

In contrast with the common hematogenous dissemination of invasive aspergillosis (IA), we present case with a protracted course through anatomical planes in an immunocom-promised adult male. The unusual clinical features and laboratory fi ndings led to fungal genotyping and identifi cation of the mold as Aspergillus viridinutans . It appears to be the fi rst described case of IA caused by this agent in an adult patient.

Keywords Aspergillus viridinutans , adult patient , invasive aspergillosis , fungal genotyping , antifungal susceptibility

Introduction

The incidence of invasive aspergillosis (IA) has been

increasing steadily for the past few decades [1,2].

The most common etiological agent of IA is Aspergillus fumigatus , followed by Aspergillus niger , Aspergillus fl avus , Aspergillus terreus [3,4] and other members of

Aspergillus section Fumigati [5].

Aspergillus viridinutans, a mold of Aspergillus section

Fumigati , was originally isolated from rabbit dung in

Australia and from soil samples collected from around the

world [6,7]. It has subsequently been identifi ed during ret-

rospective analyses of Aspergillus species in cultures [8 – 10]

but its clinical importance has not yet been defi ned [11].

Here we describe the clinical case of a 56-year-old

immunocompromised man who died from IA which

exhibited distinct clinical manifestations compared to infec-

tions caused by A. fumigatus . We also present the morpho-

logical characteristics, genotype-based identifi cation and the

antifungal susceptibility of the isolate. To the best of our

knowledge, the case described here is the fi rst in an adult

© 2011 ISHAM DOI: 10.3109/13693786.2011.556672

Medical Mycology October 2011, 49, 755–759

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756 Coelho et al .

found to be identical to that seen on the initial X-ray and

he again underwent trans - thoracic needle aspiration biopsy.

Septate hyphae were once more observed in direct exam,

but on this occasion a fi lamentous fungus, morphologically

identifi ed as Aspergillus spp., probably A . fumigatus was

recovered in culture.

At this point the patient was started on posaconazole

therapy 400 mg/day, continuously for 5 months, with

positive clinical and radiological response.

In October 2007, following unrelated abdominal sur-

gery, posaconazole was discontinued for a month. Due to

worsening of pulmonary imaging, a bronchoscopy was

performed but no endobronchial lesions were identifi ed.

Direct examination of bronchial and bronchoalveolar

lavage revealed hyphae and cultures were again positive

for Aspergillus spp. Upon discharge from hospital in

November, the patient was maintained on voriconazole PO

200 mg/day and continued with his usual medication,

including methotrexate and prednisolone, which he took

regularly during the course of the disease.

In early July 2008, a right cervical, deeply adherent

infl ammatory swelling, with a maximum diameter of 10 cm,

was observed. Aspiration needle biopsy and cytological

exam of the aspirate revealed fungal structures suggestive

of Aspergillus . Treatment with intravenous voriconazole

resulted in clinical improvement and decrease in mass size.

The patient was discharged with oral voriconazole. How-

ever, at the end of the second month of treatment, there

was a resurgence of the right cervical swelling and the

appearance of a second one with similar characteristics in

the thoracic region at the right anterior axillary line, 6th and

7th rib. This time there was a clear clinical worsening, includ-

ing fever of 38 – 40 ° C, deterioration of general condition,

swelling enlargement and chest X-ray changes. In late

August 2008, he was admitted and observed for the fi rst

time in the Infectious Diseases Service.

Medical treatment was optimized with the combination

of posaconazole, 200 mg four times a day and caspofungin,

50 mg daily and empirical therapy with amoxicillin and

clavulanic acid. While blood cultures were negative for

bacteria, mycobacteria and fungi, an Aspergillus spp. iso-

late was recovered from bronchial secretions.

The CT scan revealed multiple lymph adenopathies and

a right cervical conglomerate beginning in the supra-

clavicular region down to the 5th rib , where a 3.5 cm nod-

ule with central necrosis was seen. A right pleural effusion

with pleural thickening and a nodular lesion with spicu-

lated margins of 5 cm, located in the posterior aspect of

the right upper lobe, in contact with the pulmonary hilum,

were also found (Fig. 2).

The fever and lesions persisted despite spontaneous and

surgical drainage of both swellings. Cancer and infection

by other fungi, mycobacteria, or bacteria were excluded.

The clinical condition of the patient progressively wors-

ened with respiratory failure and oliguric irreversible renal

failure, and he died after 39 days of hospitalization. No

post mortem study was performed.

All isolates sent to the microbiology laboratory between

May 2007 and September 2008 presented the same mor-

phological and microscopic characteristics and were

identifi ed as Aspergillus spp., probably A. fumigatus . They

Fig. 1 Direct examination of the sample from the aspirative biopsy of

the pulmonary nodule. Notice the existence of septate hyphae.

Fig. 2 CT depicting the thorax with the nodular spiculated lesion on the

upper lobe of the right lung (black arrow).

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Invasive Aspergillus viridinutans in an adult patient 757

results were similar for the fi rst and last isolate tested.

Serum levels of administered antifungal drugs were not

measured.

Discussion

The case reported here shows a form of aspergillosis with

a clinical course different from that usually observed in

neutropenic and transplanted patients. Invasive aspergil-

losis typically presents as an acute, rapidly progressive

disease with predilection for angioinvasion and hematog-

enous dissemination. In contrast, this case and the two

others previously reported in the literature [11] showed

chronic evolution and progressive spread across anatom-

ical planes.

This third case strengthens the previous assertion that

A. viridinutans is able to cause a distinct form of aspergil-

losis [11]. In fact, the patients were immunocompromised

in some manner and the disease started in the lungs, likely

after inhalation of spores (the most frequent portal of entry

in IA), before disseminating to adjacent areas. Moreover,

the infection developed over several months in all cases (3

months in the 14-year-old boy, 7 months in the 8-year-old

boy and almost 18 months in this 56-year-old man), and

did not respond to medical and surgical therapy, emphasiz-

ing the indolent, albeit progressive, features of this form

of aspergillosis. An interesting observation is that all three

cases involved male patients, although the limited number

does not allow for conclusions on gender susceptibility (there

is no such predilection in usual forms of aspergillosis).

This case further evidences the need for genotypically-

based identifi cation of isolates of members of Aspergillus ,

section Fumigati [8 – 10]. Indeed, the mold isolated in the

present case was found to belong to that group, according

to morphological and microscopic characteristics, but its

features did not fully correspond to those of A. fumigatus

(for example, differences in growth patterns), which

prompted the use of molecular biology tools to obtain

defi nite species identifi cation.

The earlier therapeutic decisions deserve attention.

When the patient presented with a solitary nodule, he was

treated with 100 mg of itraconazole each day. Due to the

lack of a positive response, posaconazole PO 400 mg/day

was prescribed and, later, after surgery for unrelated rea-

sons, he was medicated with voriconazole PO 200 mg/day.

The under dose schedule employed for the three antifungal

drugs did not meet fully the Infectious Diseases Society of

America recommendations [14]. Although they included

the oral route for administration, it may not have been

adequate in this setting.

Finally, in vitro susceptibility tests of A. viridinutans

revealed peculiar fi ndings when compared to the antifun-

gal susceptibility of other Aspergillus spp. The MIC of

showed the usual features of members of the genus Asper-gillus including: ‘ foot cells ’ ; upright, usually nonseptate,

conidiophores, with swollen vesicles at the tips; vesicles

covered entirely or in part by the phialides [12]. However,

in comparison to A. fumigatus , colonies of the isolates

grew slower in Sabouraud-gentamicin-chloramphenicol

culture medium at 25 ° C, were initially white (Fig. 3) and

required additional incubation time to develop their green

color due to conidial formation. On microscopical exam of

a lactophenol cotton-blue tease preparation, the vesicles

were smaller than those seen with A. fugmiatus , i.e., their

diameters ranged from 17 – 31 μ m (average 23.8 μ m)

whereas those of A. fumigatus measured from 28 – 51 μ m

(average 38.6 μ m). In addition, a lower proportion of

vesicles exhibited phialides (39%, compared to 46% on

Aspergillus fumigatus ), which, when present, appeared in

smaller numbers (Fig. 4).

The mold was genotypically identifi ed as A. viridinu-tans at Centro Nacional de Microbiologia Instituto de

Salud Carlos III, Madrid, Spain, according to a previously

described procedure based on partial sequencing of the

β -tubulin and rodlet A genes [8]. Susceptibility testing was

performed according to the recommendations of the Clin-

ical and Laboratory Standard Institute (CLSI), document

M38-A2 [13]. The minimal inhibitory concentrations

(MIC) were 1.0, 4.0, 0.25, � 16 μ g/ml for amphotericin B,

voriconazole, posaconazole and itraconazole respectively.

Minimal effective concentrations for caspofungin and

anidulafungin were � 0.016 μ g/ml for both. Susceptibility

Fig. 3 Image of Aspergillus viridinutans culture on Sabouraud-

gentamicin-chloramphenicol medium at 25 ° C at 5 days (A) and

13 days (B).

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758 Coelho et al .

voriconazole against A. viridinutans was consistently

higher when compared to A. fumigatus isolates [15 – 19],

a fi nding in agreement with the refractory nature of the

infection to voriconazole, observed in the patient for

almost 1 ½ years. A. viridinutans does appear to be gen-

erally susceptible to posaconazole and the echinocan-

dins, according to our results and those described in the

two previous reports [8,11]. However, it should be

pointed out that MIC criteria for susceptibility may dif-

fer. In fact, the MIC we found for posaconazole indicates

susceptibility according to CLSI guidelines, but, accord-

ing to European Committee for Antimicrobial Suscepti-

bility Testing [20], it is at the uppermost limit of

susceptibility and is probably intermediate.

Therefore, this case emphasizes the peculiar clinical

features of an unusual Aspergillus species infection caused

by A. viridinutans . It further highlights the importance of

employing specifi c diagnostic tools to achieve the correct

identifi cation of molds and the need to perform antifungal

susceptibility testing, using reference procedures and

breakpoints established by expert committees [13,20].

This is particularly important in those clinical isolates whose

microbiological examination presents unexpected features

and the empirical therapy proves to be ineffective. Further

epidemiological and antifungal susceptibility data are neces-

sary before defi nitive practice recommendations may be

generated to guide the clinical management of IA by

A. viridinutans , other A. fumigatus -like molds, or rare and

emerging species.

Acknowledgements

We acknowledge the courtesy of Professor Manuel Cuenca-

Estrella, from Centro Nacional de Microbiologia, Instituto

de Salud Carlos III, Madrid, Spain.

Lu í s Vale-Silva acknowledges the Foundation for Sci-

ence and Technology (FCT, Portugal) for his postdoctoral

grant (SFRH/BPD/29112/2006).

Declaration of interest: The authors report no confl icts of

interest. The authors alone are responsible for the content

and writing of the paper.

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This paper was fi rst published online on Early Online on 11 February 2011.

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