Occurrence of Albifimbria verrucaria in the Blood of a Female Child With Neuroblastoma

Abstract

We report for the first time the occurrence of a filamentous fungus, Albifimbria verrucaria, in the blood of a pediatric neuroblastoma patient. The Albifimbria genus comprises common soil-inhabiting and saprophytic fungi and has been isolated as a plant pathogen in Northern and Southern Italy. As a human pathogen, A. verrucaria has been implicated in keratitis and can produce trichothecene toxins, which are weakly cytotoxic for mammalian cell lines. A. verrucaria was isolated from blood during the follow-up of a previous coagulase-negative Staphylococcus catheter-related infection. Lung nodules, compatible with fungal infection, had been observed on a CT scan 6 months earlier; they still persist. Possible routes of transmission were considered to be airborne, catheter related, or transfusion dependent, as the patient had undergone platelet and red blood cell transfusions during rescue chemotherapy. No filamentous fungi were isolated from sputum or CVCs. In conclusion, we describe an unprecedented fungemia caused by A. verrucaria and show how an unexpected pathogen may be acquired from the environment by patients at high risk due to immunosuppression. The route of transmission remains unknown.

Full text

CASE REPORT
published: 14 February 2020
doi: 10.3389/fmed.2020.00013
Frontiers in Medicine | www.frontiersin.org 1February 2020 | Volume 7 | Article 13
Edited by:
Aleksandra Barac,
University of Belgrade, Serbia
Reviewed by:
Hamid Badali,
The University of Texas Health Science
Center at San Antonio, United States
Jacques F. Meis,
Canisius Wilhelmina
Hospital, Netherlands
*Correspondence:
Monica Cricca
monica.cricca3@unibo.it
Specialty section:
This article was submitted to
Infectious Diseases - Surveillance,
Prevention and Treatment,
a section of the journal
Frontiers in Medicine
Received: 04 October 2019
Accepted: 13 January 2020
Published: 14 February 2020
Citation:
Masetti R, Prodi A, Liberatore A,
Carfagnini F, Cappelletti E, Leardini D,
Pession A, De Carolis E and Cricca M
(2020) Occurrence of Albifimbria
verrucaria in the Blood of a Female
Child With Neuroblastoma.
Front. Med. 7:13.
doi: 10.3389/fmed.2020.00013
Occurrence of Albifimbria verrucaria
in the Blood of a Female Child With
Neuroblastoma
Riccardo Masetti 1, Antonio Prodi 2, Andrea Liberatore 3, Filomena Carfagnini 4,
Eleonora Cappelletti 2, Davide Leardini 1, Andrea Pession 1, Elena De Carolis 5and
Monica Cricca 3
*
1“Lalla Seràgnoli”, Hematology-Oncology Unit, Department of Pediatrics, University of Bologna, Bologna, Italy, 2Department
of Agricultural and Food Sciences, University of Bologna, Bologna, Italy, 3Department of Experimental, Diagnostic and
Specialty Medicine, University of Bologna, Bologna, Italy, 4Pediatric Radiology, Sant’Orsola Malpighi Hospital, Bologna, Italy,
5Institute of Microbiology, IRCCS, Catholic University of the Sacred Heart, Polyclinic University Foundation Agostino Gemelli,
Rome, Italy
We report for the first time the occurrence of a filamentous fungus, Albifimbria verrucaria,
in the blood of a pediatric neuroblastoma patient. The Albifimbria genus comprises
common soil-inhabiting and saprophytic fungi and has been isolated as a plant pathogen
in Northern and Southern Italy. As a human pathogen, A. verrucaria has been implicated
in keratitis and can produce trichothecene toxins, which are weakly cytotoxic for
mammalian cell lines. A. verrucaria was isolated from blood during the follow-up of a
previous coagulase-negative Staphylococcus catheter-related infection. Lung nodules,
compatible with fungal infection, had been observed on a CT scan 6 months earlier;
they still persist. Possible routes of transmission were considered to be airborne,
catheter related, or transfusion dependent, as the patient had undergone platelet and
red blood cell transfusions during rescue chemotherapy. No filamentous fungi were
isolated from sputum or CVCs. In conclusion, we describe an unprecedented fungemia
caused by A. verrucaria and show how an unexpected pathogen may be acquired
from the environment by patients at high risk due to immunosuppression. The route of
transmission remains unknown.
Keywords: blood, neuroblastoma, immunodeficiency, invasive infection, filamentous fungus, Albifimbria
verrucaria, climatic change adaption
INTRODUCTION
Invasive fungal infections are an emerging problem worldwide. Hyaline molds, such as Fusarium
and Scedosporium spp., are the filamentous fungi which can most frequently cause fungemia in
humans (1). The occurrence of invasive infections is predominantly driven by the increasing use
of invasive devices such as central venous catheters (CVC), especially in immunocompromised
patients. Another critical factor is the emergence of saprophytic environmental fungi and their
acquisition of thermotolerance, due to global warming, which is a key step toward opportunistic
human infections (2,3). Emerging pathogens are sometimes resistant to the antifungals available,
increasing the threat of new fungal diseases (4). Albifimbria verrucaria is a saprophytic fungus that
belongs to the Stachybotryaceae family, found mainly in soil and as a plant pathogen. More than 30
species of Albifimbria have been described worldwide (5), and occurrence of itraconazole-tolerant

Masetti et al. Albifimbria verrucaria and Bloodstream Infection
species in soil and food products has been reported. The
importance of A. verrucaria in humans arose from the
observation that it produces a potent mycotoxin, which may
produce numerous adverse effects, such as the inhibition of
protein synthesis, immune suppression, and impairment of
alveolar macrophage function (6). Some authors have recently
reported Myrotecium spp., also called Albifimbria spp., as the
causative agents of keratitis in immunocompetent patients (7).
To our knowledge, ours is the only report of A. verrucaria as a
causative agent of infection in humans. Here, we report, for the
first time, the occurrence of A. verrucaria in the blood of a child
with neuroblastoma (NBL).
CASE PRESENTATION
We report the case of a female child diagnosed with high-risk
NBL at the age of 2 years and 5 months (Figure 1). Written
informed consent was obtained from the legal representatives
of the patient for the publication of this case report. At
disease onset, the primary lesion was a left adrenal mass
associated with multiple bone secondary localizations and
bone marrow infiltration. She was treated according to the
SIOPEN NB-HR-01 protocol (8), which consists of induction
chemotherapy, peripheral blood stem cell harvest, attempted
complete excision of the primary tumor, myeloablative therapy
followed by peripheral blood stem cell rescue, radiotherapy,
and immunotherapy. The patient was resistant to this first-
line chemotherapy protocol, so she was given rescue treatment
of Temozolamide and Irinotecan (9). She received a 5-day
course of chemotherapy every 3 weeks for 18 months. Owing
to the side effects of the chemotherapy, she became severely
immunocompromised and transfusion dependent, receiving at
least two to three filtered and irradiated erythrocyte and platelet
transfusions per month. During this 18-month period, at the
age of 5, she experienced an episode of fever and chills at the
end of a chemotherapy infusion. Blood samples were collected
and sent to the Microbiology Lab, Sant’Orsola Malpighi Hospital,
Bologna, Italy, for routine diagnostic procedures. Coagulase-
negative Staphylococcus was isolated from one of the lines of
the CVC; the other was negative. Concomitant blood analysis
showed a slight increase in C-reactive protein (CRP) (6.62
mg/dl). Suspecting a CVC-related infection, both lines were
closed, and lock therapy with 0.8 ml of a solution of 16 mg
of Vancomycine and 1,613 UI of Urokinase was administered.
After treatment, the cultures of blood drawn from the CVC
and peripheral blood became negative. Over the following 6
months, she had sporadic episodes of fever without any other
clinical signs with a variable increase in C-reactive protein
(maximum value, 11.32 mg/dl). In consideration of these clinical
findings, the blood cultures from the CVC lines were serially
repeated every 3 weeks before the beginning of chemotherapy.
At the age of 5 years and 9 months, a filamentous fungus
was isolated from two serial CVC blood cultures, one collected
from each of the lines over a 2-week interval. The blood was
inoculated into BD BACTECTM Peds PlusTM media and incubated
in the Bactec FX Instrument. On direct microscopic examination
of blood cultures, we observed the presence of conidia and
septate hyphae (time to positivity was ∼52 h for the first blood
culture and 26 h for the second). After 2 days of incubation
on horse blood agar and Sabouraud Chloramphenicol agar
tubes (Vakutainer Kima, Padova, Italy) at 30◦C, a slow-growing
mold appeared on both media (Figure 2). Identification was not
possible with matrix-assisted laser desorption/ionization time of
flight analysis (MALDI Biotyper Instrument, Bruker Daltonics,
Bremen, Germany) because the spectrum of A. verrucaria
(Figure 3) was not present in the filamentous fungi reference
database, which includes 430 mass spectrum profiles developed
at the Polyclinic Gemelli Hospital (10). Thus, sequencing
was used to identify the mold. Total genomic DNA was
extracted with Dneasy Plant Mini Kit (Qiagen, Milan, Italy).
First, the isolate was mechanically broken down with glass
beads (G8772-100G, Sigma-Aldrich, Milan, Italy) and bead
beater; then, it was treated according to the manufacturer’s
instructions. Part of the β-tubulin (Tub) gene was amplified
using primers BT-2a/BT-2b (11); the sequences obtained showed
99.06% agreement with the extype of A. verrucaria CBS 328.52
(GeneBank KU845969.1) and 100% with A. verrucaria strain
CBS 189.46 (GeneBank KU845965.1). Nucleotide sequences of
the isolate were deposited in the GeneBank database under
the accession number (GeneBank MN428067). Sequencing with
ITS1-4 primers was not conclusive for the identification at
species level.
Finally, we performed antifungal susceptibility testing
using the commercial microdilution method Sensititre
YeastOne (SYO ITAMYUCC, Thermo Scientific, Italy),
following the manufacturer-recommended protocol (12).
After 2 days of incubation at 30◦C, we found the following
minimum inhibitory concentrations (MIC): amphotericin B,
0.25 µg/ml; posaconazole, 0.5 µg/ml; isavuconazole, 0.5 µg/ml;
voriconazole, 0.5 µg/ml; itraconazole, 2 µg/ml; fluconazole,
256 µg/ml; and echinocandins (Caspofungin, Anidulafungin
and Micafungin), >8µg/ml. We also tested some drugs
(Voriconazole, Posaconazole, Itraconazole) with the reference
method for conidia-forming molds EUCAST (definitive
document E.DEF 9.3.), and at 30◦C, the same MIC values
were obtained.
Shortly after finding the second positive blood culture, the
CVC was replaced, as it was the suspected source of fungemia.
The catheter was treated with dithiothreitol (Thermo Fisher,
Italy), the solution seeded into Sabouraud-CAF agar tubes as well
as brain heart infusion broth (Biolife, Italy), and incubated for
3 and 2 weeks, respectively. No fungal or bacterial growth was
observed. Thus, a catheter-related infection has been excluded.
Routine abdomen and chest CT scans were performed
during rescue therapy to follow-up treatment of the NBL
masses. These revealed multiple small bilateral lesions, mainly
at the bases, with a nodular shape and ground glass features
(Figure 4). However, the patient did not present any concomitant
respiratory symptoms. The lung is not a preferential site
for NBL metastases (3.3%) (13), and the diameter of the
lesions was not influenced by chemotherapy. Hence, after two
positive blood cultures, we hypothesized the involvement of
A. verrucaria in the pathogenesis of the lung lesions. To test
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Masetti et al. Albifimbria verrucaria and Bloodstream Infection
FIGURE 1 | Patient medical history timeline. (1) First-line chemotherapy: SIOPEN NB-HR-01 protocol consisting of 10 weeks of induction chemotherapy (COJEC
scheme), surgery on the primary tumor, auto HSCT with Busulfan and Melphalan, 21 Gys of radiotherapy, and six courses of anti-GD2 antibody. (2) Rescue therapy:
temozolamide 100 mg/m2and Irinotecan 50 mg/m2for five consecutive days every 3 weeks. The patient received 36 courses of chemotherapy and several blood
and platelet transfusions due to the myelotoxicity of the chemotherapy. CoNS, coagulase-negative Staphylococcus (Staphylococcus haemolyticus).
FIGURE 2 | Presence of conidia (A, black arrows and inset a) and hyphae (B, black arrow) in the blood culture (20×magnification, Gram stain). The mold isolated
from blood culture was subcultured on Sabouraud dextrose at 30◦C. A. verrucaria colonies were flat, smooth, moist, white to cream, with regular and sharp margins
(C–E growth over 10 days). The colonies developed a black pigment represented more and more along the days of culture and produced serous vesicles (F, black
arrows). The reverse was yellow to orange (G). Septate hyphae (H, black arrow) and grapes of conidia (H) were present on microscopic examination (lactophenol
cotton blue, 40×magnification).
this hypothesis, we cultured a sputum specimen, but no fungus
was isolated. No invasive procedures have been performed, and
no antifungal therapy has been administered, as the patient
has not presented any respiratory or systemic clinical signs or
symptoms. Furthermore, the patient has received very high doses
of chemotherapy, and both the antifungal therapy and invasive
procedure to obtain a byoptic sample could produce more side
effects than in a healthy subject. The patient is now being
followed up at our clinic for treatment of NBL, as the disease is
still in progress and she is waiting to receive more radiotherapy.
DISCUSSION
To our knowledge, this is the first case of A. verrucaria in an
immunocompromised child with NBL. A. verrucaria belongs
to the Stachybotryaceae family. Members of this family include
important plant and human pathogens, as well as several
species used industrially and commercially as biodegraders and
biocontrol agents (14). Some species of Albifimbria have been
isolated in extreme environments such as hot desert sands
from Saudi Arabia and Jordan (15). In Northern and Southern
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Masetti et al. Albifimbria verrucaria and Bloodstream Infection
FIGURE 3 | Spectrum of Albifimbria verrucaria collected within a mass range of 2,000–20,000 Da. The spectrum was analyzed with Bruker Biotyper 3.1 software and
compared with those of the reference database.
FIGURE 4 | High-resolution CT scan highlights a 6-mm cavitated nodule (arrow) in the anteromedial segment of the left inferior lobe (A) and a 5-mm subpleural
nodule (arrow) in the anterior segment of the right superior lobe (B). Maximum intensity projection (MIP) reconstruction image (C,D) shows the presence of multiple
disseminated parenchymal and subpleural nodules ( max. 5 mm).
Italy, A. verrucaria has been described as a causative agent
of severe leaf necrosis and plant decay and has recently been
observed on leafy vegetable crops such as spinach, lamb’s
lettuce, and wild rocket (16). Its diffusion as a plant pathogen
in Italy may increase the risk of opportunistic infections in
immunocompromised hosts. Some authors claim that the recent
emergence of environmental pathogens due to global warming
and the acquisition of thermotolerance may represent a threat
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Masetti et al. Albifimbria verrucaria and Bloodstream Infection
for human health, especially for patients with weakened immune
systems (2).
A. verrucaria was isolated from our patient’s blood while
monitoring a prior coagulase-negative Staphylococcus catheter-
related infection. Lung nodules compatible with fungal infection
had been visible on CT scans 6 months earlier and still persist.
We hypothesized that our patient had a primary infection
in the respiratory tract and that the fungus had invaded the
bloodstream. To test this hypothesis, we analyzed a sputum
specimen which was negative for mold infection and all
other microorganisms. We speculated that the catheter could
be another possible route of transmission, but CVC culture
examination was negative, so this route was excluded. The
presence of A. verrucaria in the blood could be due to the
transfusions carried out during rescue chemotherapy over an
18-month period, even though transfused blood is filtered and
irradiated. Inoculation by drug infusion cannot be excluded
either. No cases of A. verrucaria have been described in human
pathology, except for two cases of keratitis in immunocompetent
hosts. These patients were treated with topical natamycin and
voriconazole. One patient completely recovered, but the other
did not, suggesting potential resistance to azoles (7). In our case,
A. verrucaria was resistant to fluconazole and echinocandins,
and elevated MIC values for itraconazole were observed. The
recent observation of azole-resistant strains of environmental
Aspergillus spp. due to fungicide use in agricultural settings are
in line with our findings (17).
So far, our patient has not developed invasive infections
in deep organs, even though she has not received antifungal
therapy. This may be due to the weak pathogenic potential of
A. verrucaria, or its poor adaptation to human body temperature,
in the case of direct inoculation into the blood via medical
products. This report highlights not only the emergence of
unusual pathogens in human immunocompromised hosts but
also the need to improve diagnostic procedures and in vitro
antifungal sensitivity testing for the prompt identification and
management of fungal infections in immunocompromised hosts.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Sant’Orsola Malpighi Hospital Ethics Committee.
Written informed consent to participate in this study was
provided by the participants’ legal guardian/next of kin. Written
informed consent was obtained from the individual(s), and
minor(s)’ legal guardian/next of kin, for the publication of any
potentially identifiable images or data included in this article.
AUTHOR CONTRIBUTIONS
RM and MC: conception and design of the work. RM, APr, AL,
FC, EC, DL, ED, and MC: data collection. RM, APr, ED, and MC:
data analysis and interpretation, manuscript writing, and critical
revision of the article. RM, APe, AL, FC, EC, DL, ED, and MC:
approval of the final version of the article.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Masetti, Prodi, Liberatore, Carfagnini, Cappelletti, Leardini,
Pession, De Carolis and Cricca. This is an open-access article distributed under the
terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) and
the copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
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