A bronchoalveolar lavage-driven antimicrobial treatment improves survival in hematologic

malignancy patients with lung infiltrates detection: a prospective multicenter study of the
SEIFEM group

Francesco Marchesi,1 Chiara Cattaneo,2 Marianna Criscuolo,3 Mario Delia,4 Michelina Dargenio,5
Maria Ilaria Del Principe,6 Antonio Spadea,1 Nicola Stefano Fracchiolla,7 Lorella Melillo,8 Katia
Perruccio,9 Caterina Alati,10 Domenico Russo,11 Mariagrazia Garzia,12 Marco Brociner,13
Mariagiovanna Cefalo,14 Daniele Armiento,15 Simone Cesaro,16 Nunzia Decembrino,17 Andrea
Mengarelli,1 Mario Tumbarello,3,18 Alessandro Busca,19 and Livio Pagano3,20 on behalf of the
Sorveglianza Epidemiologica Infezioni nelle Emopatie (SEIFEM) Group

1
Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute (Rome, Italy)
2
Hematology Division, ASST-Spedali Civili di Brescia (Brescia, Italy);
3
Fondazione Policlinico Universitario Agostino Gemelli - IRCCS (Roma, Italy);
4
Hematology and Bone Marrow Transplantation Unit, Department of Emergency and Organ Transplantation,
University of Bari (Bari, Italy);
5
Hematology and Stem Cell Transplantation Unit, 'Vito Fazzi' Hospital (Lecce, Italy);
6
Hematology, Department of Biomedicine e Prevention, Tor Vergata University (Rome, Italy);
7
Hematology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico (Milan, Italy);
8
UO of Hematology, Fondazione IRCSS Casa Sollievo della Sofferenza (San Giovanni Rotondo, Italy);
9
Pediatric Hematology Oncology, Azienda Ospedaliera Universitaria, Ospedale Santa Maria della Misericordia
(Perugia, Italy);
10
Hematology Unit, Bianchi-Melacrino-Morelli Hospital (Reggio Calabria, Italy);
11
Bone Marrow Transplant Unit, University of Brescia and ASST-Spedali Civili (Brescia, Italy);
12
Hematology, San Camillo-Forlanini Hospital (Rome, Italy);
13
Division of Hematology, Foundation IRCCS Policlinico San Matteo, University of Pavia (Pavia, Italy);
14
Hematology, San Eugenio Hospital (Rome, Italy);
15
Hematology and Stem Cell Transplantation Unit, University Campus Bio-Medico (Rome, Italy);
16
Pediatric Hematology Oncology, Azienda Ospedaliera Universitaria Integrata (Verona, Italy);
17
Pediatric Hematology Oncology, IRCCS Policlinico San Matteo, University of Pavia (Pavia, Italy);

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/ajh.25585

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18
Istituto di Malattie Infettive, Università Cattolica del Sacro Cuore (Roma, Italy);
19
Stem Cell Transplant Center, AOU Citta' della Salute e Della Scienza (Turin, Italy);
20
Istituto di Ematologia, Università Cattolica del Sacro Cuore (Roma, Italy)

Running title: BAL in hematologic malignancies

Keywords: bronchoalveolar lavage fluid (BAL), lung infiltrate, hematologic malignancies,

antimicrobial treatment, invasive fungal disease

Corresponding author:
Francesco Marchesi, MD
Hematology and Stem Cell Transplant Unit
IRCCS Regina Elena National Cancer Institute
Via E. Chianesi, 53 00144 - Rome (Italy)
Email: francesco.marchesi@ifo.gov.it
Phone: +39 06-52665022
Fax: +39 06-52662849

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ABSTRACT

Bronchoalveolar lavage (BAL) is recommended for diagnosing lung infiltrates (LI) in patients with
hematologic malignancy (HM). Prospective data on the impact of BAL on survival are still lacking.
We conducted a prospective observational study on patients who performed BAL for LI among
3.055 HM patients hospitalized from January to September 2018. BAL was performed in 145 out of
434 patients who developed LI at a median time of 4 days from LI detection. The median age was
60 (1-83). Most patients had an acute myeloid leukaemia/myelodisplastic syndrome (81), followed
by lymphoma (41), acute lymphoblastic leukaemia (27), and other types of HM (36). A putative
causal agent was detected in 111 cases (76%) and in 89 cases (61%) where the BAL results allowed
to guide antimicrobial treatment. We observed a significantly improved outcome of LI at day +30 in
patients who could receive a BAL-driven antimicrobial treatment (improvement/resolution rate:
71% vs 55%; P=0,04). Moreover, we observed a significantly improved outcome in 120d-OS (78%
vs 59%; P=0,009) and 120d-AM (11% vs 30%; P=0,003) for patients who could receive a BAL-
driven treatment. The multivariate analysis showed that BAL-driven antimicrobial treatment was
significantly associated with better 120d-OS and lower 120d-AM. We did not observe any severe
adverse events. In conclusion BAL allows to detect a putative agent of LI in about 75% of cases, it
resulted feasible and well tolerated in most cases, demonstrating that a BAL-driven antimicrobial
treatment allows to improve clinical outcome and survival.

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INTRODUCTION

Detection of lung infiltrates (LI) is a relevant concern in patients with hematologic malignancy
(HM), occurring in about 12-15% of severely neutropenic patients1 and significantly affecting
survival.2-4 Moreover, infectious and less frequently non-infectious LI occur in up to 70% and 25%
of patients undergoing allogeneic hematopoietic stem cell transplant (alloSCT) and autologous
hematopoietic stem cell transplant (ASCT), respectively. Despite advances in the diagnostic yield, a
putative microbiological agent of LI remains undetected because of the low sensibility of available
non-invasive diagnostic assays. Moreover, microbiologically undiagnosed LI may reflect a deferred
diagnostic approach where clinicians prefer to adopt an empirical treatment strategy and use more
invasive approaches only in cases of persistence of fever and when there is an appearance or
worsening of respiratory distress.2 In case of LI of undefined etiology, bronchoscopy coupled with
bronchoalveolar lavage (BAL) are used to diagnose putative microbiological agents in order to
perform guided-antimicrobial therapy. This procedure is associated with a significantly lower
complication rate compared with trans-bronchial or transthoracic needle aspirate.5 In HM or allo-
SCT patients, the yield of BAL was of 15-67% before the advent of non-culture-based methods.
The yield was significantly higher when the BAL was performed early after LI detection (up to 87%
of positive results), allowing to change the on-going empirical antimicrobial treatment in about 50-
65% of patients.6-13 Nevertheless, several issues are still under debate and investigation, these
include: the diagnostic yield of BAL, the gold-standard used to properly evaluate the positive and
negative predictive value of BAL, as well as the impact of using a BAL-guided antimicrobial
strategy on clinical outcome and survival.1,14-15 Based on these considerations, we designed a
prospective multicenter study in 18 hematology institutes participating in the Sorveglianza
Epidemiologica Infezioni nelle Emopatie (SEIFEM) Group on adult and pediatric HM patients with
LI detection in order to evaluate the efficacy and the safety of BAL as well as the impact on clinical
outcome and survival of a BAL-guided antimicrobial strategy.

METHODS

Study design

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This is a prospective observational multicenter study that was carried out in 18 hematology centers
from January 1 to September 30, 2018. Adult and pediatric severely immunocompromised patients
with HM at any phase of treatment were enrolled according to the following criteria: 1) the presence
of a host risk factor for invasive fungal disease (IFD) according to the EORTC/MSG 2008
criteria;16 2) the absence of any microbiological documentation at standard non-invasive diagnostic
work-up; 3) any fever or respiratory distress not responding to broad-spectrum antimicrobial
therapy; 4) LI detection at computed tomography (CT) scan. According to the investigators, the
patients fulfilling these criteria underwent a bronchoscopy with BAL.
The study was approved by the Ethical Committee of the coordinating center (IRCCS Regina Elena
National Cancer Institute, prot. R.S. 976/17) and by each participating institute. The study was
conducted in accordance with the Helsinki declaration and all patients signed a written informed
consent before study enrolment.

End-points
The primary study end-point was to evaluate the efficacy of BAL in identifying a putative
microbiological agent. The secondary endpoints were to evaluate the folowing: 1) the clinical
response to a BAL-driven antimicrobial treatment at day +30 from LI detection; 2) the overall
survival (OS) and attributable mortality (AM) at day +120 from LI detection; 3) the frequency of
BAL positivity for GM in patients with LI radiologically atypical for IFD; 4) the safety of BAL.

Procedures and definition

Patients experiencing a fever of unknown origin underwent empiric broad-spectrum antibiotic
treatment, according the international guidelines17-18 and the local policy. A standard diagnostic
work-up including serial blood and urine cultures, nasopharyngeal swab for respiratory viruses,
serum GM (three consecutive checks), 1,3-beta-D-glucan and CT scan was performed in those
patients who did not respond to empiric broad-spectrum antibiotic treatment and/or who presented
respiratory distress in the subsequent 48-72 hours from the onset of fever. Patients with LI were
classified according to the standard glossary of CT imaging and to the EORTC/MSG 2008 criteria,
dividing them into typical and atypical criteria.16,19 Patients experiencing fever or respiratory
distress who did not respond to broad-spectrum antibiotics and those who did not have any

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microbiological and/or serological documentation at diagnostic work-up were eligible candidates to
perform BAL, ideally within 3-4 days from LI detection.
Demographic, hematological and microbiological data were registered in an electronic form. All
patients were evaluated for severity of their illness by the APACHE II scoring system.20 Response
to antimicrobial treatment at day +30 from LI detection was evaluated by CT scan. Resolution was
defined as the complete disappearance of radiological features in the presence of a significant
clinical improvement, whereas improvement was defined as a decrease in size of previous described
radiological features without complete disappearance. Finally, a worsening of LI was defined as an
increase in size of previously described radiological features or the appearance of new lesions,
associated with the deterioration of clinical conditions. OS was defined as the time from LI
detection to death due to any causes, whereas AM was defined as the time from LI detection to
death directly caused by lung infection. The observation time was of 120 days from LI detection.
Bronchoscopy with BAL was performed according to the standard procedures published by the MD
Anderson Cancer Center group.11 The recommended diagnostic panel on BAL included: a) bacterial
and fungal growth; b) cytologic exam; c) GM; d) multiplex PCR for respiratory bacteria (S.
pneumoniae, L. pneumophila, C. pneumoniae, M. pneumoniae, B. pertussis, B. parapertussis, H.
influenziae); e) PCR for respiratory viruses (including Cytomegalovirus); f) PCR for P. jiroveci,
adding evaluation of immunofluorescence in case of PCR-positivity to confirm diagnosis;21 g) PCR
for M. tuberculosis complex; h) PCR for Aspergillus spp. Available commercial kits were used
according to the manufacturer's instructions, with the only exception of PCR for Aspergillus spp
(homemade). A positive BAL-GM was defined as an optical density index > 0,5.22-25 CMV findings
were interpreted according to the current published guidelines.26 Acceptable adherence to the
recommended diagnostic panel was reached if the single test was performed in at least 75% of
enrolled patients.
Invasive pulmonary aspergillosis (IPA) was defined according to the EORTC/MSG 2008 criteria.16
Post-procedure adverse effects were defined and graded according to the Common Toxicity Criteria
(version 4.02).

Statistical analysis

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The estimated sample size was approximately 150 patients and was calculated on the basis of a
previous study in which the efficacy of BAL identifying a putative agent of LI was of 65% with a
confidence interval (CI) of 58-73%.13 Even if we consider a drop-out rate of 10%, this CI would
have remained the same whether 150 patients had been enrolled into the study. Data were analyzed
by the Statistical Package of Social Sciences software (version 20, Chicago, IL, USA). Continuous
variables were compared with Student’s t-test (for normally distributed variables) or the Mann–
Whitney U-test (for non-normally distributed variables). Categorical variables were evaluated with
the two tailed Fisher’s exact test. Results are expressed as median (continuous variables) or as
percentages of the group from which they were derived (categorical variables). The two-tailed tests
were used to determine statistical significance. The OS and AM was estimated with Kaplan-Meier
method and was compared to the two-sided log-rank test. The multivariate analysis was used in
order to identify independent risk factors for survival and mortality. For this analysis, Cox
proportional hazard models were built and variables found to be significant in univariate testing
were incorporated by using a stepwise approach. The related estimates were reported as hazard
ratios (HR) and 95% CI. P-values ≤ 0,05 were considered statistically significant.

RESULTS

Epidemiological data
During the study period, a total of 3.055 patients were hospitalized in participating centers. The
underlying disease was as follows: lymphoma 861, acute myeloid leukaemia (AML) 742, myeloma
567, acute lymphoblastic leukaemia (ALL 383 patients with), chronic lymphocytic leukaemia
(CLL) 92, other HM 410 patients. CT-confirmed LI were diagnosed in 434 out of 3.055 patients
(14%). A LI was detected after a median time of 3 days after fever onset. Baseline demographic
characteristics and flow-chart of enrolled patients is shown in Table 1 and Figure 1 (panel A),
respectively. Among 434 patients with LI, 145 out of 177 (82%) patients matching the study
criteria performed BAL whereas 32 patients (18%) did not undergo the procedure for the following
reasons: clinical instability n=11, severe thrombocytopenia n=8, local clinical decision n=7, patient
refusal n=6). At the time of BAL, 58 patients (33%) were receiving glucocorticoids at the time of

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enrolment (> 0,3 mg/Kg/day for at least 3 weeks or higher doses for less long time) with a median
administration time of 10 days. Median absolute neutrophil count (ANC) was of 0,46 x 10/9L, and
106 patients (60%) had experienced prolonged and severe neutropenia (ANC < 0,1 x 10/9L for a
median of 12 days); median platelets (PLT) count was of 40 x 109/L. Twenty-nine patients (16%)
presented severe respiratory failure (p02 < 60 mmHg) and the median APACHE II score was 16
(range: 5-27). The adherence of clinicians to the recommended diagnostic panel on BAL was
acceptable (≥ 75%) for all tests with the only exception of Aspergillus PCR, that was performed in
less than 50% of patients.

Identification of a putative microbiological agent (primary end-point)

A putative causal agent of LI was detected in 111 out of 145 patients who underwent BAL (76%).
In 41 cases, positive BAL-GM was found, 36 for bacteria, 6 for Pneumocystis and in the remaining
28 cases for respiratory viruses. In 10 cases, a mixed infection was diagnosed; no cases of
tuberculosis were detected. As shown in Figure 1, the results of BAL allowed to guide the ongoing
empiric antimicrobial therapy in 89 cases (61%) (Table 2), whereas in the remaining 56 cases (39%)
BAL did not permit to modify it (no further microbiological data n=43; virus-positive-BAL for
which a specific therapy is not possible to perform n=13). Table 1S (supplementary material) shows
a comparative analysis between patients undergoing or not a BAL-driven antimicrobial treatment:
we did not find any significant differences in terms of clinical features and empiric antimicrobial
strategy between cohort of patients undergoing or not a BAL-driven treatment. In particular, among
89 patients in which a BAL-driven antimicrobial treatment was possible on the basis of BAL
results, 7 patients (8%) were treated with empiric antifungal treatment at the time of LI detection
and in other 36 (40%) an empirical antifungal treatment was started on the basis of radiological
findings. Similar results were obtained in patients in which a BAL-driven antimicrobial treatment
was not possible: 3 patients (5%) were treated with empiric antifungal treatment at the time of LI
detection and in other 21 (37%) empirical antifungal treatment was started in the basis of
radiological findings. Even though GM was positive in 41 cases (28%), a diagnosis of probable IPA
according to the EORTC/MSG 2008 criteria was possible only in 18 cases with "typical"
radiological signs. Alternatively, in 23 cases (56%) a positive BAL-GM in patients with "atypical"
radiological signs was detected. However, in almost all cases in which a GM was positive on BAL

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(40/41), these findings allowed clinicians to guide the antimicrobial treatment by starting patients
on specific antifungal therapy and/or modifying the on-going empirical therapy. Cases with
“atypical” radiological signs and with positive BAL-GM but not fulfilling the EORTC/MSG 2008
criteria were more frequently reported in patients with chronic lymphoproliferative diseases
(P=0,039) and with less severe neutropenia evaluated at the time of LI detection (median ANC
0,95 vs 0,17 x 109/L; P<0,001). Interestingly, a BAL driven antimicrobial treatment approach was
possible in a similar amount of patients in both cohorts (96% vs 100%; P=0,965) and clinical
outcome was not significantly different between the two patient cohorts (P=0,878).

Overall survival and attributable mortality (secondary end-points)

We observed a significant better outcome of LI at day +30 from its detection in patients where a
BAL-driven antimicrobial treatment was possible (resolution/improvement rate: 71% vs 55%;
P=0,04). We observed a significantly improved outcome in terms of 120d-OS (78% vs 59%;
P=0,009) and 120d-AM (11 vs 30%; P=0,003) for patients in which a BAL-driven antimicrobial
treatment was possible (Figure 1, panel B and Figure 1S, supplementary material). Finally, the
univariate and multivariate analyses were performed in order to identifying the factors that were
significantly associated with the risk of OS and AM (Table 3). Advanced age (HR 2,33 95%CI:
1,03-5,21; P=0,04) and high value of APACHE II (HR 2,28 95%CI: 1,03-5,05; P=0,04) were found
to be independent factors negatively affecting survival, whereas a BAL-driven antimicrobial
treatment was related to better 120d-OS (HR 0,24 95%CI: 0,11-0,52; P<0,001). Moreover, a BAL-
driven antimicrobial treatment was the only independent factor related to a lower 120d-AM in our
study patient-population (HR 0,35 95%CI: 0,16-0,79; P=0,011).

Safety (secondary end-point)

We did not observe any severe adverse effects, but only 5 cases of grade 1-2 adverse events
occurred after the procedure and were easily managed and resolved (respiratory distress n=2, fever
n=2, epistaxis n=1).

DISCUSSION

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Bronchoscopy with BAL is useful in HM patients particularly when performed early, allowing to
guide on-going antimicrobial treatment based on results from 50-60% of cases.6-7 However, most of
these studies were designed as retrospective analyses, but large prospective studies with rigorous
inclusion criteria suggesting the impact of BAL execution on clinical outcome are very few. Lucena
and collaborators14 conducted a prospective single institution study on patients undergoing ASCT
and allo-SCT identifying 73 cases of pulmonary complications among 169 patients in the study.
The diagnostic yield for infectious pulmonary complications was 78%, showing a more frequent
viral (28%) and bacterial (26%) etiology of pneumonia. A fungal infection was diagnosed in about
16% of cases. Interestingly, an early bronchoscopy (≤5 days from LI detection) had significantly
higher diagnostic yield when compared to late bronchoscopy (78 vs 23%; P=0,02). In this study, the
authors reported that data obtained by BAL results allowed to change the empirical antimicrobial
treatment in about half of the patients, demonstrating the utility of this procedure in the transplant
setting, leading to better patient management. Overall, the mortality rate after 1 year follow-up was
22%, with about one third of all deaths due to pulmonary complications, however the clear
correlation between changes of empirical antimicrobial treatment and survival was not shown by
the authors. The strength of our study is the prospective design and the relatively stringent inclusion
criteria (severely immunocompromised HM patients with LI detection at CT assessment without
any non-invasive diagnostic documentation and unresponsive to broad-spectrum antimicrobial
therapy) that allowed us to analyze an homogenous patient population. Moreover, we established
primary and secondary end-points, including clinical outcomes in term of both treatment response
and survival. This was an observational study because the final choice on performing or not
bronchoscopy in eligible patients was up to the local physicians. Our data show that a LI was
detected in about 14% of HM patients admitted to hospital, which is in line with other published
data.1 Out of 177 eligible patients, BAL was feasible in 82% of cases within a median time from LI
detection of 4 days. Our patients were severely immunocompromised, most frequently affected by
acute leukaemia or undergoing allo-SCT, with a median ANC at LI detection of about 0,4 x 10/9L
and most having experienced (60%) a prolonged and severe neutropenia or received steroid therapy
(33%). Moreover, some patients presented severe respiratory failure (16%) and the median value of
APACHE II score was 16, suggesting a “severely ill population”. Despite the multicenter design,
our data appeared quite homogeneous because of how the recommended diagnostic panel was

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planned, including culture-based and non-culture-based exams, obtaining an acceptable adherence
rate for all diagnostic assays, except for Aspergillus PCR. Using this recommended panel, the
diagnostic yield was of 76%: in 41 cases positive BAL-GM was found, 36 for bacteria, 5 for
Pneumocystis and the remaining cases for respiratory viruses. In 10 cases, a mixed infection was
diagnosed. Our diagnostic yield seems to be better than the other published data,1-2,6-7,11 but almost
identical to those reported in the few prospective studies with a similar design.13-14 In our study, the
BAL results allowed to guide the on-going empirical treatment in about 60% of cases, according to
the data reported in the literature that are more heterogeneous but reported an overall percentage
between 20 and 70%.2,6-7,13,15,27-31 We showed a significantly better clinical outcome in terms of
response to antimicrobial treatment, 120d-OS (78% vs 59%; P=0,009) and 120d-AM (11% vs 30%;
P=0,003) for patients in which a BAL-driven antimicrobial treatment was possible. However, it is
important to underline that the most relevant weakness of our study is the non-randomized design,
that could negatively affects the reliability of these findings. Nevertheless, our data are very
relevant because only a few studies prospectively evaluated the impact of diagnostic BAL on
survival. Interestingly, we showed that BAL results permitted to adding or modifying antibiotic or
antiviral treatment in about half of patients, avoiding unnecessary prolonged antifungal treatment.
Azoulay and collaborators did not find that BAL had any influence on mortality in a prospective
experience on 128 critically ill cancer patients, including transplant recipients, suggesting however
that performing an early BAL (within 4 days from symptoms onset) was significantly associated
with better survival compared with late BAL.15 Similar results were obtained by another interesting
study that failed to show an overall impact on survival, but highlighted that BAL-driven antibiotic
changes within 7 days from LI presentation significantly correlated with a lower mortality.27
However, the results obtained in both these studies are not completely comparable with our data, as
they were carried out several years ago and focused on a more heterogeneous and critical patient
population. To the best of our knowledge, our study is the first prospective study in which a BAL-
driven antimicrobial approach is found to have a positive impact on clinical outcome and mortality.
These results are relevant because they were obtained in the context of a prospective multicenter
study. As for safety, similarly to previously cited studies, we did not observe any grade 3 adverse
events, but only five case of grade 1-2 adverse events that were easily managed, confirming that
BAL execution is safe also in this population of patients. In our study, a positive BAL-GM was

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defined as an optical density index > 0,5 according to the recent clinical guidelines of the European
Society for Clinical Microbiology and Infectious Diseases, the European Confederation of Medical
Mycology and the European Respiratory Society Joint .22-25 The optimal cut-off of BAL GM has not
been established yet. Some published studies suggested a higher positive predictive value of
1,0,25,32-33 others instead showed the best diagnostic performance of 0,523 while some reports
highlighted the excellent sensitivity and specificity of 1,5 in the HM setting.27 By using the 0,5 cut-
off, we found 41 positive cases of BAL-GM. In 40 cases, the local investigators considered this
value reliable and modified the on-going antimicrobial treatment accordingly, whereas one case
only was interpreted as a "false positive". Only about half of the cases, were we able to diagnose a
probable IPA according to the EORTC/MSG 2008 criteria, as only these presented "typical"
radiological signs needing formal diagnosis of a probable IPA. Interestingly, by comparing cases
with "typical" and "atypical" radiological signs, we found that positivity of BAL-GM in the absence
of typical radiological lesions of IFD can be detected mostly in patients with chronic
lymphoproliferative disorders and with less severe neutropenia and that clinical outcome and
survival between the two group of patients were similar, probably due to the prompt start of a
specific antifungal treatment. These data are interesting and suggest the need to revise the
EORTC/MSG radiological criteria that seem to be too stringent. The EORTC/MSG criteria were
proposed with the aim to establish a standardized set of criteria that allow to easily compare the
results of clinical trials concerning IFD, but failed to be applied extensively in clinical practice
where the application of these criteria could underestimate these severe complications.34-39 As a
result, the revision of these criteria with the inclusion of a new category of IPA granting a greater
weight on microbiological criteria was proposed by Nucci and collaborators in 2010.38 Based on
these considerations, in our study 40 over 41 cases of BAL-GM positivity were considered, treated
and managed as a probable IPA and the clinical outcome of cases without "typical" radiological
criteria was substantially equivalent to others, suggesting a higher positive predictive value of a
positive BAL-GM irrespective of the presence of typical radiological findings.
In conclusion, in severely immunocompromised HM patients with LI detection at high risk of
invasive fungal disease without any microbiological and/or serological documentation nor fever or
respiratory distress not responding to broad-spectrum antimicrobial therapy we found that: 1) BAL
allows to detect a putative agent of LI in about 75% of cases; 2) BAL is a feasible procedure in

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almost all cases; 3) cases with positive BAL-GM without radiological criteria according to
EORTC/MSG show similar features and clinical outcome of probable IPA completely fulfilling
EORTC/MSG criteria; 4) a BAL-driven antimicrobial treatment is feasible in 61% of patients who
underwent BAL and allows to improve clinical outcome and survival. Further randomized studies
are warrant to confirm these results.

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ACKNOWLEGMENTS
We thank Federica Falcioni and Elena Papa for the technical support during protocol submission to
Ethical Committee and Diana Giannarelli for the support in statistical study design. We thank
thoracic surgeons, pneumologists and microbiologists for their support during study carrying out.
We thank Tania Merlino (English native speaker, scientific clinical writer) for revising the
manuscript.

FUNDING
None. The study has been carried out as part of our routine work.

CONFLICT OF INTEREST
All authors: none to declare.

AUTHOR CONTRIBUTIONS
FM, CC, MD and LP: conception and design of the study; FM, CC, MC, MD, MD, MIDP, AS,
NSF, LM, KP, CA, DR, MG, MB, MC, DA, SC, ND, AB: patients management and data
collection; FM, CC, AM, LP: data analysis and results interpretation; FM and CC: manuscript
writing; SC, MT, AM and LP: critical manuscript review.

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Figure legends

Figure 1. Flow chart of enrolled patients (panel A); attributable mortality (AM) of 145 patients
undergoing BAL evaluated at 120 days from LI detection (panel B).

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Accepted Article

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 Table 1. Demographic characteristics of 145 patients undergoing BAL.

Demographic characteristics at enrollment N (%)

Median age (range) 61 (1-83)
Sex
M 85 (59%)
F 60 (41%)
Diagnosis
Accepted Article

AML/MDS 67 (46%)
Lymphoma 34 (24%)
ALL 19 (13%)
Myeloma 14 (10%)
CLL 5 (3%)
Other* 6 (4%)
Disease status
Newly diagnosed 48 (33%)
CR 41 (28%)
PR 41 (28%)
R/R 15 (11%)
Last treatment received
Induction 53 (36%)
Salvage 37 (25%)
AlloSCT 27 (19%)
Cytoreduction/pre-phase 11 (8%)
Consolidation 10 (7%)
ASCT 7 (5%)
Radiologic features of LI **
Not well circumscribed or diffuse consolidations 66 (45%)
Dense, well circumscribed lesions with or without halo sign 46 (32%)
Ground glass opacity 20 (14%)
Micronodules 4 (3%)
Interstitial lung abnormalities 3 (2%)
Cavity 3 (2%)
Tree in bud 3 (2%)

AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; ALL: acute lymphoblastic leukemia;
CLL: chronic lymphocytic leukemia; CR: complete response; PR: partial response; R/R: relapsed/refractory;
alloSCT: allogeneic hematopoietic stem cell transplant; ASCT: autologous hematopoietic stem cell transplant.
*
Other: aplastic anemia (n=2), hairy cell leukemia (n=2), idiopathic myelofibrosis (n=1), POEMS syndrome (n=1).
**
Radiologic feature of LI: the prevalent aspect at thoracic computed tomography assessment has been reported.

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 Table 2. Detailed report of pathogens detected by BAL in 89 patients undergoing a BAL-
driven antimicrobial treatment.

Isolation (*) No. cases Percentage

Galactomannan 40 45%
Without fungal growth 35
Aspergillus fumigatus 3
Aspergillus terreus 1
Aspergillus niger 1
Bacteria 32 36%
Klebsiella pneumoniae 6
Accepted Article

Stenotrophomonas maltophilia 5
Pseudomonas aeruginosa 5
VRE 4
Staphylococcus aureus 3
Acinetobacter baumanii 2
Escherichia coli 2
Other gram positive (**) 2
Haemophilus influenziae 1
Legionella pneumophila 1
Clamydia pneumoniae 1
Viruses 11 12%
Cytomegalovirus 7
Influenza A (H1N1) 4 (3)
Pneumocystiis jirovecii 6 7%

(*) in 10 cases we detected mixed infections

(**) Streptococcus pneumoniae (1); Rothia mucillaginosa pneumonia diagnosed and confirmed by MALDI (2).
VRE: vancomycin-resistant Enterococci

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 Table 3. Univariate and multivariate analysis for 120d-OS and 120d-AM in patients undergoing BAL (n=145).

Variable (120d-OS) Univariate Multivariate

P HR (95%CI) P
Accepted Article

Age < vs ≥ median (60 years) 0,009 2,33 (1,03-5,21) 0,04

Diagnosis (AML vs ALL vs lymphoma vs myeloma vs other) 0,905 - -
Treatment (induction vs alloSCT vs salvage vs other) 0,169 - -
Disease status (CR/PR vs newly diagnosed vs R/R) 0,124 2,67 (0,33-21,6) 0,122
APACHE II score < vs ≥ median (16) 0,042 2,28 (1,03-5,05) 0,04
BAL-driven antimicrobial treatment (yes vs no) 0,009 0,24 (0,11-0,52) < 0,001
Variable (120d-AM) Univariate Multivariate
P HR (95%CI) P
Age < vs ≥ median (60 years) 0,046 2,18 (0,94-5,07) 0,068
Diagnosis (AML vs ALL vs lymphoma vs myeloma vs other) 0,982 - -
Treatment (induction vs alloSCT vs salvage vs other) 0,098 - -
Disease status (newly diagnosed vs R/R vs CR/PR) 0,494 - -
APACHE II score < vs ≥ median (16) 0,249 - -
BAL-driven antimicrobial treatment (yes vs no) 0,003 0,35 (0,16-0,79) 0,011

120d-OS: overall survival after 120 days from lung infiltrate detection; HR: hazard ratio; CI: confidence interval;
AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; R/R: relapsed/refractory; CR: complete remission;
PR: partial remission.

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