Thirty of 46 (65%) BALs performed on COVID-19 patients tested positive for SARS-CoV 2 RNA, of which 3/79 were diagnostic (3.8%) for three patients who tested negative for NPS. No BAL performed in 76 patients without COVID-19 pneumonia was positive for SARS-CoV2 (Table 3).

In 229 patients, 111 microorganisms were isolated with BAL (48% of cases), apart from viruses: 41 identified in the 86 BAL of COVID-19 patients (52%); 70 in the 150 BAL of non-COVID patients (48%) (Table 1). It should be noted that 9 (11%) of the 79 patients with COVID-19 pneumonia presented Enterobacteriaceae infection versus 5 (3%) of the 150 non-COVID patients (RR=3.41; 95%CI 1.19-9.85; P=0.0229). Pseudomonas/Stenotrophomonas infections were also more frequent: 10% vs 4% in COVID patients (RR=2.53; 95%CI 0.91-7.04; P=0.075).

Mycobacterial and P. jirovecii infections were more frequent in the non-COVID group. We documented positivity for Aspergillus in 17 cases, with a comparable frequency in the two groups. Remarkably, pulmonary tuberculosis was observed in both groups: 5/79 cases (6%) versus 21/150 cases non-COVID (14%) (RR=0.45; 95%CI 0.18-1.15; P=0.097).

We compared the incidence rate of respiratory disease diagnoses and complications per case (Table 1), and we saw that in the COVID-19 patients’ group each patient had on average 2.4 diagnoses, compared to 1.4 diagnoses in the non-COVID group (186 diagnoses/79 COVID-19 positive cases versus 208 diagnoses/150 COVID-negative patients: IRR=1.7 (95% CI 1.392.08; P<0.001.

Sepsis was documented in 19/79 (24%) COVID-19 patients and in 14/150 (9%) non-COVID patients (RR 2.58; 95%CI: 1.37- 4.86; P=0.003). Atelectasis / obstruction were more frequent in COVID-19 patients: 15/79 (19%) versus 2/150 (1%) of nonCOVID (Fisher’s exact test; probability<0.001) and ARDS 18/79 (23%) COVID-19 vs 9/150 (6%) in patients non-COVID (RR=3.8; 95% CI 1.79-8.06; P<0.001).

Hemoptysis was more frequent among non-COVID patients:

(14/150 patients, 0/79 in COVID patients, Fisher’s exact test; probability=0.003).

We believe this data correlates with the observed greater clinical severity of cases with COVID-19. In fact, if we look at the number of deaths, we find 14 deaths among the 79 cases (22%), versus 9 deaths (6%) among the 150 cases without COVID-19 pneumonia (RR=2.95; 95% CI 1.34-6.52; P=0.007).

Discussion

We examined a cohort of 229 patients, 79 with SARS-CoV2 infection and 150 without, all hospitalized for lung pathologies and subjected to bronchoscopy from March 2020 to January 2022. Our patients with COVID-19 pneumonia were characterized by a greater severity of clinical picture compared to non-COVID, evident both for the higher percentage of admissions to intensive care (27% vs 7%) and for the higher mortality observed during hospitalization (22% vs 6%).

The two groups differed significantly in the total number of respiratory disease diagnoses and complications, higher in the COVID19 group (2.4 vs 1.4 diagnoses/patient), interstitial pneumonia (100% vs 15%); atelectasis/obstruction (19% vs 1%); ARDS (23% vs 6%); sepsis (24% vs 9%) (Table 1).

In clinical practice, the NPS for SARS-CoV2 RT-PCR performed upon entry was confirmed to be very sensitive, testing positive in 92% of cases and allowing a diagnosis in 73 out of 79 cases. In one case a second NPS allowed the diagnosis of SARS-CoV2 infection. Induced sputum test for SARS-CoV2 in our study group had a sensitivity of 62%, allowing the diagnosis of 2 of the 79 cases (3%). BAL tested positive for SARS-CoV2 in 30/46 COVID positive cases (65%), being diagnostic in three of the 79 cases (4%).

NPS, tested with RT-PCR assay, is confirmed as the reference diagnostic test, because it is faster, easily repeatable, and sensitive. Induced sputum and bronchoscopy are often performed with a certain delay because they require dedicated spaces, specific equipment and trained medical personnel to avoid nosocomial transmission of infectious agents during their execution. Normally, the execution of induced sputum and FBS with BAL in COVID-19 patients is dictated by the need to identify concomitant respiratory pathologies. However, in our case series, these two types of respiratory sample allowed the diagnosis of SARS-CoV2 infection in 5 out of 79 COVID patients (7% of cases).

The time of collection of respiratory samples certainly influences the result of the PCR for SARS-CoV2. Wang and colleagues in 2020 [6] found SARS-CoV2 RNA in 14 out of 15 (93%) BAL samples but in only 126 out of 398 (32%) pharyngeal swabs from patients with COVID-19. We confirm their observation on BAL, because also in our series, out of 18 BAL performed within the 5th day of hospitalization of COVID patients, 16 tested positive for SARS CoV2 (89%). For IS it is almost the same thing, in fact out of 15 induced sputa collected within the fifth day of hospitalization, 13 tested positive for SARS-CoV2 RNA (86.7%). Regarding nasopharyngeal swabs, our data are quite different, probably because the technique for performing respiratory swabs has improved compared to the beginning of the pandemic. In fact, nasopharyngeal swabs have been shown to be more sensitive than nasal-only or throat-only swabs [7]. Furthermore, due to the development of a rapid referral system for hospitalization of COVID cases in our region, it is possible that the COVID-positive patients in our series were hospitalized at an earlier stage of the disease than in Wang’s case series.

The research of SARS-CoV2 on other respiratory specimens such as BAL and IS is justified with the intent to definitively exclude the presence of SARS-CoV2 in doubtful cases and to document the elimination or persistence of the virus in the lower respiratory tract or compartmentalization [8], with the possibility of prolonging or repeating antiviral therapy, in other selected cases.

Furthermore, BAL allows to diagnose bacterial or fungal superinfections (52% in COVID-19 patients; 48% in nonCOVID), allowing targeted treatment. Enterobacteriaceae lung infections and Pseudomonas / Stenotrophomonas infections were more frequent in COVID-19 patients: 11% vs 5% and 10% vs 4%, respectively.

We did not observe a significant difference between the two groups regarding the number of pulmonary aspergillosis and pulmonary thromboembolism. It should also be said that, following the appearance of COVID-19, radiological research of TEP and research of Aspergillus on BAL are performed much more frequently even in our non-COVID series.

Tuberculosis was diagnosed in 6% of patients hospitalized for COVID-19 by bronchoscopy, which is confirmed as the best procedure for diagnosing tuberculosis in patients who have difficulty producing spontaneous or induced sputum and in the noncavitary pulmonary form. Bronchoscopy has been also crucial to manage tracheal complications and obstructive atelectasis, which have been frequently encountered among COVID-19 patients also in our series.

More than three years have passed since the appearance of the SARS-CoV2 epidemic and since then many things have changed, both in terms of diagnosis and in terms of prevention and treatment of the disease. The virus continues to circulate in the population, posing a continuous threat to the most vulnerable, who continue to fall ill and often require hospitalization. It also happens that the presence of asymptomatic infection is found in patients hospitalized for other pathologies, or who access outpatient services, with the risk that healthcare facilities become places of contagion for healthcare personnel and the patients themselves.

Since 2021, multiple Authors have documented how the negativity of NPS, administered to patients at low risk for COVID-19, is always corroborated by the negativity of BAL for SARS-CoV2 [9,10]. This finding supported the use of NPS tests, associated with symptom screening questionnaires, as an appropriate screening method for aerosol-generating procedures, like bronchoscopy and IS.

On the other hand, there are also numerous reports of symptomatic patients with NPS negative for SARS-CoV2, in which the BAL tested positive for SARS-CoV2 [11-13]. Indeed, we observed three symptomatic cases, NPS negative for SARS-CoV2, in which the BAL tested positive. It is therefore necessary to ask ourselves what the risk is that medical-health personnel run in carrying out the procedure on suspected or known positive patients and whether it is worth it.

Conclusions

Our case series documents a greater severity and clinical complexity of COVID-19 patients compared to non-COVID, with a greater need for diagnostic investigations, so we are of the idea that the best role for bronchoscopy in suspect COVID patients would be when less invasive testing to confirm SARS-CoV2 infection, as NPS1, NPS2, and IS, are inconclusive, or if there is suspicion for an alternative diagnosis that would impact clinical management, or when an urgent life-saving intervention is needed [3,14].

CDC Guidelines for Collecting and Handling of Clinical Specimens for COVID-19 Testing [4], in the last update of July 2022, do not recommend the induction of sputum in COVID-19 patients and they reiterate that “collection of specimens other than sputum from the lower respiratory tract may be limited to patients presenting with more severe disease, including people admitted to the hospital and/or fatal cases.”

In our institution FBS and IS have proven to be safe for healthcare personnel and patients, thanks to the absolute compliance with respiratory, droplet and contact precautions and the availability of negative pressure rooms for carrying out the procedures. In fact, from the beginning of the epidemic to today, we did not observe secondary cases of COVID-19 in our staff members and non-COVID patients undergoing FBS and IS. It should also be emphasized that precautions have been used universally for all patients, suspected or not for COVID-19, also because, in most cases, IS and FBS at INMI are performed to diagnose pulmonary infectious diseases, including tuberculosis and pulmonary mycobacteriosis, for which we are in favour of their generalization. Likewise, thanks to the development of an institutional internal protocol for IS adapted to the risk of airborne and droplet transmission, so far, no cases of COVID-19 have been observed in healthcare personnel and patients attributable to the sputum induction procedure.

Institutional procedures for performing sputum induction in hospitalized patients have proven effective in preventing nosocomial transmission of SARS-CoV2 and have therefore also been applied to outpatient cases, for example, to obtain control samples from patients being treated for pulmonary tuberculosis.

Consent for Publication

Written informed consent was obtained from patients for this paper to be published. A copy of the written consent is available for review by the Editor of this journal.

Authors Contributions

PC, MA, MP, GG, and FP conceived the study. MP wrote the paper. PC, MA, MP, MMC, LMAV, CS, D’AS and MG supplied the clinical data. FP, GG and AN reviewed the paper. All authors reviewed and approved the final version of the paper submitted to the journal.

Funding

This work was supported by funding from Italian Ministry of Health, grant Ricerca Corrente IRCCS, Research program no. 4 Tuberculosis and by liberal donation funding for COVID-19 research from Compagnia Italiana Surgelati S.p.A. Progetto “Spallanzani Porte Aperte” (grant number 62/2022). The funder had no role in study design, data collection, analysis, interpretation, and redaction of the manuscript.

Ethical Approval

Epidemiological, demographic, clinical, and laboratory data and information on treatments and outcomes of all patients with a confirmed COVID-19 diagnosis were collected and recorded using a standardized electronic database (ReCOVery study). The study was approved by our local ethics committee.

Conflicts of Interest

All authors declare no conflict of interest.

Acknowledgements

Members of the INMI ReCOVeRI Study Group: Maria

Alessandra Abbonizio, Amina Abdeddaim, Elisabetta Agostini, Chiara Agrati, Fabrizio Albarello, Gioia Amadei, Alessandra Amendola, Andrea Antinori, Maria Assunta Antonica, Mario Antonini, Tommaso Ascoli Bartoli, Francesco Baldini, Raffaella Barbaro, Barbara Bartolini, Rita Bellagamba, Martina Benigni, Nazario Bevilacqua, Gianluigi Biava, Michele Bibas, Licia Bordi,

Veronica Bordoni, Evangelo Boumis, Marta Branca, Rosanna Buonomo, Donatella Busso, Marta Camici, Paolo Campioni, Flaminia Canichella, Maria Rosaria Capobianchi, Alessandro Capone, Cinzia Caporale, Emanuela Caraffa, Ilaria Caravella, Fabrizio Carletti, Concetta Castilletti, Adriana Cataldo, Stefano

Cerilli, Carlotta Cerva, Roberta Chiappini, Pierangelo Chinello, Maria Assunta Cianfarani, Carmine Ciaralli, Claudia Cimaglia, Nicola Cinicola, Veronica Ciotti, Stefania Cicalini, Francesca Colavita, Angela Corpolongo, Massimo Cristofaro, Salvatore Curiale, Alessandra D’Abramo, Cristina Dantimi, Alessia De Angelis, Giada De Angelis, Maria Grazia De Palo, Federico De Zottis, Virginia Di Bari, Rachele Di Lorenzo, Federica Di Stefano, Gianpiero D’Offizi, Davide Donno, Francesca

Evangelista, Francesca Faraglia, Anna Farina, Federica Ferraro, Lorena Fiorentini, Andrea Frustaci, Matteo Fusetti, Vincenzo

Galati, Roberta Gagliardini, Paola Gallì, Gabriele Garotto, Ilaria Gaviano, Saba Gebremeskel Tekle, Maria Letizia Giancola,

Filippo Giansante, Emanuela Giombini, Guido Granata, Maria Cristina Greci, Elisabetta Grilli, Susanna Grisetti, Gina Gualano, Fabio Iacomi, Marta Iaconi, Giuseppina Iannicelli, Carlo Inversi, Giuseppe Ippolito, Eleonora Lalle, Maria Elena Lamanna, Simone Lanini, Daniele Lapa, Luciana Lepore, Raffaella Libertone, Raffaella Lionetti, Giuseppina Liuzzi, Laura Loiacono, Andrea Lucia, Franco Lufrani, Manuela Macchione, Gaetano Maffongelli, Alessandra Marani, Luisa Marchioni, Andrea Mariano, Maria Cristina Marini, Micaela Maritti, Annelisa Mastrobattista,

Ilaria Mastrorosa, Giulia Matusali, Valentina Mazzotta, Paola Mencarini, Silvia Meschi, Francesco Messina, Sibiana Micarelli, Giulia Mogavero, Annalisa Mondi, Marzia Montalbano, Chiara Montaldo, Silvia Mosti, Silvia Murachelli, Maria Musso, Michela Nardi, Assunta Navarra, Emanuele Nicastri, Martina

Nocioni, Pasquale Noto, Roberto Noto, Alessandra Oliva, Ilaria

Onnis, Sandrine Ottou, Claudia Palazzolo, Emanuele Pallini, Fabrizio Palmieri, Giulio Palombi, Carlo Pareo, Virgilio Passeri, Federico Pelliccioni, Giovanna Penna, Antonella Petrecchia, Ada Petrone, Nicola Petrosillo, Elisa Pianura, Carmela Pinnetti, Maria Pisciotta, Pierluca Piselli, Silvia Pittalis, Agostina Pontarelli, Costanza Proietti, Vincenzo Puro, Paolo Migliorisi Ramazzini, Alessia Rianda, Gabriele Rinonapoli, Silvia Rosati, Dorotea Rubino, Martina Rueca, Alberto Ruggeri, Alessandra Sacchi, Alessandro Sampaolesi, Francesco Sanasi, Carmen Santagata, Alessandra Scarabello, Silvana Scarcia, Vincenzo Schininà, Paola

Scognamiglio, Laura Scorzolini, Giulia Stazi, Giacomo Strano,

Fabrizio Taglietti, Chiara Taibi, Giorgia Taloni, Tetaj Nardi, Roberto Tonnarini, Simone Topino, Martina Tozzi, Francesco Vaia, Francesco Vairo, Maria Beatrice Valli, Alessandra Vergori, Laura Vincenzi, Ubaldo Visco-Comandini, Serena Vita, Pietro Vittozzi, Mauro Zaccarelli, Antonella Zanetti and Sara Zito.

We sincerely thank S. Di Lorenzi and all the nursing staff and socio-health operators of INMI who provided expertise and care for the patients.

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