In their efforts to provide care for patients with novel coronavirus (SARS-CoV-2) disease
(COVID-19) infection, many healthcare workers around the globe exposed to SARS-CoV-2 got
infected and died over the past two months. Quarantined nurses and physicians have become
the norm in regions with COVID-19 patients, putting at risk the overall functionality of
the regional healthcare system. Other than strict contact-precautions, no proven
vaccination or targeted therapy is available to prevent COVID-19 in healthcare workers.
Inhaled nitric oxide gas (NO) has shown in a small clinical study to have antiviral
activity against a Coronavirus during the 2003 SARS outbreak. We have designed this study
to assess whether intermittent inhaled NO in healthcare workers might prevent their
infection with SARS-CoV-2.

Background: After almost two months of fight against COVID-19 infection, on February 24,
more than 3,000 physicians and nurses were reported as contracting COVID-19 disease in
Wuhan (China). Fatalities among those healthcare workers were reported to be related to
SARS-CoV-2 infection. Implementation of strict contact protections for all healthcare
personnel is essential to decrease and contain the risks of exposure. However, despite
best efforts, dozens of thousands of healthcare providers have been quarantined for at
least 14 consecutive days in Wuhan alone. Similarly data have been reported in Italy,
several healthcare providers have been quarantined, developed pneumonia and died. Most
recent information from Italy reported that 12% of healthcare workers are infected.

The shortage of hospital personnel, especially in the critical care and anesthesiology
domains, led many hospitals to postpone indefinitely scheduled surgical procedures,
including cardiac surgery or oncological procedures. Only urgent and emergent cases are
performed in patients without symptoms (i.e., absence of fever, cough or dyspnea), no
signs (i.e., negative chest CT for consolidations, normal complete blood count) and a
negative test on SARS-CoV-2 reverse transcriptase (rt)-PCR. If time does not allow for
thorough screening (i.e., after traumatic injury), such patients are considered to be
infected and medical staff in the OR are fully protected with third degree protections
(i.e., N95 masks, goggles, protective garments and a gown and double gloving).

Rationale. In 2004 in a collaborative study between the virology laboratory at the
University of Leuven (Belgium), the Clinical Physiology Laboratory of Uppsala University
(Sweden) and the General Airforce Hospital of China (Beijing, China), nitric oxide (NO)
donors (e.g. S-nitroso-N-acetylpenicillamine) greatly increased the survival rate of
infected eukaryotic cells with the coronavirus responsible for SARS (SARS-CoV-1),
suggesting direct antiviral effects of NO. These authors suggest that oxidation is the
antiviral mechanism of nitric oxide. A later work by Akerstrom and colleagues showed that
NO or its derivatives reduce palmitoylation SARS-CoV spike (S) protein affecting its
fusion with angiotensin converting enzyme 2. Furthermore, NO or its derivatives reduce
viral RNA synthesis in the infected cells. Future in-vitro studies should confirm that NO
donors are equally effective against SARS-CoV-2, as the current virus shares 88% of its
genome with the SARS-CoV [3]. However, at present it is reasonable to assess that a high
dose of inhaled NO might be anti-viral against SARS-CoV-2 in the lung. The virus is
transmitted by human-to-human contact and occurs primarily via respiratory droplets from
coughs and sneezes within a range of about 1.5 meters. The incubation period ranges from
1 to 14 days with an estimated median incubation period of 5 to 6 days according to the
World Health Organization [1]. COVID-19 disease is mainly a respiratory system disease,
but in the most severe forms can progress to impair also other organ function (i.e.,
kidneys, liver, heart). Nitric oxide gas inhalation has been successfully and safely used
for decades (since 1990) in thousands of newborns and adults to decrease pulmonary artery
pressure and improve systemic oxygenation.

Recently at the Massachusetts General Hospital, a high dose of inhaled NO (160 ppm) for
30 - 60 minutes was delivered twice a day to an adolescent with cystic fibrosis and
pulmonary infection due to multi-resistant Burkholderia cepacia. There were no adverse
events to this patient, blood methemoglobin remained below 5% and lung function and
overall well-being improved.

Clinical Gap. Thousands of healthcare workers have been infected with SARS-CoV-2 and
contracted COVID-19 despite their best efforts to prevent contamination. No proven
vaccine is available to protect healthcare workers against SARS-CoV-2.

Hypothesis. Due to genetic similarities with the Coronavirus responsible for SARS, it is
expected that inhaled NO gas retains potent antiviral activity against the SARS-CoV-2
responsible for COVID-19.

Aim. To assess whether intermittent delivery of inhaled NO gas in air at a high dose may
protect healthcare workers from SARS-CoV-2 infection.

Observational group: daily symptoms and body temperature monitoring. SARS-CoV-2 RT-PCR
test will be performed if fever or COVID-19 symptoms.

Treatment group: the subjects will breathe NO at 160 parts per million (ppm) for two
cycles of 15 minutes each at the beginning of each shift and before leaving the hospital.
Daily symptoms and body temperature monitoring. SARS-CoV-2 RT-PCR test will be performed
if fever or COVID-19 symptoms. Safety: Oxygenation and methemoglobin levels will be
monitored via a non-invasive CO-oximeter. If methemoglobin levels rise above 5% at any
point of the gas delivery, inhaled NO will be stopped. NO2 gas will be monitored and
maintained below 5 ppm.

Blinding. The treatment is not masked.