The study was approved by the Ethics Committee from “Hospital Español - ASSE”, a COVID-19 reference public hospital in Montevideo, Uruguay. Fragments of lung tissue were collected during clinical autopsy performed on patients with COVID-19 (n = 8) between November 2020 and February 2021 admitted in the ICU. Confirmation of infection by SARS-CoV-2 was made by RT-qPCR during ICU stay and informed consent was signed by direct family members before autopsy. All samples were obtained in the first 2 h post-mortem and stored at -80°C.
On the other hand, lung fragments from non-COVID-19 deceased patients were collected between December 2016 and June 2018 also at Hospital Español – ASSE. This group includes microbiological and serological positive results for Klebsiella pneumoniae, Leptospira interrogans, and respiratory syncytial virus (n = 6). In all cases, lung tissues were obtained immediately after confirmation of death, and were stored immediately at -80°C for further studies.
The inclusion criteria for both groups were adults older than 18 years, admitted in the ICU with respiratory sepsis, septic shock or multi-organ failure, and all patients were mechanically ventilated.
An adaptation of previously published methods6 was performed and lung fragment samples were homogenized and extracted with CHCl3/MeOH/H2O. Briefly, lung tissue was homogenized in water and methanol in a Bullet Blender (Next Advance, USA). Subsequently, chloroform was added for the final ratio of 8:4:3 CHCl3:MeOH:H2O respectively, mixed in a vortex for 5 minutes, and centrifuged for 5 minutes at 5000g. Aqueous phase was lyophilized and resuspended in buffer phosphate in deuterium water (D2O) pH 7,4. Water-suppressed 1D-NOESY 1H NMR spectra of aqueous tissue extracts were obtained at 500 MHz in a Bruker AVANCE III 500. A spectral width of 10 KHz, a data size of 32 K, and a total of 128 scans were employed to record each spectrum, using a relaxation delay of 4 s between scans. When required, gradient enhanced heteronuclear single quantum correlation (HSQC) protocol and 1D total correlated spectroscopy (TOCSY) spectra were acquired using standard pulse sequences provided with the spectrometer.