Our article has several take-home messages for the management of patients with COVID-19 pneumonia admitted to ICU: first, approximately a quarter of patients with COVID-19 pneumonia admitted to ICU have bacterial co-infection; second, a negative FA-PNEU result prevents the inappropriate empirical use of antibiotics in these patients as a stewardship strategy in COVID-19 and third the overall concordance was 90.1%, and it was between 92.7% and 100% by microorganisms.
Bacterial co-infection in critically-ill COVID-19 patients occurred in 24.54% and 17.27% by FA-PNEU and conventional cultures, respectively. In the most recent meta-analysis on the identification of bacterial co-infections by FA-PNEU in ICU-hospitalized COVID-19 patients, four of the seven studies reported on the timing of specimen collection within the first 48 hours of ICU admission. In total, 221 patients were included and the pooled incidence of co-infections by FA-PNEU was 33% (95% CI 0.25 to 0.41) and 18% by conventional cultures (95% CI 0.02 to 0.45) [6–9]; the incidence is higher than the reported in inpatient services, which ranges between 3.5-8% [3, 15].The largest study by Kolenda et al  included 99 patients admitted to 3 ICUs from France and the samples were taken in absence of mechanical ventilation or within 48 hours after this was initiated; cultures identified 17 bacteria in 15 of 99 samples (15.1%).
The two most frequently detected bacteria were Staphylococcus aureus (37.5%) and Streptococcus agalactiae (20%) by FA-PNEU and Staphylococcus aureus (34.78%) and Klebsiella pneumoniae (26.08%) by culture. Verroken et al. , reported the results of 32 respiratory samples in 41 COVID 19 patients in the ICU; FA-PNEU identified 13/32 (40.6%) patients with a bacterial co-infection, where Staphylococcus aureus (38.46% -60% methicillin-sensitive-), Haemophilus influenzae (23.07%), and Moraxella catarrhalis (15.38%) were the main pathogens identified. Kreitmann et al., documented bacterial co-infection in 13 of 47 subjects (27.7%) from samples taken within 24 hours of tracheal intubation, with three bacterial species representing ≥ 90% of those identified: Staphylococcus aureus 69.2% -all methicillin-sensitive-), Haemophilus influenzae (38.5%), and Streptococcus pneumoniae (23.1%). Kolenda et al. analyzed 99 patients with respiratory samples taken in the absence of mechanical ventilation or their first 48 hours; conventional cultures detected bacterial co-infection in 15%, being Staphylococcus aureus (46.6% -all methicillin-sensitive-), Haemophilus influenzae (26.66%), and Streptococcus pneumoniae (13.33%) the most prevalent pathogens.
When comparing the aforementioned studies with our work, three aspects are worth highlighting: firstly, in all the studies, including ours, Staphylococcus aureus was the most prevalent microorganism; secondly, in the present study, methicillin resistance was higher (40% of the FA-PNEU isolates had MecA / C / MREJ, while in the cultures no methicillin resistance was found); and lastly, unlike other studies, Klebsiella pneumoniae was the second most prevalent microorganism in the current study by culture, with no ESBL or KPC resistance mechanisms.
Regarding the qualitative agreements between FA-PNEU and conventional cultures, in our study we found that the PPV was between 50% and 100%, being lower for Enterobacter cloacae and Staphylococcus aureus; NPV were high (between 99.1% and 100%). Caméléna et al. demonstrated that the results of FA-PNEU are consistent (sensitivity 95%, specificity 99%, PPV 82%, and NPV 100%) with those of conventional culture for bacterial pathogens of 96 samples from 43 intubated patients with suspected bacterial co-infection or superinfection; Staphylococcus aureus, as opposed to ours, did have a good PPV (91%). Kolenda et al., reported a FA-PNEU sensitivity of 100%, since all isolated bacteria in culture were also detected using FA-PNEU, with a specificity of 98.7%, being the lowest for Haemophilus influenza (< 88.4%); the specificity for Staphylococcus aureus was 93.5%.
In our study, Staphylococcus aureus had a sensitivity of 100%, a PPV of 53.3%, a specificity of 93.1%, and a NPV of 100%, since 6 of the 9 patients with FA-PNEU positive and culture-negative microorganisms were Staphylococcus aureus. Moreover, in 6 FA-PNEU samples, the MecA/C/MREJ resistance mechanism was detected, not identified by conventional cultures. Fontana et al.  used FA-PNEU to assess co-infection in 152 respiratory specimens from inpatient COVID-19 individuals; 23 of them required assisted ventilation in the ICU. The most representative species was Staphylococcus aureus in both BAL (21; 16 mecA positive) and sputum (27; 14 mecA positive), with the majority being mecA positive (30/44, 62%). Although most of the patients were not in the ICU, their results are consistent with our findings.
Concerning the quantitative agreement, in our study, microorganisms in cultures of ETA samples with > 105 CFU, 84.21% had a count of ≥ 105 copies/mL in FA-PNEU; of the culture-negative, 40.9% had microorganisms with a count < 105 copies/mL in FA-PNEU. In the study of Kolenda et al  among 16 bacteria reported in culture, 15 (93.8%) showed ≥ 106 copies/mL using FA-PNEU; in contrast, amidst 26 bacteria detected using FA-PNEU yet culture-negative, 20 (76.9%) had ≤ 105 copies/mL using FA-PNEU. We can conclude that most positive samples in FA-PNEU, with negative cultures, have low DNA copies/mL. These findings raise the following questions: is it possible that in these cases it is not strictly a coinfection and rather a contamination by the endogenous flora? What is the clinical impact of this finding? Could antibiotic treatment be discontinued in these cases? In future studies, we will try to give some answers by comparing the results of the cultures and FA-PNEU with the lung microbiome through the extraction of DNA and RNA for sequencing the same samples for metagenomics and metatranscriptomics, which will allow us to determine the functional profiles of the virulence and resistance genes of microorganisms.
We did not find that having co-infection by culture or by FA-PNEU, was associated with an increase in mortality. Another Latin American study, conducted by Soto et al. evaluated ninety-three hospitalized patients with a diagnosis of COVID-19 who were analyzed with FA-PNEU. Co-infection was evidenced in 38 (40.86%) cases and no association with mortality was found (OR 1.63; 95% CI 0.45–5.82).
We acknowledge some limitations within our study: primarily, we did not perform an analysis of the empirical antibiotic therapies received by the patients, nor their modifications according to FA-PNEU or culture results. However, we established, as exclusion criteria, not having received an antibiotic before taking the samples of LRTI to not alter the results of FA-PNEU and cultures. Kolenda  found that the FA-PNEU positivity rate was 19.4% (14 of 72) and 51.9% (14 of 27) in patients with or without prior administration of antibiotics, respectively (p = 0.001), and the percentage of FA-PNEU positive results concordant with culture was not affected by antimicrobial administration. Besides, samples were collected either by endotracheal aspirate or mini-BAL and not through BAL; however, it is our usual practice due to the lack of availability of pulmonary professionals 24 hours a day.
There are several strengths in this study: first, the high NPV of FA-PNEU was demonstrated; therefore, we can conclude that if we find a negative result, bacterial co-infection is practically excluded. Novy et al. proposed an algorithm for rational use of FA-PNEU in critically-ill ventilated COVID-19 patients; this would allow 65.6% antibiotic spare in bacterial co-infection and better adequacy of empirical antibiotic therapy . Secondly, as far as we know, this is the study with the largest number of patients included whose objective is the diagnostic concordance of FA-PNEU with culture, in subjects with COVID-19 pneumonia admitted to ICUs. Finally, this is the first Latin American study with this purpose; most of the studies have been carried out in Europe.