Rapid System to Detect Variants of SARS-CoV-2 in Nasopharyngeal Swabs.

Currently, the reference method for identifying the presence of variants of SARS-CoV-2 is whole genome sequencing. Although it is less expensive than in the past, it is still time-consuming, and interpreting the results is difficult, requiring staff with specific skills who are not always available in diagnostic laboratories. The test presented in this study aimed to detect, using traditional real-time PCR, the presence of the main variants described for the spike protein of the SARS-CoV-2 genome. The primers and probes were designed to detect the main deletions that characterize the different variants. The amplification targets were deletions in the S gene: 25-27, 69-70, 241-243, and 157-158. In the ORF1a gene, the deletion 3675-3677 was chosen. Some of these mutations can be considered specific variants, while others can be identified by the simultaneous presence of one or more deletions. We avoided using point mutations in order to improve the speed of the test. Our test can help clinical and medical microbiologists quickly recognize the presence of variants in biological samples (particularly nasopharyngeal swabs). The test can also be used to identify variants of the virus that could potentially be more diffusive as well as not responsive to the vaccine.

A new qualitative RT-PCR assay detecting SARS-CoV-2.

The world is facing an exceptional pandemic caused by SARS-CoV-2. To allow the diagnosis of COVID-19 infections, several assays based on the real-time PCR technique have been proposed. The requests for diagnosis are such that it was immediately clear that the choice of the most suitable method for each microbiology laboratory had to be based, on the one hand, on the availability of materials, and on the other hand, on the personnel and training priorities for this activity. Unfortunately, due to high demand, the shortage of commercial diagnostic kits has also become a major problem. To overcome these critical issues, we have developed a new qualitative RT-PCR probe. Our system detects three genes-RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N)-and uses the ß-actin gene as an endogenous internal control. The results from our assay are in complete agreement with the results obtained using a commercially available kit, except for two samples that did not pass the endogenous internal control. The coincidence rate was 0.96. The LoD of our assay was 140 cp/reaction for N and 14 cp/reaction for RdRp and E. Our kit was designed to be open, either for the nucleic acid extraction step or for the RT-PCR assay, and to be carried out on several instruments. Therefore, it is free from the industrial production logics of closed systems, and conversely, it is hypothetically available for distribution in large quantities to any microbiological laboratory. The kit is currently distributed worldwide (called MOLgen-COVID-19; Adaltis). A new version of the kit for detecting the S gene is also available.

Co-infections observed in SARS-CoV-2 positive patients using a rapid diagnostic test.

Rapid diagnostic tests are tools of paramount impact both for improving patient care and in antimicrobial management programs. Particularly in the case of respiratory infections, it is of great importance to quickly confirm/exclude the involvement of pathogens, be they bacteria or viruses, while obtaining information about the presence/absence of a genetic target of resistance to modulate antibiotic therapy. In this paper, we present our experiences with the use of the Biofire® FilmArray® Pneumonia Panel Plus (FAPP; bioMérieux; Marcy l'Etoile, France) to assess coinfection in COVID-19 patients. A total of 152 respiratory samples from consecutive patients were examined, and 93 (61%) were found to be FAPP positive, with the detection of bacteria and/or viruses. The patients were 93 males and 59 females with an average age of 65 years who were admitted to our hospital due to moderate/severe acute respiratory symptoms. Among the positive samples were 52 from sputum (SPU) and 41 from bronchoalveolar lavage (BAL). The most representative species was S. aureus (most isolates were mecA positive; 30/44, 62%), followed by gram-negative pathogens such as P. aeruginosa, K. pneumoniae, and A. baumannii. Evidence of a virus was rare. Cultures performed from BAL and SPU samples gave poor results. Most of the discrepant negative cultures were those in which FAPP detected pathogens with a microbial count ≤ 105 CFU/mL. H. influenzae was one of the most common pathogens lost by the conventional method. Despite the potential limitations of FAPP, which detects a defined number of pathogens, its advantages of rapid detection combined with predictive information regarding the antimicrobial resistance of pathogens through the detection of some relevant markers of resistance could be very useful for establishing empirical targeted therapy for the treatment of patients with respiratory failure. In the COVID era, we understand the importance of using antibiotics wisely to curb the phenomenon of antibiotic resistance.