RESUMO
We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.
RESUMO
Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.