Identification of Novel Non-Nucleoside Inhibitors of Zika Virus NS5 Protein Targeting MTase Activity.

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors.

Use of saliva and RT-PCR screening for SARS-CoV-2 variants of concern: Surveillance and monitoring.

Genomic surveillance has been applied since the beginning of the COVID-19 pandemic to track the spread of the virus, leading to the characterization of multiple SARS-CoV-2 variants, including variants of concern (VOC). Although sequencing is the standard method, a rapid molecular test for screening and surveillance of VOC is considered for detection. Furthermore, using alternative saliva as specimen collection facilitates the implementation of a less invasive, self-collected sample. In this study, we applied a combinatory strategy of saliva collection and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 VOC detection. Saliva samples from patients attending a tertiary hospital with suspected COVID-19 were collected and SARS-CoV-2 RNA was detected using SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer). Positive saliva samples were screened for SARS-CoV-2 VOC with previously described RT-PCR for Alpha, Beta, and Gamma variants. Saliva samples were positive in 171 (53%) of 324 tested. A total of 108 (74%) from positive samples were also positive for VOC by RT-PCR screening. Those samples were found between January and August 2021. This approach allowed us to successfully use an alternative and complementary tool to genomic surveillance to monitor the circulation of SARS-CoV-2 VOC in the studied population.

Plasma Torquetenovirus (TTV) microRNAs and severity of COVID-19.

BACKGROUND: Torquetenovirus (TTV), a widespread anellovirus recognized as the main component of the healthy human virome, displays viremia that is highly susceptible to variations in immune competence. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Among TTV-encoded miRNAs, miRNA t1a, t3b, and tth8 have been found in biological fluids. Here, the presence of TTV DNA and TTV miRNAs in the plasma of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected subjects was investigated to monitor the possible association with coronavirus disease 2019 (COVID-19) severity. METHODS: Detection of TTV DNA and miRNA t1a, t3b, and tth8 was investigated in plasma samples of 56 SARS-CoV-2-infected subjects with a spectrum of different COVID-19 outcomes. TTV DNA and TTV miRNAs were assessed with a universal single step real-time TaqMan PCR assay and miRNA quantitative RT-PCR miRNA assay, respectively. RESULTS: The TTV DNA prevalence was 59%, whereas at least one TTV miRNA was found in 94% of the patients tested. miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNAt1a (30%). Remarkably, although TTV DNA was unrelated to COVID-19 severity, miRNA tth8 was significantly associated with the degree of disease (adjusted incidence rate ratio (IRR) 2.04, 95% CI 1.14-3.63, for the subjects in the high severity group compared to those in the low severity group). CONCLUSIONS: Our findings encourage further investigation to understand the potential role of TTV miRNAs in the different outcomes of COVID-19 at early and late stages.

Volatiles and Antifungal-Antibacterial-Antiviral Activity of South African Salvia spp. Essential Oils Cultivated in Uniform Conditions.

Spontaneous emissions of S. dentata Aiton and S. scabra Thunb., as well as the essential oil (EO) composition of the cited species, together with S. aurea L., were investigated. The chemical profile of the first two species is reported here for the first time. Moreover, in vitro tests were performed to evaluate the antifungal activity of these EOs on Trichophyton mentagrophytes, Microsporum canis, Aspergillus flavus, Aspergillus niger, and Fusarium solani. Secondly, the EO antibacterial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus pseudointermedius was examined, and their antiviral efficacy against the H1N1 influenza virus was assessed. Leaf volatile organic compounds (VOCs), as well as the EOs obtained from the arial part of Salvia scabra, were characterized by a high percentage of sesquiterpene hydrocarbons (97.8% and 76.6%, respectively), mostly represented by an equal amount of germacrene D (32.8% and 32.7%, respectively). Both leaf and flower spontaneous emissions of S. dentata, as well as the EO composition, showed a prevalence of monoterpenes divided into a more or less equal amount of hydrocarbon and oxygenated compounds. Interestingly, its EO had a non-negligible percentage of oxygenated sesquiterpenes (29.5%). S. aurea EO, on the contrary, was rich in sesquiterpenes, both hydrocarbons and oxygenated compounds (41.5% and 33.5%, respectively). S. dentata EO showed good efficacy (Minimal Inhibitory Concentration (MIC): 0.5%) against M. canis. The tested EOs were not active against E. coli and S. aureus, whereas a low inhibition of S. dentata EO was observed on S. pseudointermedius (MIC = 10%). Once again, S. dentata EO showed a very good H1N1 inhibition; contrariwise, S. aurea EO was completely inactive against this virus. The low quantity of S. scabra EO made it impossible to test its biological activity. S. dentata EO exhibited interesting new perspectives for medicinal and industrial uses.

Co-circulation of the two influenza B lineages during 13 consecutive influenza surveillance seasons in Italy, 2004-2017.

BACKGROUND: Since 1985, two antigenically distinct lineages of influenza B viruses (Victoria-like and Yamagata-like) have circulated globally. Trivalent seasonal influenza vaccines contain two circulating influenza A strains but a single B strain and thus provide limited immunity against circulating B strains of the lineage not included in the vaccine. In this study, we describe the characteristics of influenza B viruses that caused respiratory illness in the population in Italy over 13 consecutive seasons of virological surveillance, and the match between the predominant influenza B lineage and the vaccine B lineage, in each season. METHODS: From 2004 to 2017, 26,886 laboratory-confirmed influenza cases were registered in Italy, of which 18.7% were type B. Among them, the lineage of 2465 strains (49%) was retrieved or characterized in this study by a real-time RT-PCR assay and/or sequencing of the hemagglutinin (HA) gene. RESULTS: Co-circulation of both B lineages was observed each season, although in different proportions every year. Overall, viruses of B/Victoria and B/Yamagata lineages caused 53.3 and 46.7% of influenza B infections, respectively. A higher proportion of infections with both lineages was detected in children, and there was a declining frequency of B/Victoria detections with age. A mismatch between the vaccine and the predominant influenza B lineage occurred in eight out of thirteen influenza seasons under study. Considering the seasons when B accounted for > 20% of all laboratory-confirmed influenza cases, a mismatch was observed in four out of six seasons. Phylogenetic analysis of the HA1 domain confirmed the co-circulation of both lineages and revealed a mixed circulation of distinct evolutionary viral variants, with different levels of match to the vaccine strains. CONCLUSIONS: This study contributes to the understanding of the circulation of influenza B viruses in Italy. We found a continuous co-circulation of both B lineages in the period 2004-2017, and determined that children were particularly vulnerable to Victoria-lineage influenza B virus infections. An influenza B lineage mismatch with the trivalent vaccine occurred in about two-thirds of cases.

Multiple Signatures of the JC Polyomavirus in Paired Normal and Altered Colorectal Mucosa Indicate a Link with Human Colorectal Cancer, but Not with Cancer Progression.

The JC polyomavirus (JCV) has been repeatedly but discordantly detected in healthy colonic mucosa, adenomatous polyps, and colorectal cancer (CRC), and proposed to contribute to oncogenesis. The controversies may derive from differences in JCV targets, patient's cohorts, and methods. Studies of simultaneous detection, quantification, and characterization of JCV presence/expression in paired samples of normal/altered tissues of the same patient are lacking. Therefore, we simultaneously quantified JCV presence (DNA) and expression (mRNA and protein) of T-antigen (T-Ag), Viral Protein 1 (Vp1), and miR-J1-5p in paired normal/altered tissues of CRC or polyps, and from controls. JCV signatures were found in most samples. They increased in patients, but were higher in normal mucosa than in corresponding polyp or CRC lesions. JCV non-coding control region (NCCR) DNA rearrangements increased in CRC patients, also in normal mucosa, thus before the onset of the lesion. A new ∆98bp NCCR DNA rearrangement was detected. T-Ag levels were higher in normal mucosa than in adenoma and adenocarcinoma lesions, but decreased to levels of controls in established CRC lesions. In CRC, miR-J1-5p expression decreased with CRC progression. Vp1 expression was not detected. The data indicate a JCV link with the disease, but possible JCV contributes to oncogenesis should occur at pre-polyp stages.

Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples.

BACKGROUND: Torquetenovirus (TTV) belongs to Anelloviridae family, infects nearly all people indefinitely without causing overt disease establishing a fine and successful interaction with the host. Increasing evidence have shown some human viruses exploit extracellular vesicles thereby helping viral persistence in the host. Here, the presence of TTV in extracellular vesicles circulating in human plasma was investigated. METHODS: TTV DNA was quantified in plasma-derived exosomes from 122 samples collected from 97 diseased patients and 25 healthy donors. Exosomes enriched vesicles (EEVs) were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81 and annexin II protein and, finally, by electron microscopy (EM). Presence and quantitation of TTV DNA were assessed with an universal single step real-time TaqMan PCR assay. RESULTS: Preliminary investigation showed that the human plasma extracted extracellular vesicles exhibited a main size of 70 nm, had concentration of 2.5 × 109/ml, and scored positive for tetraspanin CD63, CD81 and annexin II, typical characteristic of the exosomes vesicles. EEVs extracted from pooled plasma with TTV DNA viremia of 9.7 × 104 copies/ml showed to contain 6.3 × 102 TTV copies/ml, corresponding to 0.65% of total viral load. Important, TTV yield changed significantly following freezing/thawing, detergents and DNAse treatment of plasma before EEVs extraction. EEVs purified by sucrose-density gradient centrifugation and analysis of gradient fraction positive for exosomes marker CD63 harbored 102 TTV copies/ml. Moreover, EM evidenced the presence of TTV-like particles in EEVs. Successive investigation of plasma EEVs from 122 subjects (37 HIV-positive, 20 HCV infected, 20 HBV infected, 20 kidney transplant recipients, and 25 healthy) reported TTV DNA detection in 42 (34%) of the viremic samples (37 were from diseased patients and 5 from healthy people) at a mean level of 4.8 × 103 copies/ml. The examination of EEVs selected samples reported the presence of TTV genogroup 1, 3, 4 and 5, with genogroup 3 highly observed. CONCLUSIONS: Collectively, although these observations should be confirmed by further studies, circulation of TTV particles in EEVs opens new avenues and mechanistic insights on the molecular strategies adopted by anelloviruses to persist in the host.

Polyomavirus microRNAs circulating in biological fluids during viral persistence.

Increasing evidence suggests that microRNA-mediated gene silencing, detected during exosome intercellular communication between cells, may be exploited by persistent human viruses. Recently, it has been reported that human polyomaviruses encode microRNAs that downregulate large T expression and target host factors, helping the virus to escape immune elimination. Consequently, viral microRNAs and their genetic variability may have roles in the induction of polyomavirus reactivation, the success of persistence or replication and the development of diseases. In vitro experiments have detected polyomavirus JC (JCPyV) microRNAs in exosomes obtained from cell supernatants after viral infection and showed that they can be carried into uninfected cells. JCPyV and BKPyV microRNAs have been sought in clinical samples obtained from patients with or at risk of severe polyomavirus-associated diseases and from healthy subjects. Variable expressions of JCPyV and BKPyV microRNAs circulating in blood, urine, and cerebrospinal fluid samples were found in patients who were polyomavirus DNA positive and were also observed in negative subjects. Differences in the relationship between the JCPyV and BKPyV microRNA expressions and viral DNA load have been observed. All the data point towards a potential role of polyomavirus exosome microRNAs in viral persistence and suggest that further work is warranted to define their role in viral reactivation and to identify potential new antiviral strategies targeting these viruses.

Detection of JCPyV microRNA in blood and urine samples of multiple sclerosis patients under natalizumab therapy.

Polyomavirus JC (JCPyV) reactivation and development of progressive multifocal leukoencephalopathy is a health concern in multiple sclerosis patients under natalizumab therapy. Here, the JCPyV microRNA-J1-3p and microRNA-J1-5p expressions and genomic variability were investigated in blood and urine samples of multiple sclerosis patients before and under natalizumab therapy and in healthy controls. The two JCPyV microRNAs were detected in the JCPyV-DNA-positive peripheral blood mononuclear cell samples and in the exosomes derived from plasma and urine obtained from JCPyV-DNA-positive and JCPyV-DNA-negative patients. In particular, the increased JCPyV microRNA expression in samples of multiple sclerosis patients under natalizumab therapy was consistent with the high JCPyV-DNA positivity observed in these samples. Moreover, JCPyV microRNA genomic region showed few nucleotide differences in samples obtained from blood and urine of multiple sclerosis patients and healthy controls. Overall, these data suggest a potential role of the JCPyV microRNA expression in counteracting the viral reactivation to maintain JCPyV asymptomatic persistence in the host.

Investigation of Oral Shedding of Torquetenovirus (TTV) in Moderate-to-Severe COVID-19 Hospitalised Patients.

BACKGROUND: Torquetenovirus (TTV) is a small DNA virus constituting the human virome. High levels of TTV-DNA have been shown to be associated with immunosuppression and inflammatory chronic disorders. AIM: To assess the possible association between the salivary viral load of TTV-DNA in patients hospitalised due to COVID-19 and disease severity. METHODS: Saliva samples collected from 176 patients infected with SARS-CoV-2 were used to investigate the presence of SARS-CoV-2 and TTV-DNA by use of real-time RT-PCR. RESULTS: The majority of patients were male with severe COVID-19. Presence of SARS-CoV-2 was observed in the saliva of 64.77% of patients, showing TTV-DNA in 55.68% of them. Patients with impaired clinical conditions (p < 0.001), which evolved to death (p = 0.003), showed a higher prevalence of TTV-DNA. The median viral load in patients with severe condition was 4.99 log10 copies/mL, in which those who were discharged and those evolving to death had values of 3.96 log10 copies/mL and 6.27 log10 copies/mL, respectively. A statistically significant association was found between the distribution of TTV-DNA viral load in saliva samples and severity of COVID-19 (p = 0.004) and disease outcomes (p < 0.001). CONCLUSIONS: These results indicate that TTV-DNA in saliva could be a useful biomarker of COVID-19 severity and prognosis.

Antiviral Activity against SARS-CoV-2 of Conformationally Constrained Helical Peptides Derived from Angiotensin-Converting Enzyme 2.

Despite the availability of vaccines, COVID-19 continues to be aggressive, especially in immunocompromised individuals. Therefore, the development of a specific therapeutic agent with antiviral activity against SARS-CoV-2 is necessary. The infection pathway starts when the receptor binding domain of the viral spike protein interacts with the angiotensin converting enzyme 2 (ACE2), which acts as a host receptor for the RBD expressed on the host cell surface. In this scenario, ACE2 analogs binding to the RBD and preventing the cell entry can be promising antiviral agents. Most of the ACE2 residues involved in the interaction belong to the α1 helix, more specifically to the minimal fragment ACE2(24-42). In order to increase the stability of the secondary structure and thus antiviral activity, we designed different triazole-stapled analogs, changing the position and the number of bridges. The peptide called P3, which has the triazole-containing bridge in the positions 36-40, showed promising antiviral activity at micromolar concentrations assessed by plaque reduction assay. On the other hand, the double-stapled peptide P4 lost the activity, showing that excessive rigidity disfavors the interaction with the RBD.

SARS-CoV-2 inhibitory activity of a short peptide derived from internal fusion peptide of S2 subunit of spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a great concern in human population. To fight coronavirus emergence, we have dissected the conserved amino acid region of the internal fusion peptide in the S2 subunit of Spike glycoprotein of SARS-CoV-2 to design new inhibitory peptides. Among the 11 overlapping peptides (9-23-mer), PN19, a 19-mer peptide, exhibited a powerful inhibitory activity against different SARS-CoV-2 clinical isolate variants in absence of cytotoxicity. The PN19 inhibitory activity was found to be dependent on conservation of the central Phe and C-terminal Tyr residues in the peptide sequence. Circular dichroism spectra of the active peptide exhibited an alpha-helix propensity, confirmed by secondary structure prediction analysis. The PN19 inhibitory activity, exerted in the first step of virus infection, was reduced after peptide adsorption treatment with virus-cell substrate during fusion interaction. Additionally, PN19 inhibitory activity was reduced by adding S2 membrane-proximal region derived peptides. PN19 showed binding ability to the S2 membrane proximal region derived peptides, confirmed by molecular modelling, playing a role in the mechanism of action. Collectively, these results confirm that the internal fusion peptide region is a good candidate on which develop peptidomimetic anti SARS-CoV-2 antivirals.

Severe acute respiratory syndrome by influenza and factors associated with death in older adults: a population study.

INTRODUCTION: Influenza is characterized by an acute viral infection, which can lead to severe conditions and death, especially in vulnerable populations, such as older adults. Therefore, we sought to analyze cases of severe acute respiratory syndrome (SARS) due to influenza in older adults registered in Brazil and investigate the factors related to death due to this disease. METHODOLOGY: This is a cross-sectional, population-based study that used secondary data from the Influenza Epidemiological Surveillance Information System (IESIS-Influenza). Older adults aged 60 years and above with laboratory diagnosis of influenza were included. RESULTS: A total of 3,547 older adults with SARS due to influenza were included, out of which 1,185 cases with death as the outcome were identified. Among older adults with death as the outcome, 87.4% were not vaccinated against influenza. The main risk factors for death were invasive ventilatory support use, intensive care unit admission, brown skin color and dyspnea (p < 0.001). CONCLUSIONS: This study described the profile of older adults with SARS due to influenza in Brazil. Factors associated with death in this population were identified. Moreover, the need to encourage compliance with vaccination among older adults is evident in order to prevent severe cases and unfavorable outcomes related to influenza.

Assessment of the risk of polyomavirus JC reactivation in patients with immune-mediated diseases during long-term treatment with infliximab.

Polyomavirus JC (JCV) reactivation causing progressive multifocal leukoencephalopathy is a main concern during biological therapies. Here, JCV reactivation in patients suffering from immune-mediated diseases after a long-term treatment with anti-tumor necrosis factor alpha (TNF-α) inhibitor infliximab was investigated. Peripheral mononuclear blood cells (PBMC), plasma and urine samples were obtained from 61 immune-mediated diseases patients treated or not with infliximab in combination with steroid and other immunomodulators and from 20 healthy donors. JCV DNA was transiently detected in 12 PBMC of 40 patients at different doses of infliximab with a higher prevalence than that of the 21 patients untreated. Conversely, a stable JCV positivity in urine of treated and untreated patients was detected. Sequencing the noncoding control region (NCCR), all samples exhibited the archetype structure with few mutations in transcriptional factor binding regions. The consequence of anti-TNF-α treatment on viral persistence was examined monitoring Torquetenovirus viremia and investigating the TNF-α-induced microRNA regulators of transcriptional factors, with a binding site on NCCR. Although infliximab treatment in this study did not affect directly JCV reactivation, further investigation on host factor(s) regulated by it will be of warranty in the understanding the mechanism(s) that may affect viral persistence.

Screening of the essential oil effects on human H1N1 influenza virus infection: an in vitro study in MDCK cells.

Nineteen essential oils (EOs) obtained from different plants have been evaluated for their potential in vitro anti-H1N1 influenza virus efficacy. Both multivariate analyses and bivariate correlation were performed to better understand how the composition influences the activity. The results evidenced that for the laboratory distilled EOs both rosemary hybrids (S. x lavandulaceus and S. x mendizabalii) showed a good antiviral activity with low cytotoxic effect. Concerning the commercial ones: Eucalyptus globulus and Juniperus communis EOs exhibited virtuous effects on influenza virus. These results were confirmed by the multivariate analyses and only eucalyptol showed a positive correlation with cell viability. On the contrary, o-cymene and terpinolene correlated to the inhibitory effect. Rosemary hybrids, E. globulus and J. communis could be considered as promising candidate to develop new alternative anti-H1N1 natural agent.

Antiviral Activity of Oligonucleotides Targeting the SARS-CoV-2 Genomic RNA Stem-Loop Sequences within the 3'-End of the ORF1b.

Increased evidence shows vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited no long-term efficacy and limited worldwide availability, while existing antivirals and treatment options have only limited efficacy. In this study, the main objective was the development of antiviral strategies using nucleic acid-based molecules. To this purpose, partially overlapped 6-19-mer phosphorothioate deoxyoligonucleotides (S-ONs) designed on the SARS-CoV-2 genomic RNA stem-loop packaging sequences within the 3' end of the ORF1b were synthetized using the direct and complementary sequence. Among the S-ONs tested, several oligonucleotides exhibited a fifty percent inhibitory concentration antiviral activity ranging from 0.27 to 34 µM, in the absence of cytotoxicity. The S-ON with a scrambled sequence used in the same conditions was not active. Moreover, selected 10-mer S-ONs were tested using different infectious doses and against different SARS-CoV-2 variants, showing comparable antiviral activity that was abrogated when the central sequence was mutated. Experiments to evaluate the intracellular functional target localization of the S-ON inhibitory activity were also performed. Collectively the data indicate that the SARS-CoV-2 packaging region in the 3' end of the ORF1b may be a promising target candidate for further investigation to develop innovative nucleic-acid-based antiviral therapy.

Factors associated with death due to severe acute respiratory syndrome caused by influenza: Brazilian population study.

INTRODUCTION: Influenza infection is characterized by acute viral infection of high transmissibility. Worsening of the case can lead to the need for hospitalization, severe acute respiratory syndrome (SARS) and even death. METHOD: This is a cross-sectional population-based study that used secondary database from the Brazilian Influenza Epidemiological Surveillance Information System. Only cases of adults with diagnosis of influenza by RT-PCR and case evolution recorded were included. RESULTS: We identified 2273 adults with SARS by influenza, 343 of which had death as an outcome. The main risk factors for death were lack of hospitalization, not having cough and age, both with p < 0.001. In addition, without asthma, having black skin color, not receiving flu vaccine, having brown skin color and not having a sore throat (p ≤ 0.005) were risk factors too. CONCLUSION: Factors associated with death due to SARS caused by influenza in Brazil, risk factors and protective factors to death were identified. It was evident that those who did not receive the flu vaccine presented twice the risk of unfavorable outcome, reinforcing the need to stimulate adherence to vaccination adhering and propose changes in public policies to make influenza vaccines available to the entire population, in order to prevent severe cases and unfavorable outcomes.

Quantification of torque teno virus (TTV) DNA in saliva and plasma samples in patients at short time before and after kidney transplantation.

BACKGROUND: Several reports have proposed that the viral load of torque teno virus (TTV) in plasma is a biomarker of immune function in solid organ transplantation (SOT) and in allogeneic hematopoietic stem cell transplantation. Additionally, for the latter one, TTV-DNA quantification in saliva has also been suggested. AIM: to investigate the correlation between the TTV viral load and immune function in paired saliva and plasma samples in patients on kidney transplantation. MATERIALS AND METHODS: TTV-DNA viral load was quantified in paired samples of saliva and plasma from 71 patients before and a short-time after renal-transplantation by real-time PCR. RESULTS: The data obtained from 213 paired samples showed a slight consistency in the comparison between saliva and plasma, with prevalence of TTV-DNA being 58%, 52% and 60% in saliva samples and 60%, 73% and 90% in plasma samples before and at 15-20 and 45-60 days after transplantation, respectively. Additionally, a high TTV viral load was observed in plasma at 15-20 and 45-60 days after transplantation compared to that observed in saliva at the same time. CONCLUSIONS: Overall, monitoring TTV-DNA in saliva samples could be an additional fast non-invasive option to assess the immune functionality in SOT populations.

MALDI-TOF mass spectrometry of saliva samples as a prognostic tool for COVID-19.

BACKGROUND: The SARS-CoV-2 infections are still imposing a great public health challenge despite the recent developments in vaccines and therapy. Searching for diagnostic and prognostic methods that are fast, low-cost and accurate are essential for disease control and patient recovery. The MALDI-TOF mass spectrometry technique is rapid, low cost and accurate when compared to other MS methods, thus its use is already reported in the literature for various applications, including microorganism identification, diagnosis and prognosis of diseases. METHODS: Here we developed a prognostic method for COVID-19 using the proteomic profile of saliva samples submitted to MALDI-TOF and machine learning algorithms to train models for COVID-19 severity assessment. RESULTS: We achieved an accuracy of 88.5%, specificity of 85% and sensitivity of 91.5% for classification between mild/moderate and severe conditions. When we tested the model performance in an independent dataset, we achieved an accuracy, sensitivity and specificity of 67.18, 52.17 and 75.60% respectively. CONCLUSION: Saliva is already reported to have high inter-sample variation; however, our results demonstrates that this approach has the potential to be a prognostic method for COVID-19. Additionally, the technology used is already available in several clinics, facilitating the implementation of the method. Further investigation using a larger dataset is necessary to consolidate the technique.