•PCR assays to detect key mutations of SARS-CoV-2 spike protein are described•Discrimination of Variants of Concern (VOCs) and other variants is achieved•The assays detected key mutations of ...20I/501Y.V1 and 20 J/501Y.V3 in sewage in Italy•The method allows rapid and cost-effective detection of VOCs in sewage for WBE•The described approach can be used for rapid screening in clinical samples
New SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response.
We describe a nested RT-PCR assay (~1500 bps) to detect multiple nucleotide changes resulting in key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20 J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (~300 bps) were also designed, targeting portions of the region spanned by the long nested assay.
The three newly-designed assays were then tested on field samples, including 31 clinical samples (7 fully-sequenced swab samples, and 24 uncharacterized ones) and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported.
The long assay successfully amplified 29 of the 31 swabs (93%), allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel of previously characterized samples. The Spanish variant was detected in 14/24 of the uncharacterized samples as well. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20 J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity.
The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.
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Cardiovascular disease (CVD) is still the leading cause of death worldwide. Despite successful advances in both pharmacological and lifestyle strategies to fight well-established risk factors, the ...burden of CVD is still increasing. Therefore, it is necessary to further deepen our knowledge of the pathogenesis of the disease for developing novel therapies to limit even more its related morbidity and mortality. Oxidative stress has been identified as a common trait of several manifestations of CVD and could be a promising target for innovative treatments. Mitochondria are a major source of oxidative stress and sirtuins are a family of enzymes that generate different post-translational protein modifications, thus regulating important cellular processes, including cell cycle, autophagy, gene expression, and others. In particular, three sirtuins, SIRT3, SIRT4, and SIRT5 are located within the mitochondrial matrix where they regulate energy production and antioxidant pathways. Therefore, these sirtuins are strongly involved in the balance between oxidant and antioxidant mechanisms. In this review, we summarize the activities of these sirtuins with a special focus on their role in the control of oxidative stress, in relation to energy metabolism, atherosclerosis, and CVD.
Seabirds are extensively used as environmental biomonitors and feathers are among the most analyzed matrices because they are one of the main excretory pathways to detoxify the bird's body of ...environmental contaminants. Still, there is a variation in contamination level between the different feathers of seabird species, driven by diet and physiology, such as molt strategy and feather formation sequence. We measured total mercury (THg) concentration in different types of feathers (wing, tail, ventral and dorsal) of the same individual in adults and juveniles of brown boobies (Sula leucogaster) from the northern coast of Rio de Janeiro state, Brazil. Brown booby had higher mean THg concentration (μg.g−1 d. w.) in ventral (adults: 6.46 ± 1.19, 4.79 to 8.34; juveniles: 4.23 ± 0.60, 3.07 to 5.07) and wing (adults: 5.85 ± 1.10, 4.66 to 8.32; juveniles: 3.86 ± 0.54, 3.23 to 4.63), compared to dorsal (adults: 4.52 ± 1.33, 3.01 to 6.44; juveniles: 3.51 ± 0.19, 3.29 to 3.8) and tail feathers (adults: 2.94 ± 0.45, 2.32 to 3.46; juveniles: 2.8 ± 0.23, 2.45 to 3.08). This difference may be explained because feathers grow in a specific sequence during molts leading to different THg concentrations in each type of feather. Additionally, juveniles had significantly lower concentrations of THg than adults in all feather types, which may be explained by the shorter life span, leading to less time to bioaccumulate Hg in their body. It is essential to choose carefully which feather type is more suitable to be used as a biomonitor of THg contamination in a particular species. For brown boobies, we suggest the use of ventral feathers, which represent the highest Hg concentration, are easy to sample and do not impair the seabird's flight ability, although more studies are needed to replicate these results in other tropical seabirds species.
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•THg levels were higher in adults than juveniles probably due to bioaccumulation.•THg was lower in tail and higher in ventral feathers of adults and juveniles.•Ventral feathers are the best choice to analyze THg concentration in brown booby.
The outbreak of coronavirus infectious disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has rapidly spread throughout the world. Several studies ...have shown that detecting SARS-CoV-2 in untreated wastewater can be a useful tool to identify new outbreaks, establish outbreak trends, and assess the prevalence of infections. On 06 May 2021, over a year into the pandemic, we conducted a scoping review aiming to summarize research data on SARS-CoV-2 in sewage. Papers dealing with raw sewage collected at wastewater treatment plants, sewer networks, septic tanks, and sludge treatment facilities were included in this review. We also reviewed studies on sewage collected in community settings such as private or municipal hospitals, healthcare facilities, nursing homes, dormitories, campuses, airports, aircraft, and cruise ships. The literature search was conducted using the electronic databases PubMed, EMBASE, and Web Science Core Collection. This comprehensive research yielded 1090 results, 66 of which met the inclusion criteria and are discussed in this review. Studies from 26 countries worldwide have investigated the occurrence of SARS-CoV-2 in sewage of different origin. The percentage of positive samples in sewage ranged from 11.6 to 100%, with viral concentrations ranging from ˂LOD to 4.6 × 10
8
genome copies/L. This review outlines the evidence currently available on wastewater surveillance: (i) as an early warning system capable of predicting COVID-19 outbreaks days or weeks before clinical cases; (ii) as a tool capable of establishing trends in current outbreaks; (iii) estimating the prevalence of infections; and (iv) studying SARS-CoV-2 genetic diversity. In conclusion, as a cost-effective, rapid, and reliable source of information on the spread of SARS-CoV-2 and its variants in the population, wastewater surveillance can enhance genomic and epidemiological surveillance with independent and complementary data to inform public health decision-making during the ongoing pandemic.
Graphic Abstract
Since several seabird species have sexual size dimorphism, in which one sex is larger than the other, and may consume bigger prey, this size difference may affect the contamination concentration in ...the seabird’s tissues depending on their sex and age. In this study, mercury contamination was investigated in brown booby (Sula leucogaster) adults and juveniles during their breeding season at the Santana Archipelago, on the southeast coast of Brazil. Two hypotheses were evaluated: 1. As females consume larger prey than males due to the reverse sexual dimorphism, higher total mercury (THg) and methylmercury (MeHg) concentrations are expected in females tissues than in males; 2. Adult seabirds have more time to accumulate mercury than juveniles, so it is expected that adults will show higher THg and MeHg concentrations than juveniles in their feathers, but none in blood since the last indicates the exposure of short time (30–60 days), as it is a constantly synthesized tissue. Feathers and blood were sampled from 20 individuals of each group (males, females and juveniles). Also, 10 eggs of the brown booby and muscle tissue samples of their main prey were collected, from February to October 2018. Females and males had similar THg concentrations in the tissues with no statistical differences between sexes. Thus, the sexual size dimorphism did not influence mercury concentrations among the tissues and both genders can be used as a biomonitor. Brown booby juveniles had low THg and MeHg concentrations compared to adults due to a shorter time of exposure for mercury to bioaccumulate in their tissues. This is the first study, to the best of our knowledge, analyzing methylmercury in feathers, blood and eggs of a tropical seabird, which can be a useful baseline for future studies on the effects of contaminants on this species in tropical regions.
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•Brown boobies are good monitors to indicate the presence of mercury.•Despite a reverse sexual dimorphism, Hg concentrations were similar for both sexes.•In blood, Hg concentrations of adults and juveniles were similar due the same diet.•This seabird population had low Hg concentrations, below to the threshold limit.•This is the first study to analyze methylmercury in tissues of a tropical seabird.
Tropical seabird species, with reverse sexual dimorphism had similar Hg concentrations between sexes, while adults showed higher Hg concentrations than juveniles in feathers, but not in blood.
Intracellular pathogens are a critical challenge for antimicrobial therapies. Staphylococcus aureus (S. aureus) causes approximately 85% of all skin and soft tissue infections in humans worldwide and ...more than 30% of patients develop chronic or recurrent infections within three months, even after appropriate antibacterial therapies. S. aureus is also one of the most common bacteria found in chronic wounds. Recent evidences suggest that S. aureus is able to persist within phagolysosomes of skin cells (i.e. keratinocytes, phagocytic cells), being protected from both the immune system and a number of antimicrobials. To overcome these limits, nano-formulations that enable targeted therapies against intracellular S. aureus might be developed. Herein, the biodistribution and intracellular localisation of hyaluronan (HA) and HA-based nanoparticles (nanogels, NHs) are investigated, both after intravenous (i.v.) injections (in mice) and topical administrations (in ex vivo human skin). Results indicate HA and NHs accumulate especially in skin and liver of mice after i.v. injection. After topical application on human skin explants, no penetration of both HA and NHs was detected in skin with intact stratum corneum. By contrast, in barrier-disrupted human skin (with partial removal and loosening of stratum corneum), HA and NHs penetrate to the viable epidermis and are taken up by keratinocytes. In mechanically produced wounds (skin without epidermis) they accumulate in wound tissue and are taken up by dermis cells, e.g. fibroblasts and phagocytic cells. Interestingly, in all cases, the cellular uptake is CD44-mediated. In vitro studies confirmed that after CD44-mediated uptake, both HA and NHs accumulate in lysosomes of dermal fibroblasts and macrophages, as previously reported for keratinocytes. Finally, the colocalisation between intracellular S. aureus and HA or NHs is demonstrated, in macrophages. Altogether, for the first time, these results strongly suggest that HA and HA-based NHs can provide a targeted therapy to intracellular S. aureus, in persistent skin or wound infections.
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•Hyaluronan (HA) and its self-assembled nanogels (NHs) mainly accumulate in skin and liver of mice after i.v. injection•HA and NHs do not penetrate human skin with intact stratum corneum•In barrier-disrupted human skin HA and NHs penetrate to the viable epidermis and are mostly taken up by keratinocytes•In wounds HA and NHs are taken up by dermis cells. In all cases, the cellular uptake is CD44-mediated•HA and NHs accumulate within lysosomes of skin cells and colocalise with intracellular S. aureus
•Our study compares the clinical usefulness of nerve ultrasound and nerve conduction study in patients with Bell’s palsy.•Nerve Ultrasound may show abnormally increased facial nerve diameter in the ...acute phase of Bell’s palsy.•The predictive value of facial nerve ultrasound for incomplete recovery is lower than that of the nerve conduction study.
This longitudinal study aims at assessing the predictive value of facial nerve high-resolution ultrasound (HRUS) for incomplete clinical recovery in patients with Bell’s palsy, the most common facial nerve disease.
We prospectively enrolled 34 consecutive patients with Bell’s palsy. All patients underwent neurophysiological testing (including facial nerve conduction study) and HRUS evaluations 10–15 days (T1), one month (T2), and three months (T3) after the onset of Bell’s palsy. Patients who did not experience complete recovery within three months were also evaluated after six months (T4). We have then compared the accuracy of HRUS with that of the facial nerve conduction study in predicting incomplete clinical recovery at three and six months.
At T1, the facial nerve diameter, as assessed with HRUS, was larger on the affected side than on the normal side, particularly in patients with incomplete recovery at T2, T3 and T4. ROC curve analysis, however, showed that the facial nerve diameter at T1 had a lower predictive value than the facial nerve conduction study for an incomplete clinical recovery at three (T3) and six (T4) months. Still, the facial nerve diameter asymmetry, as assessed with HRUS, had a relatively high negative predictive value (thus indicating a strong association between normal HRUS examination and a good prognosis).
Although HRUS shows abnormally increased facial nerve diameter in patients in the acute phase of Bell’s palsy, the predictive value of this technique for incomplete clinical recovery at three and six months is lower than that of the nerve conduction study.
Nerve ultrasound has a low predictive value for incomplete clinical recovery in patients with Bell’s Palsy.
Saffold virus (SAFV) is an emerging human cardiovirus associated with respiratory and gastrointestinal infection, and, more recently, to symptoms related to the endocrine, cardiovascular, and ...neurological systems. Information about SAFV circulation in Italy is scarce. In order to provide insights into the epidemiology of SAFV in Italy, 141 raw sewage samples collected throughout Italy were tested using broad‐range nested RT‐PCR primers targeting the 5′‐NC region. Seven samples (5·0%) were confirmed as SAFV in samples collected in North, Centre and Southern Italy. Typing was attempted through amplification of the VP1 coding region, using both published and newly designed primers, and one sample was characterized as SAFV‐2.
Significance and Impact of the Study
Prevalence, genetic diversity and geographic distribution of SAFV in Italy is currently unknown. This study represents the first detection of SAFV in sewage samples in Italy, suggesting that it is circulating in the population despite lack of clinical reporting. Whether the virus is associated with asymptomatic cases or with undetected gastroenteritis or respiratory illness is unknown. Further studies are needed to investigate on the occurrence and persistence of SAFV in water environments and its waterborne transmission potential.
Significance and Impact of the Study: Prevalence, genetic diversity and geographic distribution of SAFV in Italy is currently unknown. This study represents the first detection of SAFV in sewage samples in Italy, suggesting that it is circulating in the population despite lack of clinical reporting. Whether the virus is associated with asymptomatic cases or with undetected gastroenteritis or respiratory illness is unknown. Further studies are needed to investigate on the occurrence and persistence of SAFV in water environments and its waterborne transmission potential.
Objectives
To characterize swallowing deficits in amyotrophic lateral sclerosis (ALS); investigate the delay in dysphagia onset; estimate correlations between dysphagia severity and patients' ...functional status; identify the symptom(s) most likely to predict dysphagia.
Materials and methods
A group of 49 consecutive patients with ALS, 14 with bulbar onset and 35 with spinal onset, underwent swallowing evaluation including bedside and fiberoptic endoscopic examination to detect dysphagia.
Results
Patients with dysphagia were more likely than those without to have bulbar onset ALS (P = 0.02); more severely impaired chewing (P = 0.01); and tongue muscle deficits (P = 0.001). The only variable measured at first examination significantly associated with dysphagia was a more than mild tongue muscle deficit. The only variable useful in predicting dysphagia was a chewing deficit. In 10 of the 49 patients studied, swallowing evaluation disclosed an impaired cough reflex.
Conclusions
Dysphagia in patients with ALS correlates significantly with bulbar onset and with oral swallowing impairment. Fiberoptic swallowing evaluation is a useful tool for detecting swallowing deficits and laryngeal sensitivity in patients with ALS. An impaired cough reflex is an unexpected finding in many patients with ALS.
Hepatitis E is a liver disease caused by the hepatitis E virus (HEV), primarily transmitted through contaminated water or food. There are four different HEV genotypes in humans, with genotypes 1 and ...2 being the most widespread. Genotypes 3 and 4 are found in animals and can also infect humans. Genotype 4 is prevalent in Asia, mainly in China. In Italy, only one outbreak of HEV-4 has been documented, which occurred in 2011, involving five patients. In 2013, HEV G4 was also detected in a pig farm. Since then, no further evidence of HEV genotype 4 has been found in the country. This study describes the first detection of HEV genotype 4, subtype d, in wastewater in central Italy, despite a lack of any clinical case reported in the area. By using a multiplex PCR protocol and two sequencing strategies, Illumina and ONT, the virus's complete genome was sequenced and characterized as subtype 4d. These findings shed light on the potential of environmental surveillance for infectious agents to improve our understanding of epidemiology and support public health efforts.
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•First detection of HEV genotype 4, subtype d, in wastewater in central Italy•Complete genome of HEV-4d was obtained by Illumina and ONT sequencing.•Importance of environmental monitoring in epidemiology and public health
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