•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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Maintaining people living with HIV (PLWHIV) in clinical care is a global priority. In the Metro Detroit area of Michigan, approximately 30% of PLWHIV are out of care. To re-engage lost-to-follow-up ...patients, Wayne Health Infectious Disease clinic launched an innovative Homecare program in 2017. In addition to home healthcare delivery, the program included links to community resources and quarterly community meetings. We aimed to evaluate Homecare's impact on participants' ability to stay engaged in HIV care and reach viral suppression. We included data from PLWHIV and their healthcare workers.
We used a convergent mixed-methods design, including first year program record review, semi-structured interviews, and a validated Likert scale questionnaire rating illness perception before and after Homecare. Interview data were collected from 15 PLWHIV in Metro Detroit and two healthcare workers responsible for program delivery. Semi-structured interviews focused on obstacles to clinic-based care, support networks, and illness perceptions. Interview data were transcribed and analyzed using a thematic approach. A fully coded analysis was used to create a conceptual framework of factors contributing to Homecare's success. Means in eight categories of the Brief Illness Perception (IPQ) were compared using paired T-tests.
In the first year of Homecare, 28 of 34 participants (82%) became virally suppressed at least once. The program offered (1) social support and stigma reduction through strong relationships with healthcare workers, (2) removal of physical and resource barriers such as transportation, and (3) positive changes in illness perceptions. PLWHIV worked towards functional coping strategies, including improvements in emotional regulation, acceptance of their diagnosis, and more positive perspectives of control. Brief-IPQ showed significant changes in six domains before and after Homecare.
Homecare offers an innovative system for successfully re-engaging and maintaining lost-to-follow-up PLWHIV in care. These findings have implications for HIV control efforts and could inform the development of future programs for difficult to reach populations.
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
Microbial safety of recreational waters is a significant public health issue. In this study we assessed the occurrence and quantity of enteric viruses in bathing and non-bathing waters in Italy, in ...parallel with microbial faecal indicators, somatic coliphages and Vibrio spp.
Enteric viruses (aichivirus, norovirus and enterovirus) were detected in 55% of bathing water samples, including samples with bacterial indicator concentrations compliant with the European bathing water Directive. Aichivirus was the most frequent and abundant virus. Adenovirus was detected only in non-bathing waters. Somatic coliphages were identified in 50% bathing water samples, 80% of which showed simultaneous presence of viruses.
Vibrio species were ubiquitous, with 9 species identified, including potential pathogens (V. cholerae, V. parahaemoylticus and V. vulnificus).
This is the first study showing the occurrence and high concentration of Aichivirus in bathing waters and provides original information, useful in view of a future revision of the European Directive.
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•Enteric viruses were detected in both bathing and non-bathing water samples.•Aichivirus was the most abundant and frequently detected virus.•80% of samples with somatic coliphages were positive for enteric viruses.•Vibrio species were ubiquitous and numerous, with higher counts in August and September.
The diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires the detection of viral RNA by reverse transcription–polymerase chain reaction (RT-qPCR) performed ...mainly using nasopharyngeal swabs. However, this procedure requires separate analysis per each individual, performed in advanced centralized laboratory facilities with specialized medical personnel. In this study, an alternative approach termed “solid waste-based surveillance (SWBS)” was explored, in order to investigate SARS-CoV-2 infection in small communities through the indirect sampling of saliva left on waste. Sampling was performed at 20 different sites in Italy during the second peak of COVID-19. Three swabs were positive for SARS-CoV-2 using a published RT-qPCR protocol targeting the non-structural protein 14 region, and the viral load ranged 4.8 × 103–4.0 × 106 genome copies/swab. Amino acid substitutions already reported in SARS-CoV-2 sequences circulating in Italy (A222V and P521S) were detected in two positive samples. These findings confirmed the effectiveness of SWBS for non-invasive and dynamic SARS-CoV-2 surveillance.
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•New community surveillance method based on saliva swab and solid waste was proposed.•RNA of SARS-CoV-2 was detected on several single use glasses and bottles.•Specific variances of genomic sequences of the RNA were also detected.•Solid waste based surveillance (SWBS) resulted suitable for SARSC-CoV-2 detection.
Recent studies documented the detection of viruses strongly associated with human cancer in urban sewages and other water environments worldwide. The aim of this study was to estimate the occurrence ...of human oncogenic viruses in environmental samples (sewage, river, marine, and pool/spa water) using highly sensitive and specific multiplex bead-based assays (Luminex technology). A total of 33 samples were analysed for 140 oncogenic viral agents, including mucosal and cutaneous human papillomaviruses (HPVs), human polyomaviruses (HPyV), human herpesviruses (HHV) and mouse mammary tumour virus (MMTV).
Eighty-eight percent of the samples tested positive for at least one viral pathogen and the simultaneous presence of more than one virus was frequent (mean number of positivities/sample = 3.03). A total of 30 different Alpha, Beta and Gamma HPVs were detected, including mucosal and cutaneous types. The high-risk type HPV16 was the most frequently detected virus, identified in 73% of the samples. Of the 12 HPyVs tested, only two (BKPyV and MCPyV) were detected. At least one of these two was present in 48% of the samples. MMTV was detected in 21% of the samples, while herpesviruses - HHV-6 and HHV-1 - were detected in two samples (6%).
The present study is the first to provide a comprehensive picture of the occurrence of oncogenic viruses belonging to different families and species in diverse water environments, and the first to successfully use, in environmental samples, a Luminex-based multiplex platform for high throughput screening of infectious agents. Our findings, showing that oncogenic viruses are ubiquitous in water environments, pave the way for future studies on the fate of these pathogens in water environments as well as on their potential for transmission via the waterborne route.
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•A multiplex bead-based assay to detect oncogenic viruses in water.•Widespread viral contamination detected in waters.•Simultaneous detection of different oncogenic viruses.•Human papillomavirus as the most frequently detected virus.
Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. ...The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 10
6
) > treated sewage (2.9 × 10
5
) > river waters (6.1 × 10
2
) > estuarine waters (4.8 × 10
2
) > bathing waters (8.5 × 10
1
) > groundwater (5.9 × 10
1
). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.
AIMS: Viral outbreaks associated with swimming pools have been described worldwide. The objective of this study was to examine the extent of viral contamination in indoor and outdoor swimming pools. ...METHODS AND RESULTS: Pools were examined for the presence of human enteric viruses (adenovirus, norovirus and enterovirus) and nonenteric viruses (papillomavirus and polyomavirus—BK, JC, KI, WU and Merkel cell). Bacteriological parameters were also evaluated. The analysed pool waters met microbiological quality standards. Enteric viruses were not detected. On the other hand, papillomaviruses (HPV8, 12, 23, 25, 120 and unclassified HPVs) and polyomaviruses (JC and Merkel cell polyomaviruses) were detected in 9/14 samples (64%). The number of HPV DNA copies in pool waters, measured by quantitative Real‐time PCR, ranged from 1.27E+04 to 1.13E+05/10L. CONCLUSION: Results show that a variety of nonenteric viruses may be discharged in pool waters by various secretions and excretions from infected individuals or asymptomatic carriers. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on human papillomaviruses and polyomaviruses in swimming pools. The likelihood that these viruses can be transmitted by recreational activities deserves to be explored in future studies.
Sanitary tampons have been in existence for over 60 years. Their use may present certain health risks, potentially associated with an abnormal change of microbial flora in the vagina (e.g., toxic ...shock syndrome). Tampon production and marketing are regulated differently in different countries. In Australia, Canada and the USA, tampons are classified as Class-II medical devices and their marketing requires pre-clinical and clinical studies, including microbiological trials. In Europe, tampons are considered consumer products and safety-related data are provided only if the manufacturer deems them to be useful. Sterility of these products is not requested by law; thus they may represent a potential vehicle for microorganisms. Due to the lack of data on microbial characteristics of tampons, an analytical investigation was carried out to characterize and quantify the microbial flora present on sealed tampons of various brands present on the market in Italy.
Traditional cultural methods were used to characterize and quantify bacteria and fungi. Identification of colonies was performed with biochemical techniques.
Results showed low microbial concentrations in 93% of the positive samples. A rare presence of opportunistic pathogens was detected and a few samples (6%) were characterized by bacterial species of human origin.
In the light of these data, the examined tampons were found to have good hygienic quality. Nevertheless, to minimize the microbial risks linked to the use of these products, strict hygienic rules during their production and manipulation have to be adopted.
The presence of microorganisms in waters is of great public concern and the trouble of detecting them in water is even well known. The traditional culture methods used for detection of environmental ...and pathogenic microorganisms are often not very sensitive and tend to underestimate the actual microbial load. Furthermore, the laboratory techniques used for their isolation and quantification commonly give results within some days after sampling. Thus, all the commonest methods for the microbiological examination of water are retrospective. In most cases, the time needed for results can represent a substantial problem, since possible microbial contamination can be confirmed when public health has been already jeopardized. Consequently, the use of procedures that provide rapid results becomes of fundamental importance.
A variety of analytical approaches have been proposed for the rapid detection of microorganisms in water and the development of new and improved methods for microbiological water analysis is a continuous process. Advances in molecular biology for 20 years have increased the knowledge of microorganisms in water and molecular biology-based methods, notably those utilizing qualitative and quantitative PCR technology, have shown particular promise. These non-cultural methods are of great interest because their high throughput allows the detection of not cultivable bacterial indicators and pathogens and are less time-consuming.
For a rapid bacterial detection, biosensors have also been developed in recent years. Even techniques such as flow cytometry and immuno-magnetic separation are rapid, quantitative and versatile methods because can be combined with nucleic acid probes and immune fluorescence to monitor viability. The advent of next generation sequencing (NGS) platforms has revolutionized DNA sequencing by allowing massively parallel sequencing with millions of reactions running in the same experiment. New platforms (e.g., Illumina, Ion Torrent) have been introduced during the last decade, and NGS has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. The growing accessibility of NGS methods will greatly increase our understanding of microbial diversity in water environments.
In this review, some of the most innovative and promising analytical techniques are described.
•Microorganisms in waters are of great public concern and the trouble of detecting them in water is even well known.•Traditional culture methods are retrospective and tend to underestimate the actual microbial load.•Consequently, the use of procedures that provide rapid results are of fundamental importance.•A variety of analytical approaches has been proposed for the rapid detection of microorganisms in water.•The development of new and improved methods for microbiological water analysis is a continuing process.•Some of the most interesting and promising analytical techniques for water microbiological analysis are described.
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