Improved SARS-CoV-2 RNA recovery in wastewater matrices using a CTAB-based extraction method.

Wastewater-based epidemiology has allowed tracking the magnitude and distribution of SARS-CoV-2 in communities, allowing public health officials to prepare for impending outbreaks. While many factors influence recovery of SARS-CoV-2 from wastewater, proper extraction, concentration, and purification of RNA are key steps to ensure accurate detection of viral particles. The aim of this study was to compare the efficiency of four commonly used RNA extraction methods for detection of the SARS-CoV-2 RNA genome in sewage samples artificially inoculated with the virus, in order to identify a protocol that improves viral recovery. These methods included CTAB-based, TRIzol-based, and guanidinium thiocyanate (GTC)-based extraction procedures coupled with silica spin column-based purification, and an automated extraction/purification protocol using paramagnetic particles. Following RNA extraction, virus recovery rates were compared using RT-qPCR-based detection. The CTAB-based approach yielded the highest recovery rates and was the only method to consistently demonstrate stable virus recovery percentages regardless of the specific physicochemical characteristics of the samples tested. The TRIzol method proved to be the second most effective, yielding significantly higher recovery rates compared to both the GTC-based and the automated extraction methods. These results suggest that the CTAB-based approach could be a useful tool for the recovery of viral RNA from complex wastewater matrices.

Meta-analysis of the prevalence of norovirus and hepatitis a virus in berries.

Norovirus (NoV) and hepatitis A virus (HAV) stand as the predominant agents associated with viral foodborne infections. Outbreaks have been documented to be caused by various types of food items, including fresh and/or frozen berries. Comprehensive data concerning crucial viral pathogens in berries remain limited and are not currently available in aggregate form. Consequently, the present study aimed to compile the existing information regarding the prevalence of NoV and HAV in this matrix. Records of foodborne viruses were systematically extracted from database repositories up to December 2022, adhering to PRISMA standards and were subjected to a multilevel random effect meta-analysis model to determine the mean occurrence rate of NoV and HAV. A high heterogeneity across studies was observed (I2 = 82 %), reflecting variations in the prevalence of sampling locations, years, berry types, and sample conditions, among other potential contributing factors. The prevalence of NoV and HAV in berries was calculated at 2.12 % (95 % CI 1.74-2.59 %), and no statistically differences were observed among the viral types or genogroup categories. However, it is important to clarify that this estimate should be taken with caution given the high heterogeneity. There was no discernible correlation between viral prevalence and any particular berry type. However, there was a temporal correlation observed with the year of sampling, revealing a significantly decreasing trend throughout the study period. A significant influence of the sample condition (fresh or frozen) was observed in relation to the prevalence of NoV GII and HAV. Overall higher viral prevalences were identified in berries originating from African countries as compared to those sourced from other continents. It was also noted that the prevalence of NoV GI was significantly higher in samples collected directly from farms compared to those obtained from retailers. The outcomes of this comprehensive meta-analysis propose that while viral contamination of berries is diminishing in more recent times, the prevalence remains substantial in certain African countries, having a significant risk for foodborne infections. It is imperative to implement intervention strategies in these regions to enhance product safety.

Meta-analysis of the prevalence of the main human pathogens in vegetables, with emphasis on lettuce.

This meta-analysis aims to summarize the available information on the prevalence of the main human pathogenic microorganisms in vegetables, with emphasis on lettuce (Lactuca sativa). The database searches included scientific papers from 1980 to 2019, without language restrictions. Inclusion criteria were prevalence or incidence studies published in peer-reviewed journals reporting the total number of vegetable samples studied and the number of samples positive for the presence of the studied pathogens. The target pathogens were grouped into the following categories: bacteria, parasites and viruses. Results of different vegetable types, years of sampling, analyzed regions or species of microorganisms reported in the same article were considered as different studies. Therefore, each scientific article may contain several studies. Multilevel random-effect meta-analysis models were fitted to estimate the mean occurrence rate of pathogenic microorganisms and to compare them with different factors potentially associated with the outcome. Overall, the prevalence of bacterial, parasitic and viral pathogens in vegetables was relatively low. The mean prevalence of bacterial hazards was < 0.023, with the exception of S. aureus, whose prevalence was estimated at 0.096. The mean occurrence rates of parasites and viruses were 0.067 (95 % CI: 0.056-0.080) and 0.079 (95 % CI: 0.054-0.113), respectively. The prevalence of pathogenic E. coli and parasites increased as the year of publication of the scientific articles progressed, whereas the prevalence of the other bacterial pathogens and enteric viruses was steady. The types of vegetables evaluated did not affect pathogen prevalence. The prevalence of pathogenic microorganisms differed according to the continent of origin, except for E. coli O157:H7 and parasites. The prevalence of pathogens in vegetables is of public health importance, especially in vegetable types that are eaten raw, without thermal treatment to inactivate pathogens. This meta-analysis results show the need to apply proper sanitation methods to treat raw vegetables in order to avoid foodborne infections.

Occurrence of Norovirus, Rotavirus, Hepatitis a Virus, and Enterovirus in Berries in Argentina.

Berries have been implicated as the probable vehicle of infection in multiple outbreaks of norovirus and hepatitis A virus (HAV). These foods often receive minimal or no processing and may be exposed to virus contamination at each stage of production. In an increasingly globalized world, berries have a wide distribution and can give rise to the spread of diseases in distant parts of the world. With the aim of describing the virological quality of the berries cultivated in Argentina, a total of 184 soft fruits of different varieties (strawberries, blueberries, raspberries, blackberries, currants, pomegranate arils, cassis, and elder) were collected during the periods 2016-2018 and 2020. Viral particles were eluted and concentrated by polyethylene glycol precipitation according to ISO 15216-2:2019 guidelines. Genome detection of norovirus (NoV) genogroups I (GI) and II (GII), HAV, rotavirus, and enterovirus was performed by real-time RT-PCR with TaqMan probes. Positive samples were amplified by conventional RT-PCR and the amplicons were purified and sequenced in both directions. Phylogenetic analysis was performed using the Neighbor-Joining method based on the evolutionary model Kimura-2-parameters. NoV GII.6 was detected in 1/184 (0.5%) of the soft fruits, corresponding to a raspberry sample obtained during the fall of 2017. No presence of other human enteric viruses was found in the other berries analyzed. The collected data are the first in Argentina in relation to the prevalence of enteric viruses in berries and is useful as reference data for a risk assessment of soft fruits as vehicles of foodborne pathogenic viruses.

Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters.

Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2% and 45.0%, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 102 GC/mL) than PEG precipitation (4.3 × 103 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.

Rotavirus contamination of surface waters from the northwest of Argentina.

Fecal pollution of water is a serious concern because it is associated with the transmission of pathogens. The aim of this study was to analyze the occurrence of group A rotavirus (RVA) in surface waters from the Arias-Arenales River in Salta, a northern city in Argentina, and to define possible sources of fecal viral pollution. A total of 116 water samples were analyzed and RVA was detected in 3.4% (95% CI: 0.1-7.0%), with concentrations ranging from 1.9 × 105 to 3.8 × 106 genome copies per liter. RVA strains were characterized as G1P[8], G4P[8] and G9P[8], which are common genotypes circulating in the local population. The Arias-Arenales River presented unusual and sporadic contamination by RVA, originated from stormwater discharges and a variety of non-identified sources, and support the essential need of viral indicators for enhanced monitoring of water quality.

Evaluation of concentration efficiency of the Pseudomonas aeruginosa phage PP7 in various water matrixes by different methods.

Enteric viruses monitoring in surface waters requires the concentration of viruses before detection assays. The aim of this study was to evaluate different methods in terms of recovery efficiencies of bacteriophage PP7 of Pseudomonas aeruginosa, measured by real-time PCR, using it as a viral control process in water analysis. Different nucleic acid extraction methods (silica-guanidinium thiocyanate, a commercial kit (Qiagen Viral RNA Kit) and phenol-chloroform with alcohol precipitation) exhibited very low recovery efficiencies (0.08-4.18 %), being the most efficient the commercial kit used for subsequent experiments. To evaluate the efficiency of three concentration methods, PBS (as model for clean water) and water samples from rivers were seeded to reach high (HC, 10(6) pfu ml(-1)) and low concentrations (LC, 10(4) pfu ml(-1)) of PP7. Tangential ultrafiltration proved to be more efficient (50.36 ± 12.91, 17.21 ± 9.22 and 12.58 ± 2.35 % for HC in PBS and two river samples, respectively) than adsorption-elution with negatively charged membranes (1.00 ± 1.34, 2.79 ± 2.62 and 0.05 ± 0.08 % for HC in PBS and two river samples, respectively) and polyethylene glycol precipitation (15.95 ± 7.43, 4.01 ± 1.12 and 3.91 ± 0.54 %, for HC in PBS and two river samples, respectively), being 3.2-50.4 times more efficient than the others for PBS and 2.7-252 times for river samples. Efficiencies also depended on the initial virus concentration and aqueous matrixes composition. In consequence, the incorporation of an internal standard like PP7 along the process is useful as a control of the water concentration procedure, the nucleic acid extraction, the presence of inhibitors and the variability of the recovery among replicas, and for the calculation of the sample limit of detection. Thus, the use of a process control, as presented here, is crucial for the accurate quantification of viral contamination.