Prevalence, distribution and environmental effects on faecal indicator bacteria and pathogens of concern in commercial shellfish production areas in a subtropical region of a developing country (Santa Catarina, Brazil).

This paper reviews recent literature on the abundance and distribution of faecal indicator bacteria and pathogens in shellfish production areas in the state of Santa Catarina, on the subtropical coast of Brazil. This state supplies > 95% of the national production of shellfish. Microbiological monitoring data were mapped using GIS and the results compared with those from other countries. Coastal human population is the main predictive parameter for faecal bacteria in the production areas. Temporal variations of the bacteria can also be predicted by solar radiation and rainfall. The prevalence of pathogens such as hepatitis A virus, human norovirus, Salmonella spp. and Vibrio spp. does not differ substantially from that in developed countries. The information reported here can be used to inform development of microbiological risk profiles for shellfish production areas.

Bioaccumulation Dynamic by Crassostrea gigas Oysters of Viruses That Are Proposed as Surrogates for Enteric Virus Contamination in Environmental Samples.

Oysters are filter-feeders and retain sewage-derived pathogens in their organs or tissues. Since most enteric viruses involved in outbreaks cannot grow in cell culture, studies using viral surrogate models are essential. Some species are proposed as surrogates for enteric viruses in environmental samples, including in bivalve mollusk samples, such as murine norovirus type 1 (MNV-1) and somatic (as φX) or F-specific coliphages (as MS2) bacteriophages. This study evaluated the tissue distribution of viral surrogates for enteric virus contamination after their bioaccumulation by Crassostrea gigas. Oyster tissues were analyzed for the distribution of viral surrogates (MNV-1, φX-174, and MS2) in digestive tissue (DT), gills (GL), and mantle (MT) after 4, 6, and 24 h of experimental bioaccumulation. MNV-1 had higher counts at 6 h in DT (1.2 × 103 PFU/g), followed by GL and MT (9.5 × 102 and 3.8 × 102 PFU/g, respectively). The bacteriophage φX-174 had a higher concentration in the MT at 4 and 6 h (3.0 × 102 PFU/g, in both) and MS2 in the GL after 24 h (2.2 × 102 PFU/g). The bioaccumulation pattern of MNV-1 by oysters was similar to the other enteric viruses (more in DT), while that of phages followed distinct patterns from these. Since the MNV-1 is bioaccumulated by C. gigas and is adapted to grow in cell culture, it is an important tool for bioaccumulation and viral inactivation tests in oysters. Although bacteriophage bioaccumulation was not similar to enteric viruses, they can be indicated for viral bioaccumulation analysis, analyzing MT and GL, since they do not bioaccumulate in DT.

Genomic Surveillance of SARS-CoV-2 in Healthcare Workers: A Critical Sentinel Group for Monitoring the SARS-CoV-2 Variant Shift.

SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa Catarina, southern Brazil, and evaluated the similarity between variants present in the population and healthcare workers (HCW). A total of 5291 sequenced genomes demonstrated the circulation of 55 strains and four variants of concern (Alpha, Delta, Gamma and Omicron-sublineages BA.1 and BA.2). The number of cases was relatively low in May 2021, but the number of deaths was higher with the Gamma variant. There was a significant increase in both numbers between December 2021 and February 2022, peaking in mid-January 2022, when the Omicron variant dominated. After May 2021, two distinct variant groups (Delta and Omicron) were observed, equally distributed among the five Santa Catarina mesoregions. Moreover, from November 2021 to February 2022, similar variant profiles between HCW and the general population were observed, and a quicker shift from Delta to Omicron in HCW than in the general population. This demonstrates the importance of HCW as a sentinel group for monitoring disease trends in the general population.

Evaluation of tropical water sources and mollusks in southern Brazil using microbiological, biochemical, and chemical parameters.

Florianópolis, a city located in the Santa Catarina State in southern Brazil, is the national leading producer of bivalve mollusks. The quality of bivalve mollusks is closely related to the sanitary conditions of surrounding waters where they are cultivated. Presently, cultivation areas receive large amounts of effluents derived mainly from treated and non-treated domestic, rural, and urban sewage. This contributes to the contamination of mollusks with trace metals, pesticides, other organic compounds, and human pathogens such as viruses, bacteria, and protozoan. The aim of this study was to perform a thorough diagnosis of the shellfish growing areas in Florianópolis, on the coast of Santa Catarina. The contamination levels of seawater, sediments, and oysters were evaluated for their microbiological, biochemical, and chemical parameters at five sea sites in Florianópolis, namely three regular oyster cultivation areas (Sites 1, 2, and oyster supplier), a polluted site (Site 3), and a heavily polluted site (Site 4). Samples were evaluated at day zero and after 14 days. Seawater and sediment samples were collected just once, at the end of the experiment. Antioxidant defenses, which may occur in contaminated environments in response to the increased production of reactive oxygen species (ROS) by organisms, were analyzed in oysters, as well as organic compounds (in oysters and sediment samples) and microbiological contamination (in oysters and seawater samples). The results showed the presence of the following contaminants: fecal coliforms in seawater samples (four sites), human adenovirus (all sites), human noroviruses GI and GII (two sites), Hepatitis A viruses (one site), JC Polyomavirus in an oyster sample from the oyster supplier, Giardia duodenalis cysts, and Cryptosporidium sp oocysts (one site). Among organochlorine pesticides, only DDT (dichlorodiphenyltrichloroethane) and HCH (hexachlorocyclohexane) were detected in some sediment and oysters samples in very low levels; site 4 had the highest concentrations of total aliphatic hydrocarbons, PAHs, and linear alkylbenzenes (LABs) found either in oysters or in sediment samples. The major concentration of fecal sterol coprostanol was found at site 4, followed by site 3. After 14 days of allocation in the four selected sites, there was a significant difference in the enzymes analyzed at the monitored spots. The detection of different contaminants in oysters, seawater, and sediment samples in the present study shows the impact untreated or inadequately treated effluents have on coastal areas. These results highlight the need for public investment in adequate wastewater treatment and adequate treatment of oysters, ensuring safe areas for shellfish production as well as healthier bivalve mollusks for consumption.

SARS-CoV-2 in Human Sewage and River Water from a Remote and Vulnerable Area as a Surveillance Tool in Brazil.

In the present study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was monitored in environmental samples from rural and vulnerable areas (a presidio, worker accommodation units, and river waters upstream and downstream of a rural community) from Minas Gerais State region, Southern Brazil, in August 2020. The sampling was performed prior to official declaration of the coronavirus disease (COVID-19) cases in those sites. SARS-CoV-2 RNA was detected in the presidio and workers accommodation units (3.0 × 104 virus genome copies (GC)/mL and 4.3 × 104 GC/mL of sewage, respectively). While SARS-CoV-2 was not detected in the river water upstream of the rural community, SARS-CoV-2 RNA was detected in downstream river waters (1.1 × 102 SARS-CoV-2 GC/mL). The results obtained in this study highlight the utility of SARS-CoV-2 monitoring in wastewater and human sewage as a non-invasive early warning tool to support health surveillance in vulnerable and remote areas, particularly in development countries.

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil.

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems.

The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil, November 2019.

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log10 SARS-CoV-2 genome copies (GC) L-1), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log10 SARS-CoV-2 GC L-1, ranging from 5.49 ± 0.02 log10 GC L-1 (27th November 2019) to 6.68 ± 0.02 log10 GC L-1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

Hepatitis E Virus in Manure and Its Removal by Psychrophilic anaerobic Biodigestion in Intensive Production Farms, Santa Catarina, Brazil, 2018-2019.

Hepatitis E virus (HEV) is an important enteric agent that can circulate in swine; it is excreted in manure, and of zoonotic interest. The present study investigated, by RT-qPCR, the circulation of HEV in swine manure from different types of pig farms (maternity, nursery, and grow-finish farms) in Santa Catarina State, the major pig production area of Brazil, and also evaluated the HEV removal efficiency of psychrophilic anaerobic biodigesters (PABs). While HEV was consistently detected in manure from grow-finish pig farms (>4 log HEV genome copies (GC) L-1), the virus was not detected in manure from maternity and nursery farms. These findings suggest a potential high biosafety status during primary-swine production, with a subsequent contamination in grow-finish production. The anaerobic biodigestion process reduced more than 2 log10 HEV GC in the processed swine manure. However, the virus concentration in final effluent remained high, with an average value of 3.85 log10 HEV GC L-1. Consequently, our results demonstrate that PABs can be a robust tool for effective inactivation of HEV, while reinforcing the need for sanitary surveillance and legislation of swine manure-derived biofertilizers, to avoid the spread of zoonotic enteric pathogens such as HEV.

Viral uptake and stability in Crassostrea gigas oysters during depuration, storage and steaming.

More stable than bacteria in environmental samples, enteric viruses are generally related to outbreaks of gastroenteritis caused by the consumption of contaminated oysters. This study evaluated: i) the dynamic processes of enteric viral models bioaccumulation by Crassostrea gigas oysters artificially contaminated; ii) the stability of these viruses in oysters in controlled temperature conditions and iii) the effect of UV light in inactivating these viruses in depurated oysters. Plaque assay (PA) was used to assess the infectivity of both viral models. Cell culture coupled with RT-qPCR (ICC-RT-qPCR) was used to measure infectious adenovirus type 2 (HAdV-2) genomes and qPCR to measure genome copies of murine norovirus (MNV-1). The virus uptake through bioaccumulation behave differently: HAdV-2 reached its peak of uptake faster than MNV-1. Both viruses showed high stability in oysters when maintained under 4 °C, but were completely inactivated in steamed oysters. The HAdV-2 was completely inactivated after 12 h of depuration with UV light and after 24 h without UV light. After 72 h of depuration, MNV-1 was still detected in both tanks, probably due to the stronger interaction of this virus with the oyster's tissues. This study demonstrated the importance of a secure depuration time in ensuring a clean and safe product, and that the steaming process is the safest way to prepare oysters for consumption.

Presence of enteric viruses, bioaccumulation and stability in Anomalocardia brasiliana clams (Gmelin, 1791).

Bivalve mollusks are filter feeders and may accumulate human pathogens in their tissues. Many studies demonstrated human diseases associated with bivalve consumption, especially oysters. Anomalocardia brasiliana clams are distributed along the Brazilian coastal area and are an exotic ingredient for some typical dishes in Brazil. Even though there are several reports describing the contamination of oysters and mussels with human pathogens, there is a lack of studies reporting contamination of A. brasiliana with human pathogens. An evaluation of natural microbiological contamination in A. brasiliana samples over a period of 18months (November 2014 to April 2016) showed that the bacteria indices were in accordance with Brazilian regulations (E. coli<230MPN and Salmonella sp. absent in 25g of meat). However, the enteric viruses evaluated were detected throughout the analysis period, with the highest result for the hepatitis A virus (HAV); followed by Rotavirus-A (RVA); Human Adenovirus (HAdV) and Norovirus GI (NoV GI). The bioaccumulation of enteric viruses by A. brasiliana during a period of 24h was performed using NoV GI and GII, HAV, RVA and HAdV as models. Interestingly the mollusk demonstrated different uptake behaviors in relation to these viruses throughout the time period. NoV GI was the most adsorbed virus after 24h. HAV concentration was <1% at 3h, but it increased to <10% at 8h, remaining unchanged until 12h, and decreasing to <3% at 24h; HAdV reached its highest concentration at 12h, being released by the animals and lowering to <3% at 24h. RVA bioaccumulation was unstable over time, reaching its highest values after 24h (<5%); NoV GII bioaccumulation remained <1%. Thermal inactivation of HAdV-2 in A. brasiliana was also evaluated. After the usual gentle cooking procedure using different times (0, 1, 1.5, 3 and 5mins), viral infectivity was evaluated using ICC-et-RT-qPCR. The temperature inside the DT remained <80°C over time and after 5min of cooking the HAdV reached a decay of 90% (1 log10). The results showed a real warn to the consumers that can be exposed to infectious human viruses if they eat these clams improperly cooked. HAV was the most detected virus in these animals, which may lead to outbreaks. A. brasiliana exhibited distinct behavior in NoV GI bioaccumulation and persistence, pointing to the need for further studies about the cellular ligands used by these viruses to become attached to these clams.

Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil.

Waterborne, food-borne and sewage-borne pathogens are a major global concern, with the annual recurrence, most notably during the summer, of outbreaks of gastroenteritis of unconfirmed etiology associated with recreational activities in marine environments. The consumption of contaminated water-based foodstuffs is also related to outbreaks of human illness. The main goals of the present study were: i) to identify the genetic assemblages of Giardia duodenalis cysts in growing and depurated oysters destined for human consumption on the southern coast of São Paulo, Brazil; ii) to verify the main circulating G. duodenalis assemblages and their subtypes in different brackish waters used for the production of mollusks and for recreational purposes; iii) to track the contamination of growing and depurated oysters by the human adenovirus and identify the infectivity of adenoviral particles recovered from oysters before and after depuration; iv) to evaluate the occurrence and genotype of the free-living amoebae of the genus Acanthamoeba in brackish water and oysters from all the sites described above. Four sampling sites in the Cananeia estuary were selected to search for pathogenic and amphizoic protozoa (Giardia and Acanthamoeba respectively): site 1: oyster growth, site 2: catchment water (before UV depuration procedure), site 3: filter backwash (filtration stage of water treatment) and site 4: oyster depuration tank. Oysters at sites 1 and 4 were evaluated for the presence of adenovirus (HAdV). Analysis consisted of conventional microbiological as well as molecular methods. Giardia duodenalis were detected in all the water sites analyzed and the molecular analysis revealed that sub-assemblage AII was the most frequently distributed throughout the estuarine environment, although one sample was identified as belonging to the assemblage C. Acanthamoeba were also isolated from different locations of the estuarine area, and were detected at all the analyzed sites. The majority of isolates belonged to the T3 genotype, while the T4 genotype was identified once. The sequencing reaction of Giardia duodenalis revealed the contamination of three batches of depurated oysters by the sub-assemblage AII. With respect to viruses, seven batches of oysters (four growing and three depurated) were found to be harboring infectious HAdV particles when submitted to plaque assay. Overall, the results of the sequencing reactions combined with the plaque assay revealed that the isolates of Giardia duodenalis and the infectious HAdV particles identified in oyster tissues have the potential to infect humans and pose a threat if consumed raw or lightly cooked. This is the first report on the sub-assemblage AII identified in oysters which are submitted to a cleaning and disinfection procedure prior to human consumption in Brazil. Acanthamoeba specific genotypes were also identified for the first time in a recreational estuarine area in Brazil, contributing to knowledge of their molecular and environmental epidemiology, which is considered scarce even in marine and estuarine areas of the world.

Retention of Rotavirus Infectivity in Mussels Heated by Using the French Recipe Moules Marinières.

To evaluate the persistence of infectious virus after heating, mussels contaminated with a rotavirus strain were prepared following the French recipe moules marinières (mariner's mussels). Rotavirus was then quantified by real-time quantitative PCR (RT-qPCR) and a cell culture infectivity assay. Results showed the persistence of infectious virus after 3 min of cooking. After 5 min, when no infectious virus could be detected, the RT-qPCR approach showed a 1-log decrease compared with concentrations detected after 1 min of cooking.

Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces.

In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.

Virus, protozoa and organic compounds decay in depurated oysters.

AIMS: (1) Evaluate the dynamic of the depuration process of Crassostrea gigas oysters using different ultraviolet doses with different amounts of contaminants (virus, protozoa and organic contaminants) and (2) investigate the morphological changes in the oysters' tissues produced by the depuration procedures. METHODS: The oysters were allocated in sites with different degrees of contamination and analyzed after 14 days. Some animals were used as positive controls by artificial bioaccumulation with HAdV2 and MNV1 and subjected to depuration assays using UV lamps (18 or 36 W) for 168 h. The following pollutants were researched in the naturally contaminated oysters, oysters after 14 days in sites and oysters during the depuration processes: virus (HAdV, HAV, HuNoV GI/GII and JCPyV), by (RT) qPCR; protozoa (Cryptosporidium and Giardia species), by immunomagnetic separation and immunofluorescence; and organic compounds (AHs, PAHs, LABs, PCBs and organochlorine pesticides-OCs), by chromatography. Changes in the oysters' tissues produced by the depuration processes were also evaluated using histochemical analysis by light microscopy. In the artificially bioaccumulated oysters, only HAdV2 and MNV1 were investigated by (RT) qPCR before the depuration procedures and after 96 and 168 h of these procedures. RESULTS: At 14 days post-allocation, HAdV was found in all the sites (6.2 × 105 to 4.4 × 107 GC g(-1)), and Giardia species in only one site. Levels of PCBs and OCs in the oyster's tissues were below the detection limit for all samples. AHs (3.5 to 4.4 µg g(-1)), PAHs (11 to 191 ng g(-1)) and LABs (57 to 751 ng g(-1)) were detected in the samples from 3 sites. During the depuration assays, we found HAdV, Giardia and Cryptosporidium species until 168 h, independent of UV treatment. AHs, PAHs and LABs were found also after 168 h of depuration (36 W and without UV lamp). The depuration procedures did not produce changes in the oysters' tissues. In the artificially contaminated and depurated oysters, we detected HAdV until 168 h and MNV1 until 96 h of depuration. CONCLUSION: The applied depuration treatments were unable to eliminate the protozoa or to degrade the HAdV genomes but were able to degrade the MNV1 genomes. Similarly, the UV water treatment was not efficient for aliphatic hydrocarbons, PAHs and LABs, as their concentrations were equivalent or higher to the concentrations of the control samples and samples from depuration tanks without UV treatment.

Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces

In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5 percent by IMS-IFA and 15.7 percent by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.