First application of a droplet digital PCR to detect Toxoplasma gondii in mussels.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/µL, 800 gc/µL, 80 gc/µL, 8 gc/µL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/µL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets-RdRp, ORF1b, and E-were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December-January and March-April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2.

Sponge Whirl-Pak Sampling Method and Droplet Digital RT-PCR Assay for Monitoring of SARS-CoV-2 on Surfaces in Public and Working Environments.

The SARS-CoV-2 can spread directly via saliva, respiratory aerosols and droplets, and indirectly by contact through contaminated objects and/or surfaces and by air. In the context of COVID-19 fomites can be an important vehicle of virus transmission and contribute to infection risk in public environments. The aim of the study was to analyze through surface sampling (sponge method) the presence of SARS-CoV-2 in public and working environments, in order to evaluate the risk for virus transmission. Seventy-seven environmental samples were taken using sterile sponges in 17 animal farms, 4 public transport buses, 1 supermarket and 1 hotel receptive structure. Furthermore, 246 and 93 swab samples were taken in the farms from animals and from workers, respectively. SARS-CoV-2 detection was conducted by real-time RT-PCR and by digital droplet RT-PCR (dd RT-PCR) using RdRp, gene E and gene N as targets. None of the human and animal swab samples were positive for SARS-CoV-2, while detection was achieved in 20 of the 77 sponge samples (26%) using dd RT-PCR. Traces of the RdRp gene, gene E and gene N were found in 17/77 samples (22%, average concentration 31.2 g.c./cm2, range 5.6 to 132 g.c./cm2), 8/77 samples (10%, average concentration 15.1 g.c./cm2, range 6 to 36 g.c./cm2), and in 1/77 (1%, concentration 7.2 g.c./cm2). Higher detection rates were associated with sampling in animal farms and on public transport buses (32% and 30%) compared to the supermarket (21%) and the hotel (no detection). The result of the study suggests that the risk of contamination of surfaces with SARS-CoV-2 increases in environments in which sanitation strategies are not suitable and/or in highly frequented locations, such as public transportation. Considering the analytical methods, the dd RT-PCR was the only approach achieving detection of SARS-CoV-2 traces in environmental samples. Thus, dd RT-PCR emerges as a reliable tool for sensitive SARS-CoV-2 detection.

Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite Toxoplasma gondii. Infection in humans has usually been related to the consumption of raw, undercooked or cured meat. The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR)-based assay for the detection and quantification of T. gondii in meat samples. To optimize the ddPCR, T.gondii reference DNA aliquots at five known concentrations: 8000 cg/µl, 800 cg/µl, 80 cg/µl, 8 cg/µl were used. Moreover, results obtained by ddPCR and quantitative PCR (qPCR) were compared using 80 known samples (40 positive and 40 negative), as well as 171 unknown diaphragm tissue samples collected at slaughterhouses. The ddPCR showed a sensitivity of 97.5% and a specificity of 100%, with a detection limit of 8 genomic copy/µl of T. gondii. A nearly perfect agreement (κ = 0.85) was found between results obtained by ddPCR and qPCR for both positive and negative known samples analysed. On the 171 diaphragm tissue samples from field, 7.6% resulted positive by ddPCR and only 1.2% by qPCR. Therefore, this innovative method could be very useful for the detection of T. gondii in meat samples, aiming to prevent human infections.

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR).

Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14).

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.

PCR detection of Neospora caninum in water buffalo foetal tissues.

The seroprevalence of Neospora caninum was surveyed by an ELISA kit on two water buffalo herds of Southern Italy. Seropositive samples were detected in 47% and 59% of individuals, respectively, thus indicating high level of exposure to the parasite even if the possibility of vertical transmission cannot be excluded. Tissue samples collected from three aborted fetuses from the same herds were investigated for N. caninum presence by PCR assays targeting the 18S and the Nc5 DNA sequences, respectively. Both methods have shown the presence of N. caninum DNA in heart and brain. Sequencing of the Nc5 genomic DNA confirmed the presence of N. caninum in the samples; phylogenetic analysis of the obtained sequences showed high homology among the Neospora recovered from different samples. The present study suggests an important role of N. caninum as a possible abortive agent for water buffaloes.