Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. METHODS: In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. RESULTS: The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen's Kappa = 0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. CONCLUSIONS: RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.

Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. METHODS: The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). RESULTS: The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. CONCLUSION: During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients' viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

Medico legal aspects of self-injection of metallic mercury in cases of suicide or self-harming.

Metallic mercury may be self-injected for suicidal or self-harm purposes or sometimes for superstitious or other inadvisable reasons. Local tissue or systemic consequences such as mercurialism can frequently occur in cases of subcutaneous or deep injection, while death due to pulmonary embolism and cardiac, brain, hepatic or renal toxicity may occur in cases of high dosage intravenous administration. The aim of this review is to focus on the diagnostic difficulties facing coroners and forensic pathologists when the courts require confirmation that evidence of self-injection of metallic mercury is the result of suicide or self-harming. Forensic examination performed on the corpses of victims who died in or out of hospital or on surviving injured or intoxicated victims showing signs of mercurialism, demands the careful evaluation of the death scene, of all related circumstances and of the clinical and autopsy data. Close interaction between forensic pathologists and toxicologists is also needed to identify and quantify mercury levels in blood, urine and tissue.

A survey of Clostridium spiroforme antimicrobial susceptibility in rabbit breeding.

Rabbit meat breeding may be heavily affected by enterotoxaemia due to Clostridium spiroforme. Data on its antimicrobial susceptibility are insufficient, presumably because of difficulties in cultivating and identifying the pathogen. Our aim is therefore to provide this information to veterinary practitioners by focusing on a panel of therapeutics used in intensive rabbit units. Lincomycin was also checked in order to investigate the origin of resistance to macrolides. Minimal inhibitory concentrations (MICs) were determined with the agar dilution method according to the CLSI M11-A7 protocol (2007). MIC(50) and MIC(90) were, respectively, 64 and 64microg/ml for tiamulin, 32 and 32microg/ml for norfloxacin, 0.063 and 0.125microg/ml for amoxicillin, and 8 and 16microg/ml for doxycycline. MIC(50) and MIC(90) were 256microg/ml for sulphadimethoxine, spiramycin and lincomycin. Our results have shown that intrinsic or acquired antimicrobial resistances are diffuse in the C. spiroforme population and suggest focusing on prevention rather than on treatment of clostridial overgrowth, by reducing risk factors and using antimicrobials prudently.