Detection of SARS-CoV-2 in wastewater is influenced by sampling time, concentration method, and target analyzed

The detection of SARS-CoV in wastewater has been proposed as a tool for monitoring COVID-19 at the community level. Although many reports have been published about detecting viral RNA in wastewater and its presence has been linked to infected people, appropriate analytical methodologies to use this approach have not yet been established. In this study, we compared ultrafiltration, polyethylene glycol precipitation, flocculation using AlCl3, and flocculation with skim milk for the recovery of SARS-CoV-2, using RNA from patients with positive diagnoses for COVID-19 and Pseudomonas phage φ6 as the control. We also evaluated the primers for detecting the E, RdRp, and N genes of the virus, as well as different storage times. Differences in the recovery efficiencies were evident with the different concentration methods, the best being ultrafiltration and precipitation with aluminum, which had recovery rates of 42.0% and 30.0%, respectively, when virus was present at high levels. Significant differences were found between the recoveries using wastewater and deionized water and between different storage times, with better recoveries for 6 and 12 h samplings. The E gene was the only one detected in all the samples analyzed. The results show that although this approach can provide important data for studying the pandemic, clear protocols are necessary for investigations to be comparable. HIGHTLIGHTS RNA of SARS-CoV-2 was used to evaluate the recovery of different methods in wastewater. The utility of phage φ6 of Pseudomonas as a control for evaluation of recovery methods of SARS-CoV-2 from wastewater was demonstrated. Differences due to the target selected for the detection of SARS was demonstrated. The results show that it is possible to detect SARS RNA in wastewater, however its epidemiological usefulness will depend on the correct validation of the technique used for detection.

Development of Moore Swab and Ultrafiltration Concentration and Detection Methods for Salmonella Typhi and Salmonella Paratyphi A in Wastewater and Application in Kolkata, India and Dhaka, Bangladesh

Enteric fever is a severe systemic infection caused by Salmonella enterica serovar Typhi (ST) and Salmonella enterica serovar Paratyphi A (SPA). Detection of ST and SPA in wastewater can be used as a surveillance strategy to determine burden of infection and identify priority areas for water, sanitation, and hygiene interventions and vaccination campaigns. However, sensitive and specific detection of ST and SPA in environmental samples has been challenging. In this study, we developed and validated two methods for concentrating and detecting ST/SPA from wastewater: the Moore swab trap method for qualitative results, and ultrafiltration (UF) for sensitive quantitative detection, coupled with qPCR. We then applied these methods for ST and SPA wastewater surveillance in Kolkata, India and Dhaka, Bangladesh, two enteric fever endemic areas. The qPCR assays had a limit of detection of 17 equivalent genome copies (EGC) for ST and 25 EGC for SPA with good reproducibility. In seeded trials, the Moore swab method had a limit of detection of approximately 0.05–0.005 cfu/mL for both ST and SPA. In 53 Moore swab samples collected from three Kolkata pumping stations between September 2019 and March 2020, ST was detected in 69.8% and SPA was detected in 20.8%. Analysis of sewage samples seeded with known amount of ST and SPA and concentrated via the UF method, followed by polyethylene glycol precipitation and qPCR detection demonstrated that UF can effectively recover approximately 8, 5, and 3 log10 cfu of seeded ST and SPA in 5, 10, and 20 L of wastewater. Using the UF method in Dhaka, ST was detected in 26.7% (8/30) of 20 L drain samples with a range of 0.11–2.10 log10 EGC per 100 mL and 100% (4/4) of 20 L canal samples with a range of 1.02–2.02 log10 EGC per 100 mL. These results indicate that the Moore swab and UF methods provide sensitive presence/absence and quantitative detection of ST/SPA in wastewater samples.

Falsely diagnosed thyrotoxicosis caused by anti-streptavidin antibodies and pre-wash procedures

Background Anti-streptavidin antibodies are causal determinants of analytical interference during Thyroid function tests, and numerous reports have detailed such interference, with anti-streptavidin antibodies attracting attention. Case presentation We conducted a straightforward investigation of interference due to anti-streptavidin antibodies, with a case of a 60-year-old Japanese man who consulted our department for inconsistencies between his clinical course and Thyroid function tests. Experiments were conducted using Cobas8000 e602, which employs assay procedures with pre-wash to evaluate FT4 and FT3 levels. Conclusions To our knowledge, this is the first published report to clearly investigate such interferences using a combination of polyethylene glycol precipitation, heterophilic blocking tube precipitation, streptavidin-coated magnetic particle precipitation, and different instruments with or without pre-wash. Clinicians should consider that interferences caused by anti-streptavidin antibodies could lead to a misdiagnosis of thyrotoxicosis. Moreover, discussions between laboratory specialists, clinicians, and manufacturers are required to identify interferences and avoid unnecessary examinations and inappropriate treatment.

Production of IgY polyclonal antibody against diphtheria toxin and evaluation of its neutralization effect by Vero cell assay

Background Diphtheria is a bacterial disease which is caused by Corynebacterium diphtheriae. The symptoms are due to the diphtheria toxin produced by the bacteria. Antibiotic therapy and the use of diphtheria antitoxin is a recommended strategy to control diphtheria. Although mammalian antibodies are used to treat patients, IgY antibody has advantages over mammalian ones, including cost-effectiveness and production through non-invasive means. Moreover, in contrast to mammalian antibodies, IgY does not bind to the rheumatoid factor and does not activate the complement system. The objective of this study was to evaluate the in vitro neutralizing effect of IgY against diphtheria toxin. Results Anti-DT IgY was produced by immunization of the laying white leghorn chickens. Indirect enzyme-linked immunosorbent assay revealed successful immunization of the animals, and the IgY was purified with a purity of 93% via polyethylene glycol precipitation method. The neutralizing activity of the purified IgY was evaluated by Vero cell viability assay. This assay confirmed that 1.95 μg (8.6 μg/ml of culture medium) of anti-DT IgY would neutralize 10 fold of cytotoxic dose 99% of DT, which was 0.3 ng (1.33 ng/ml of culture medium). Conclusion This anti-DT IgY may be applicable for diphtheria treatment and quality controls in vaccine production.

Interference Due to Heterophilic Antibody, Biotin and Thyroid Hormone Autoantibody

Purpose: Immunoassay is susceptible to interference by other substances in the serum. The main substances interfering with thyroid function testing include heterophilic antibody, biotin, thyroid hormone autoantibody, and Macro-TSH. We reported a patient with fraudulently elevated FT4 and TSH and described various common experimental methods used to explore the existence of substances interfering with thyroid function testing.Methods and Results: FT4 and TSH were significantly lower when measured on the Architect platform (FT4: 7.09 pmol/L, TSH: 58.94 µIU/ml). Polyethylene glycol precipitation showed lower FT4 and TSH, suggesting the presence of a high molecular weight interfering substance. Heterophile blocking tube study showed heterophilic antibody interfered with TSH detection. 125I-hTSH binding study and radioimmunoprecipitation assay indicated that the patient didn’t contain anti-TSH autoantibody. The new generation of Elecsys immunoassay kit indicated that biotin interfered with TSH detection. Radioimmunoprecipitation assay showed that all four kinds of thyroid hormone autoantibodies were positive. After reviewing 24 literatures, we provided the diagnostic strategy for investigation of interferences with thyroid function immunoassays.Conclusion: We reported a case with falsely elevated TSH due to the combined action of heterophilic antibody and biotin and fraudulently elevated FT4 caused by thyroid hormone autoantibody. When there is a discrepancy between thyroid function and clinical manifestation, the presence of immunoassay interference with one or more indicators needs to be considered.

Development of Moore Swab and Ultrafiltration Concentration and Detection Methods for Salmonella Typhi and Salmonella Paratyphi A in Wastewater and Application in Kolkata, India and Dhaka, Bangladesh

Enteric fever is a severe systemic infection caused by Salmonella enterica serovar Typhi (ST) and Salmonella enterica serovar Paratyphi A (SPA). Detection of ST and SPA in wastewater can be used as a surveillance strategy to determine burden of infection and identify priority areas for water, sanitation, and hygiene interventions and vaccination campaigns. However, sensitive and specific detection of ST and SPA in environmental samples has been challenging. In this study, we developed and validated two methods for concentrating and detecting ST/SPA from wastewater: the Moore swab trap method for qualitative results, and ultrafiltration (UF) for sensitive quantitative detection, coupled with qPCR. We then applied these methods for ST and SPA wastewater surveillance in Kolkata, India and Dhaka, Bangladesh, two enteric fever endemic areas. The qPCR assays had a limit of detection of 17 equivalent genome copies (EGC) for ST and 25 EGC for SPA with good reproducibility. In seeded trials, the Moore swab method had a limit of detection of approximately 0.05-0.005 cfu/mL for both ST and SPA. In 53 Moore swab samples collected from three Kolkata pumping stations between September 2019 to March 2020, ST was detected in 69.8% and SPA was detected in 20.8%. Analysis of sewage samples seeded with known amount of ST and SPA and concentrated via the UF method, followed by polyethylene glycol precipitation and qPCR detection demonstrated that UF can effectively recover approximately 8 log10 cfu, 5 log10 cfu, and 3 log10 cfu of seeded ST and SPA in 5 L, 10 L, and 20 L of wastewater. Using the UF method in Dhaka, ST was detected in 26.7% (8/30) of 20 L drain samples with a range of 0.11-2.10 log10 EGC per 100 mL and 100% (4/4) of 20 L canal samples with a range of 1.02 - 2.02 log10 EGC per 100 mL. These results indicate that the Moore swab and UF methods provide sensitive presence/absence and quantitative detection of ST/SPA in wastewater samples, and these two methods can be used jointly or separately for Salmonella Typhi environmental surveillance.

Evaluation of Two Methods to Concentrate SARS-CoV-2 from Untreated Wastewater

Use of wastewater-based epidemiology as a tool to record and manage the course of SARS-CoV-2 infections in human populations requires information about the efficiency of methods to concentrate the virus from wastewater. In the present study, we spiked untreated wastewater with quantified SARS-CoV-2 positive clinical material and enriched the virus by polyethylene glycol precipitation and ultrafiltration with Vivaspin 10 kDa MWCO columns. SARS-CoV-2 was detected and quantified by reverse transcription quantitative PCR (E- and S-gene) and droplet digital PCR. The concentration of virus with precipitation resulted in mean recoveries between 59.4% and 63.7% whereas rates from 33.0% to 42.6% after ultrafiltration of samples were demonstrated. The results suggest that the use of both methods allows an effective and practicable enrichment of SARS-CoV-2 from raw wastewater.