Nanomaterials and Biosensing Technology for the SARS-CoV-2 Detection

The outbreak of corona virus disease 2019 (COVID-19) has aroused great attention around the world. SARS-CoV-2 possesses characteristics of faster transmission, immune escape, and occult transmission by many mutation, which caused still grim situation of prevention and control. Early detection and isolation of patients are still the most effective measures at present. So, there is an urgent need for new rapid and highly sensitive testing tools to quickly identify infected patients as soon as possible. This review briefly introduces general characteristics of SARS-CoV-2, and provides recentl overview and analysis based on different detection methods for nucleic acids, antibodies, antigens as detection target. Novel nano-biosensors for SARS-CoV-2 detection are analyzed based on optics, electricity, magnetism, and visualization. In view of the advantages of nanotechnology in improving detection sensitivity, specificity and accuracy, the research progress of new nano-biosensors is introduced in detail, including SERS-based biosensors, electrochemical biosensors, magnetic nano-biosensors and colorimetric biosensors. Functions and challenges of nano-materials in construction of new nano-biosensors are discussed, which provides ideas for the development of various coronavirus biosensing technologies for nanomaterial researchers.

Characterizing SARS-CoV-2 transcription of subgenomic and genomic RNAs during early human infection using multiplexed droplet digital PCR.

BACKGROUND: Control of SARS-CoV-2 (SCV-2) transmission requires understanding SCV-2 replication dynamics. METHODS: We developed a multiplexed droplet digital PCR (ddPCR) assay to quantify SCV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from genomic RNAs (gRNAs). We applied the assay to specimens from 144 people with single nasopharyngeal samples and 27 people with >1 sample. Results were compared to qPCR and viral culture. RESULTS: sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio of sgRNA:gRNA was stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Daily testing of 6 persons revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. Further, sgRNA:gRNA is constant during infection despite changes in viral culture. CONCLUSIONS: Ct values from qPCR correlate with active viral replication. More work is needed to understand why some cultures are negative despite presence of sgRNA.

Comparison of the methods for isolation and detection of SARS-CoV-2 RNA in municipal wastewater.

Introduction: Coronavirus SARS-CoV-2 is a causative agent responsible for the current global pandemic situation known as COVID-19. Clinical manifestations of COVID-19 include a wide range of symptoms from mild (i.e., cough, fever, dyspnea) to severe pneumonia-like respiratory symptoms. SARS-CoV-2 has been demonstrated to be detectable in the stool of COVID-19 patients. Waste-based epidemiology (WBE) has been shown as a promising approach for early detection and monitoring of SARS-CoV-2 in the local population performed via collection, isolation, and detection of viral pathogens from environmental sources. Methods: In order to select the optimal protocol for monitoring the COVID-19 epidemiological situation in region Turiec, Slovakia, we (1) compared methods for SARS-CoV-2 separation and isolation, including virus precipitation by polyethylene glycol (PEG), virus purification via ultrafiltration (Vivaspin®) and subsequent isolation by NucleoSpin RNA Virus kit (Macherey-Nagel), and direct isolation from wastewater (Zymo Environ Water RNA Kit); (2) evaluated the impact of water freezing on SARS- CoV-2 separation, isolation, and detection; (3) evaluated the role of wastewater filtration on virus stability; and (4) determined appropriate methods including reverse transcription-droplet digital PCR (RT-ddPCR) and real-time quantitative polymerase chain reaction (RT-qPCR) (targeting the same genes, i.e., RdRp and gene E) for quantitative detection of SARS-CoV-2 in wastewater samples. Results: (1) Usage of Zymo Environ Water RNA Kit provided superior quality of isolated RNA in comparison with both ultracentrifugation and PEG precipitation. (2) Freezing of wastewater samples significantly reduces the RNA yield. (3) Filtering is counterproductive when Zymo Environ Water RNA Kit is used. (4) According to the specificity and sensitivity, the RT-ddPCR outperforms RT-qPCR. Discussion: The results of our study suggest that WBE is a valuable early warning alert and represents a non-invasive approach to monitor viral pathogens, thus protects public health on a regional and national level. In addition, we have shown that the sensitivity of testing the samples with a nearer detection limit can be improved by selecting the appropriate combination of enrichment, isolation, and detection methods.

Nanomaterials and Biosensing Technology for the SARS-CoV-2 Detection

The outbreak of corona virus disease 2019 (COVID-19) has aroused great attention around the world. SARS-CoV-2 possesses characteristics of faster transmission, immune escape, and occult transmission by many mutation, which caused still grim situation of prevention and control. Early detection and isolation of patients are still the most effective measures at present. So, there is an urgent need for new rapid and highly sensitive testing tools to quickly identify infected patients as soon as possible. This review briefly introduces general characteristics of SARS-CoV-2, and provides recentl overview and analysis based on different detection methods for nucleic acids, antibodies, antigens as detection target. Novel nano-biosensors for SARS-CoV-2 detection are analyzed based on optics, electricity, magnetism, and visualization. In view of the advantages of nanotechnology in improving detection sensitivity, specificity and accuracy, the research progress of new nano-biosensors is introduced in detail, including SERS-based biosensors, electrochemical biosensors, magnetic nano-biosensors and colorimetric biosensors. Functions and challenges of nano-materials in construction of new nano-biosensors are discussed, which provides ideas for the development of various coronavirus biosensing technologies for nanomaterial researchers.

Association between SARS-CoV-2 Viral Load and Patient Symptoms and Clinical Outcomes Using Droplet Digital PCR.

The association between nasopharyngeal (NP) SARS-CoV-2 viral loads and clinical outcomes remains debated. Here, we examined the factors that might predict the NP viral load and the role of the viral load as a predictor of clinical outcomes. A convenience sample of 955 positive remnant NP swab eluent samples collected during routine care between 18 November 2020 and 26 September 2021 was cataloged and a chart review was performed. For non-duplicate samples with available demographic and clinical data (i.e., non-employees), an aliquot of eluent was sent for a droplet digital PCR quantification of the SARS-CoV-2 viral load. Univariate and multivariate analyses were performed to identify the clinical predictors of NP viral loads and the predictors of COVID-19-related clinical outcomes. Samples and data from 698 individuals were included in the final analysis. The sample cohort had a mean age of 50 years (range: 19-91); 86.6% were male and 76.3% were unvaccinated. The NP viral load was higher in people with respiratory symptoms (p = 0.0004) and fevers (p = 0.0006). In the predictive models for the clinical outcomes, the NP viral load approached a significance as a predictor for in-hospital mortality. In conclusion, the NP viral load did not appear to be a strong predictor of moderate-to-severe disease in the pre-Delta and Delta phases of the pandemic, but was predictive of symptomatic diseases and approached a significance for in-hospital mortality, providing support to the thesis that early viral control prevents the progression of disease.

SARS-CoV-2 concentration in wastewater consistently predicts trends in COVID-19 case counts by at least two days across multiple WWTP scales.

Wastewater surveillance of SARS-CoV-2 has proven instrumental in mitigating the spread of COVID-19 by providing an economical and equitable approach to disease surveillance. Here, we analyze the correlation of SARS-CoV-2 RNA in influents of seven wastewater plants (WWTPs) across the state of South Carolina with corresponding daily case counts to determine whether underlying characteristics of WWTPs and sewershed populations predict stronger correlations. The populations served by these WWTPs have varying social vulnerability and represent 24% of the South Carolina population. The study spanned 15 months from April 19, 2020, to July 1, 2021, which includes the administration of the first COVID-19 vaccines. SARS-CoV-2 RNA concentrations were measured by either reverse transcription quantitative PCR (RT-qPCR) or droplet digital PCR (RT-ddPCR). Although populations served and average flow rate varied across WWTPs, the strongest correlation was identified for six of the seven WWTPs when daily case counts were lagged two days after the measured SARS-CoV-2 RNA concentration in wastewater. The weakest correlation was found for WWTP 6, which had the lowest ratio of population served to average flow rate, indicating that the SARS-CoV-2 signal was too dilute for a robust correlation. Smoothing daily case counts by a 7-day moving average improved correlation strength between case counts and SARS-CoV-2 RNA concentration in wastewater while dampening the effect of lag-time optimization. Correlation strength between cases and SARS-CoV-2 RNA was compared for cases determined at the ZIP-code and sewershed levels. The strength of correlations using ZIP-code-level versus sewershed-level cases were not statistically different across WWTPs. Results indicate that wastewater surveillance, even without normalization to fecal indicators, is a strong predictor of clinical cases by at least two days, especially when SARS-CoV-2 RNA is measured using RT-ddPCR. Furthermore, the ratio of population served to flow rate may be a useful metric to assess whether a WWTP is suitable for a surveillance program.

Quantitative Assay of SARS-CoV-2 RNA and Level of Proinflammatory Protein Gene Transcripts in Peripheral Blood Leukocytes after a Novel Coronavirus Infection.

The possibility of finding persistent SARS-CoV-2 viral particles in human peripheral blood leukocytes after a novel coronavirus infection was shown. The results of droplet digital PCR showed that 19 of 24 examined subjects had from 4 to 555 copies of the Nsp4 SARS-CoV-2 gene in 5-6 months after infection. The presence of this transcript in peripheral blood leukocytes was associated with reduced expression of FOXP3 gene and increased level of RORγ gene mRNA. The copy number of the Nsp4 gene negatively correlated with the level of FOXP3 gene mRNA (r=-0.45; p=0.028), but showed a positive correlation with the DANCR long non-coding RNA (r=0.94; p<0.001). In SARS-CoV-2-positive healthy individuals, the level of TLR2, NLRP3, and IL1B gene transcripts was higher than in SARS-CoV-2-negative donors. The presence of SARS-CoV-2 in a persistent form is probably associated with impaired immunosuppression and the development of chronic inflammation in apparently healthy volunteers after a new coronavirus infection.

Efficiency and sensitivity optimization of a protocol to quantify indoor airborne SARS-CoV-2 levels.

BACKGROUND: Development of methodologies to quantify airborne micro-organisms is needed for the prevention and control of infections. It is difficult to conclude which is the most efficient and sensitive strategy to assess airborne SARS-CoV-2 RNA levels due to the disparity of results reported in clinical settings. AIM: To improve our previously reported protocol of measuring SARS-CoV-2 RNA levels, which was based on bioaerosol collection with a liquid impinger and RNA quantification with droplet digital polymerase chain reaction (ddPCR). METHODS: Air samples were collected in COVID-19 patient rooms to assess efficiency and/or sensitivity of different air samplers, liquid collection media, and reverse transcriptases (RT). FINDINGS: Mineral oil retains airborne RNA better than does hydrophilic media without impairing integrity. SARS-CoV-2 ORF1ab target was detected in 80% of the air samples using BioSampler with mineral oil. No significant differences in effectiveness were obtained with MD8 sampler equipped with gelatine membrane filters, but the SARS-CoV-2 copies/m3 air obtained with the latter were lower (28.4 ± 6.1 vs 9 ± 1.7). SuperScript II RT allows the detection of a single SARS-CoV-2 genome RNA molecule by ddPCR with high efficiency. This was the only RT that allowed the detection of SARS-CoV-2 N1 target in air samples. CONCLUSION: The collection efficiency and detection sensivity of a protocol to quantify SARS-CoV-2 RNA levels in indoor air has been improved in the present study. Such optimization is important to improve our understanding of the microbiological safety of indoor air.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

Fate of SARS-CoV-2 coronavirus in wastewater treatment sludge during storage and thermophilic anaerobic digestion.

Since the COVID-19 outbreak has started in late 2019, SARS-CoV-2 has been widely detected in human stools and in urban wastewater. No infectious SARS-CoV-2 particles have been detected in raw wastewater until now, but it has been reported occasionally in human stools. This has raised questions on the fate of SARS-CoV-2 during wastewater treatment and notably in its end-product, wastewater treatment sludge, which is classically valorized by land spreading for agricultural amendment. In the present work, we focused on SARS-CoV-2 stability in wastewater treatment sludge, either during storage (4 °C, room temperature) or thermophilic anaerobic digestion (50 °C). Anaerobic digestion is one of the possible processes for sludge valorization. Experiments were conducted in laboratory pilots; SARS-CoV-2 detection was based on RT-quantitative PCR or RT-digital droplet PCR. In addition to SARS-CoV-2, Bovine Coronavirus (BCoV) particles were used as surrogate virus. The RNA from SARS-CoV-2 particles, inactivated or not, was close to the detection limit but stable in wastewater treatment sludge, over the whole duration of the assays at 4 °C (55 days) and at ambient temperature (∼20 °C, 25 days). By contrast, the RNA levels of BCoV and inactivated SARS-CoV-2 particles decreased rapidly during the thermophilic anaerobic digestion of wastewater treatment sludge lasting for 5 days, with final levels that were close to the detection limit. Although the particles' infectivity was not assessed, these results suggest that thermophilic anaerobic digestion is a suitable process for sludge sanitation, consistent with previous knowledge on other coronaviruses.

SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR).

BACKGROUND: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. MATERIALS AND METHODS: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. RESULTS: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. CONCLUSION: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

Assessment of Concentration, Recovery, and Normalization of SARS-CoV-2 RNA from Two Wastewater Treatment Plants in Texas and Correlation with COVID-19 Cases in the Community

The purpose of this study was to conduct a correlative assessment of SARS-CoV-2 RNA concentrations in wastewater with COVID-19 cases and a systematic evaluation of the effect of using different virus concentration methods and recovery and normalization approaches. We measured SARS-CoV-2 RNA concentrations at two different wastewater treatment plants (WWTPs) in the Bexar County of Texas from October 2020 to May 2021 (32 weeks) using reverse transcription droplet digital PCR (RT-ddPCR). We evaluated three different adsorption-extraction (AE) based virus concentration methods (acidification, addition of MgCl2, or without any pretreatment) using bovine coronavirus (BCoV) as surrogate virus and observed that the direct AE method showed the highest mean recovery. COVID-19 cases were correlated significantly with SARS-CoV-2 N1 concentrations in Salitrillo (rho = 0.75, p < 0.001) and Martinez II (rho = 0.68, p < 0.001) WWTPs, but normalizing to a spiked recovery control (BCoV) or a fecal marker (HF183) reduced correlations for both treatment plants. The results generated in this 32-week monitoring study will enable researchers to prioritize the virus recovery method and subsequent correlation studies for wastewater surveillance.

A reverse-transcription droplet digital PCR assay to detect and quantify SARS-CoV-2 RNA in upper respiratory tract specimens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Symptoms are variable and range from asymptomatic or mild to severe (i.e., pneumonia) in both healthy and immunocompromised patients. We developed a reverse-transcription droplet digital PCR (RT-ddPCR) assay for quantification of SARS-CoV-2 RNA in clinical nasopharyngeal and oropharyngeal swab specimens and evaluated the assay, including reproducibility, agreement of results, analytical measurement range, linearity, analytical sensitivity, and analytical specificity. This quantitative assay had a LoD of 218 copies/mL of viral transport media, with a linear quantification range from 500 to 5,000,000 copies/mL (R2 of 0.9817 and 0.9853 for N1 and N2 targets, respectively). Qualitative agreement of categorical results was 90.5% (57/63) between the reference and RT-ddPCR assays. Quantitative agreement between the two assays showed correlation, with R2 of 0.9726 and 0.9713 for N1 and N2 targets, respectively. No cross-reactivity with common coronavirus strains was detected. This SARS-CoV-2 quantitative RT-ddPCR assay may be a useful tool for a variety of applications including identification of patients with low viral load and serial monitoring of viral load in respiratory tracts specimens of patients for evaluation of the efficacy of therapy for COVID-19.

Application of digital PCR for public health-related water quality monitoring.

Digital polymerase chain reaction (dPCR) is emerging as a reliable platform for quantifying microorganisms in the field of water microbiology. This paper reviews the fundamental principles of dPCR and its application for health-related water microbiology. The relevant literature indicates increasing adoption of dPCR for measuring fecal indicator bacteria, microbial source tracking marker genes, and pathogens in various aquatic environments. The adoption of dPCR has accelerated recently due to increasing use for wastewater surveillance of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) - the virus that causes Coronavirus Disease 2019 (COVID-19). The collective experience in the scientific literature indicates that well-optimized dPCR assays can quantify genetic material from microorganisms without the need for a calibration curve and often with superior analytical performance (i.e., greater sensitivity, precision, and reproducibility) than quantitative polymerase chain reaction (qPCR). Nonetheless, dPCR should not be viewed as a panacea for the fundamental uncertainties and limitations associated with measuring microorganisms in water microbiology. With dPCR platforms, the sample analysis cost and processing time are typically greater than qPCR. However, if improved analytical performance (i.e., sensitivity and accuracy) is critical, dPCR can be an alternative option for quantifying microorganisms, including pathogens, in aquatic environments.

Spatial and Temporal Monitoring of SARS-CoV-2 in a Sewershed

In response to the global pandemic, Clean Water Services (Washington County, Oregon) monitored 4 wastewater treatment plants (WWTPs) and 16 manholes over a period of 15 months to determine whether spatial and temporal trends in SARS-CoV-2 concentrations corresponded with reported COVID-19 cases. A total of 738 samples were analyzed for SARS-CoV-2 using reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). SARS-CoV-2 concentrations at WWTPs appeared to be a leading indicator, with increases in wastewater observed two to three weeks before cases rose. Neighborhoods with high LatinX and high poverty populations also had higher SARS-CoV-2 concentrations. Additionally, outbreaks at local food processing plants corresponded with viral peaks in their associated manhole. Surprisingly, SARS-CoV-2 was rarely detected in hospital effluent despite the presence of known cases, and experiments revealed hospital disinfectants can destroy the RNA signal. Collectively, these findings demonstrate the value of wastewater-based epidemiology for monitoring the local burden of COVID-19. Copyright © 2021 Water Environment Federation

Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution.

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

Effect of selected wastewater characteristics on estimation of SARS-CoV-2 viral load in wastewater.

Wastewater-based epidemiology has been used as a tool for surveillance of COVID-19 infections. This approach is dependent on the detection and quantification of SARS-CoV-2 RNA in untreated/raw wastewater. However, the quantification of the viral RNA could be influenced by the physico-chemical properties of the wastewater. This study presents the first use of Adaptive Neuro-Fuzzy Inference System (ANFIS) to determine the potential impact of physico-chemical characteristics of wastewater on the detection and concentration of SARS-CoV-2 RNA in wastewater. Raw wastewater samples from four wastewater treatment plants were investigated over four months. The physico-chemical characteristics of the raw wastewater was recorded, and the SARS-CoV-2 RNA concentration determined via amplification with droplet digital polymerase chain reaction. The wastewater characteristics considered were chemical oxygen demand, flow rate, ammonia, pH, permanganate value, and total solids. The mean SARS-CoV-2 RNA concentrations ranged from 648.1(±514.6) copies/mL to 1441.0(±1977.8) copies/mL. Among the parameters assessed using the ANFIS model, ammonia and pH showed significant association with the concentration of SARS-CoV-2 RNA measured. Increasing ammonia concentration was associated with increasing viral RNA concentration and pH between 7.1 and 7.4 were associated with the highest SARS-CoV-2 concentration. Other parameters, such as total solids, were also observed to influence the viral RNA concentration, however, this observation was not consistent across all the wastewater treatment plants. The results from this study indicate the importance of incorporating wastewater characteristic assessment into wastewater-based epidemiology for a robust and accurate COVID-19 surveillance.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers.

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

Monitoring changes in COVID-19 infection using wastewater-based epidemiology: A South African perspective.

Monitoring of COVID-19 infections within communities via wastewater-based epidemiology could provide a cost-effective alternative to clinical testing. This approach, however, still requires improvement for its efficient application. In this paper, we present the use of wastewater-based epidemiology in monitoring COVID-19 infection dynamics in the KwaZulu-Natal province of South Africa, focusing on four wastewater treatment plants for 14 weeks. The SARS-CoV-2 viral load in influent wastewater was determined using droplet digital PCR, and the number of people infected was estimated using published models as well as using a modified model to improve efficiency. On average, viral loads ranged between 0 and 2.73 × 105 copies/100 ml, 0-1.52 × 105 copies/100 ml, 3 × 104-7.32 × 105 copies/100 ml and 1.55 × 104-4.12 × 105 copies/100 ml in the four wastewater treatment plants studied. The peak in viral load corresponded to the reported COVID-19 infections within the districts where these catchments are located. In addition, we also observed that easing of lockdown restrictions by authorities corresponded with an increase in viral load in the untreated wastewater. Estimation of infection numbers based on the viral load showed that a higher number of people could potentially be infected, compared to the number of cases reported based on clinical testing. The findings reported in this paper contribute to the field of wastewater-based epidemiology for COVID-19 surveillance, whilst highlighting some of the challenges associated with this approach, especially in developing countries.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.