No(cebo) Vax: COVID-19 Vaccine Beliefs Are Important Determinants of Both Occurrence and Perceived Severity of Common Vaccines' Adverse Effects.

This study highlights the role of psychological influences in triggering and amplifying the adverse effects of the COVID-19 vaccine (i.e., nocebo effects). Fear, beliefs, and expectations about the COVID-19 vaccine, trust in health and scientific institutions, and stable personality traits were measured in 315 adult Italian citizens (145 men) during the 15-min waiting time after vaccination. The occurrence and severity of 10 potential adverse effects were assessed 24 hr later. Nonpharmacological variables predicted nearly 30% of the severity of the vaccine's adverse effects. Expectations are important determinants of adverse effects from vaccines, and the results of the path analyses show that these expectations stem primarily from people's vaccine beliefs and attitudes, which can be changed. Implications for increasing vaccine acceptability and limiting the nocebo effect are discussed.

Kinetics of dried blood spot-measured anti-SARS-CoV2 Spike IgG in mRNA-vaccinated healthcare workers

Introduction One of the major criticisms facing the research community during SARS-CoV2 pandemic was the lack of large-scale, longitudinal data on the efficacy of the SARS-CoV2 mRNA vaccines. Currently, even if COVID-19 antiviral treatments have been authorized by European Medicine Agency, prevention through approved specific vaccines is the best approach available in order to contain the ongoing pandemic. Objectives Here, we studied the antibody kinetic over a one-year period from vaccination with the Pfizer-BioNTech (Pfizer) vaccines and subsequent boosting with either the BioNTech or Moderna (Spikevax) vaccines in a large cohort of 8,071 healthcare workers (HCW). We also described the impact of SARS-CoV2 infection on antibody kinetic over the same period. Methods We assessed the anti SARS-CoV2 Spike IgG antibody kinetic by the high throughput dried blood spot (DBS) collection method and the GSP®/DELFIA® Anti-SARS-CoV2 IgG assay (PerkinElmer®). Results Our data support existing models showing that SARS-CoV2 vaccination elicits strong initial antibodies responses that decline with time but are transitorily increased by administering a vaccine booster. We also showed that using heterologous vaccine/booster combinations a stronger antibody response was elicited than utilizing a booster from the same vaccine manufacturer. Furthermore, by considering the impact of SARS-CoV2 infection occurrence in proximity to the scheduled booster administration, we confirmed that booster dose did not contribute significantly to elicit higher antibody responses. Conclusion DBS sampling in our large population of HCWs was fundamental to collect a large number of specimens and to clarify the effective mRNA vaccine-induced antibody kinetic and the role of both heterologous boosters and SARS-CoV2 infection in modulating antibody responses.

SARS-CoV-2 coinfection in immunocompromised host leads to the generation of recombinant strain.

OBJECTIVES: Recombination related to coinfection is a huge driving force in determining the virus genetic variability, particularly in conditions of partial immune control, leading to prolonged infection. Here, we characterized a distinctive mutational pattern, highly suggestive of Delta-Omicron double infection, in a lymphoma patient. METHODS: The specimen was characterized through a combined approach, analyzing the results of deep sequencing in primary sample, viral culture, and plaque assay. RESULTS: Bioinformatic analysis on the sequences deriving from the primary sample supports the hypothesis of a double viral population within the host. Plaque assay on viral culture led to the isolation of a recombinant strain deriving from Delta and Omicron lineages, named XS, which virtually replaced its parent lineages within a single viral propagation. CONCLUSION: It is impossible to establish whether the recombination event happened within the host or in vitro; however, it is important to monitor co-infections, especially in the exceptional intrahost environment of patients who are immunocompromised, as strong driving forces of viral evolution.

Kinetics of dried blood spot-measured anti-SARS-CoV2 Spike IgG in mRNA-vaccinated healthcare workers.

Introduction: One of the major criticisms facing the research community during SARS-CoV2 pandemic was the lack of large-scale, longitudinal data on the efficacy of the SARS-CoV2 mRNA vaccines. Currently, even if COVID-19 antiviral treatments have been authorized by European Medicine Agency, prevention through approved specific vaccines is the best approach available in order to contain the ongoing pandemic. Objectives: Here, we studied the antibody kinetic over a one-year period from vaccination with the Pfizer-BioNTech (Pfizer) vaccines and subsequent boosting with either the BioNTech or Moderna (Spikevax) vaccines in a large cohort of 8,071 healthcare workers (HCW). We also described the impact of SARS-CoV2 infection on antibody kinetic over the same period. Methods: We assessed the anti SARS-CoV2 Spike IgG antibody kinetic by the high throughput dried blood spot (DBS) collection method and the GSP®/DELFIA® Anti-SARS-CoV2 IgG assay (PerkinElmer®). Results: Our data support existing models showing that SARS-CoV2 vaccination elicits strong initial antibodies responses that decline with time but are transitorily increased by administering a vaccine booster. We also showed that using heterologous vaccine/booster combinations a stronger antibody response was elicited than utilizing a booster from the same vaccine manufacturer. Furthermore, by considering the impact of SARS-CoV2 infection occurrence in proximity to the scheduled booster administration, we confirmed that booster dose did not contribute significantly to elicit higher antibody responses. Conclusion: DBS sampling in our large population of HCWs was fundamental to collect a large number of specimens and to clarify the effective mRNA vaccine-induced antibody kinetic and the role of both heterologous boosters and SARS-CoV2 infection in modulating antibody responses.

An Enhanced Dissolving Cyclosporin-A Inhalable Powder Efficiently Reduces SARS-CoV-2 Infection In Vitro.

This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.

Evaluation of STANDARD TM M10 SARS-CoV-2, a Novel Cartridge-Based Real-Time PCR Assay for the Rapid Identification of Severe Acute Respiratory Syndrome Coronavirus 2

Since the beginning of the pandemic, SARS-CoV-2 has caused problems for all of world's population, not only in terms of deaths but also in terms of overloading healthcare facilities in all countries. Diagnosis is one of the key aspects of controlling the spread of SARS-CoV-2, and among the current molecular techniques, real-time PCR is considered as the gold standard. The availability of tests that allow for the rapid and accurate identification of SARS-CoV-2 is therefore of considerable importance. Moreover, if these tests allow for even minimal intervention by the operator, any risk of contamination is reduced. In this study, the performances of the new STANDARDTM M10 SARS-CoV-2 (SD Biosensor Inc., Suwon, Korea) rapid molecular test, which incorporates the above-mentioned features, were characterized. The clinical and analytical performances measured by testing different variants circulating in Italy of STANDARDTM M10 SARS-CoV-2 were compared to the test already on the market and recognized as the gold standard: Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA, USA). The results obtained between the two tests are largely comparable, suggesting that STANDARDTM M10 SARS-CoV-2 can be used with excellent results in the fight against the global spread of SARS-CoV-2. [ FROM AUTHOR]

Viral Population Heterogeneity and Fluctuating Mutational Pattern during a Persistent SARS-CoV-2 Infection in an Immunocompromised Patient.

Literature offers plenty of cases of immunocompromised patients, who develop chronic and severe SARS-CoV-2 infections. The aim of this study is to provide further insight into SARS-CoV-2 evolutionary dynamic taking into exam a subject suffering from follicular lymphoma, who developed a persistent infection for over 7 months. Eight nasopharyngeal swabs were obtained, and were analyses by qRT-PCR for diagnostic purposes. All of them were considered eligible (Ct < 30) for NGS sequencing. Sequence analysis showed that all sequences matched the B.1.617.2 AY.122 lineage, but they differed by few mutations identifying three genetically similar subpopulations, which evolved during the course of infection, demonstrating that prolonged replication is paralleled with intra-host virus evolution. These evidences support the hypothesis that SARS-CoV-2 adaptive capacities are able to shape a heterogeneous viral population in the context of immunocompromised patients. Spill-over of viral variants with enhanced transmissibility or immune escape capacities from these subjects is plausible.

Omicron Sub-Lineage BA.5 and Recombinant XBB Evasion from Antibody Neutralisation in BNT162b2 Vaccine Recipients.

The recent emergence of a number of new SARS-CoV-2 variants resulting from recombination between two distinct parental lineages or sub-lineages within the same lineage has sparked the debate regarding potential enhanced viral infectivity and immune escape. Among these, XBB, recombinant of BA.2.10 and BA.2.75, has caused major concern in some countries due to its rapid increase in prevalence. In this study, we tested XBB escape capacity from mRNA-vaccine-induced (BNT162b2) neutralising antibodies compared to B.1 ancestral lineage and another co-circulating variant (B.1.1.529 BA.5) by analysing sera collected 30 days after the second dose in 92 healthcare workers. Our data highlighted an enhanced and statistically significant immune escape ability of the XBB recombinant. Although these are preliminary results, this study highlights the importance of immune escape monitoring of new and forthcoming variants and of the reformulation of existing vaccines.

Efficacy of SARS-CoV-2 Vaccination in Dialysis Patients: Epidemiological Analysis and Evaluation of the Clinical Progress.

This study investigated the impact of the fourth COVID-19 pandemic wave on dialysis patients of Romagna territory, assessing the associations of vaccination status with infection risk, clinical severity and mortality. From November 2021 to February 2022, an epidemiological search was conducted on 829 patients under dialysis treatment for at least one month. The data were then analyzed with reference to the general population of the same area. A temporal comparison was also carried out with the previous pandemic waves (from March 2020 to October 2021). The epidemiological evolution over time in the dialysis population and in Romagna citizens replicated the global trend, as the peak of the fourth wave corresponded to the time of maximum diffusion of omicron variant (B.1.1.529). Of 771 prevalent dialysis patients at the beginning of the study, 109 (14.1%) contracted SARS-CoV-2 infection during the 4-month observation period. Vaccine adherence in the dialysis population of the reference area was above 95%. Compared to fully or partially vaccinated subjects, the unvaccinated ones showed a significantly higher proportion of infections (12.5% vs. 27.0% p = 0.0341), a more frequent need for hospitalization (22.2% vs. 50.0%) and a 3.3-fold increased mortality risk. These findings confirm the effectiveness of COVID-19 vaccines in keeping infectious risk under control and ameliorating clinical outcomes in immunocompromised patients.

Mutational induction in SARS-CoV-2 major lineages by experimental exposure to neutralising sera.

The ongoing evolution of SARS-CoV-2 and the emergence of new viral variants bearing specific escape mutations responsible for immune evasion from antibody neutralisation has required a more accurate characterisation of the immune response as one of the evolutive forces behind viral adaptation to a largely immunised human population. In this work, culturing in the presence of neutralising sera vigorously promoted mutagenesis leading to the acquisition of known escape mutations on the spike as well as new presumptive escape mutations on structural proteins whose role as target of the neutralizing antibody response might have been thus far widely neglected. From this perspective, this study, in addition to tracing the past evolution of the species back to interactions with neutralising antibody immune response, also offers a glimpse into future evolutive scenarios.

Antibody Responses after Two Doses of COVID-19 mRNA Vaccine in Dialysis and Kidney Transplantation Patients Recovered from SARS-CoV-2 Infection.

Background and Objectives: Hemodialysis patients (HD) and kidney transplant recipients (KTRs) have been heavily impacted by COVID-19, showing increased risk of infection, worse clinical outcomes, and higher mortality rates than the general population. Although mass vaccination remains the most successful measure in counteracting the pandemic, less evidence is available on vaccine effectiveness in immunodepressed subjects previously infected and recovered from COVID-19. Materials and Methods: This study aimed at investigating the ability to develop an adequate antibody response after vaccination in a 2-dose series against SARS-CoV-2 in HD patients and KTR that was administered after laboratory and clinical recovery from COVID-19. Results: Comparing SARS-CoV-2 S1/S2 IgG levels measured before and after 2 doses of mRNA vaccine (BNT162b2 vaccine, Comirnaty, Pfizer-BioNTech or mRNA-1273 vaccine, Spikevax, Moderna), highly significant increases of antibody titers were observed. The antibody peak level was reached at 3 months following second dose administration, regardless of the underlying cause of immune depression and the time of pre-vaccine serology assessment after negativization. Conclusions: Our data indicate that HD patients and KTR exhibit a satisfying antibody response to a 2-dose series of mRNA vaccine, even in cases when infection-induced humoral immunity was poor or rapidly fading. Further studies are needed to evaluate the role of booster doses in conferring effective and durable protection in weak patient categories.

Importance of external quality assessment for SARS-CoV-2 antigen detection during the COVID-19 pandemic.

BACKGROUND: Antigen testing has become an essential part of fighting the ongoing COVID-19 pandemic. With the continual increase in available tests, independent and extensive comparative evaluations using data from external quality assessment (EQA) studies to evaluate test performance between different users are required. OBJECTIVES: An EQA scheme was established to assess the sensitivity of antigen tests and the potential impact of circulating SARS-CoV-2 strains on their performance. STUDY DESIGN: Panels were prepared for three challenges in 2021 containing inactivated SARS-CoV-2-positive samples of various genetic strains (including variants of concern, VOCs) at different concentrations, and negative samples. Data was analysed based on qualitative testing results in relation to the antigen test used. RESULTS: Participants registered for each individual challenge in any combination. In total, 258 respondents from 27 countries worldwide were counted submitting 472 datasets. All core samples were correctly reported by 76.7 to 83.1% at participant level and by 73.5 to 83.8% at dataset level. Sensitivity differences could be shown in viral loads and SARS-CoV-2 strains/variants including the impact on performance by a B.1.1.7-like mutant strain with a deletion in the nucleoprotein gene. Lateral flow rapid antigen tests showed a higher rate of false negatives in general compared with automated point-of-care tests and laboratory ELISA/immunoassays. CONCLUSIONS: EQA schemes can provide valuable data to inform participants about weaknesses in their testing process or methods and support ongoing assay evaluations for regulatory approval or post-market surveillance.

Organic Electrochemical Transistors as Versatile Tool for Real-Time and Automatized Viral Cytopathic Effect Evaluation.

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

Fast and real-time electrical transistor assay for quantifying SARS-CoV-2 neutralizing antibodies

Due to the SARS-CoV-2 pandemic renewed attention has been directed towards viral neutralization assays and neutralizing antibodies quantification, for vaccine pre-clinical trials and determining vaccine efficacy over time. The gold standard to assess antibody titer is the plaque reduction neutralization test, an end-point assay which evaluates the highest serum antibody dilution that neutralizes viral replication, by inspecting the cytopathic effect induced on cell cultures. Here, we use planar, PEDOT:PSS-based organic electrochemical transistors for real-time, remote-controlled, reliable and fast electrical monitoring of the cytopathic effect induced by SARS29 CoV-2 on Vero E6 cell lines, allowing the quantification of serum neutralizing titer. Our low-cost and scalable device has the potential to speed-up large-scale viral neutralization screening without the need for cancerous staining or highly specialized operators. Finally, the technology could be easily transferred to assess neutralizing antibody response towards different viruses in their permissive cell substrates.The COVID-19 pandemic highlights the importance of tests for assessing antibody titer, such as for determining vaccine efficacy. Here, a fast-operating organic electrochemical transistor is shown to assess the cytopathic effect caused by the SARS CoV-2 virus on Vero E6 cells in real-time.

Modelling RT-qPCR cycle-threshold using digital PCR data for implementing SARS-CoV-2 viral load studies.

OBJECTIVES: To exploit the features of digital PCR for implementing SARS-CoV-2 observational studies by reliably including the viral load factor expressed as copies/µL. METHODS: A small cohort of 51 Covid-19 positive samples was assessed by both RT-qPCR and digital PCR assays. A linear regression model was built using a training subset, and its accuracy was assessed in the remaining evaluation subset. The model was then used to convert the stored cycle threshold values of a large dataset of 6208 diagnostic samples into copies/µL of SARS-CoV-2. The calculated viral load was used for a single cohort retrospective study. Finally, the cohort was randomly divided into a training set (n = 3095) and an evaluation set (n = 3113) to establish a logistic regression model for predicting case-fatality and to assess its accuracy. RESULTS: The model for converting the Ct values into copies/µL was suitably accurate. The calculated viral load over time in the cohort of Covid-19 positive samples showed very low viral loads during the summer inter-epidemic waves in Italy. The calculated viral load along with gender and age allowed building a predictive model of case-fatality probability which showed high specificity (99.0%) and low sensitivity (21.7%) at the optimal threshold which varied by modifying the threshold (i.e. 75% sensitivity and 83.7% specificity). Alternative models including categorised cVL or raw cycle thresholds obtained by the same diagnostic method also gave the same performance. CONCLUSION: The modelling of the cycle threshold values using digital PCR had the potential of fostering studies addressing issues regarding Sars-CoV-2; furthermore, it may allow setting up predictive tools capable of early identifying those patients at high risk of case-fatality already at diagnosis, irrespective of the diagnostic RT-qPCR platform in use. Depending upon the epidemiological situation, public health authority policies/aims, the resources available and the thresholds used, adequate sensitivity could be achieved with acceptable low specificity.

Cold atmospheric plasma decontamination of SARS‐CoV‐2 bioaerosols

Bioaerosols (aerosolized particles with biological origin) are strongly suspected to play a significant role in the transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), especially in closed indoor environments. Thus, control technologies capable of effectively inactivating bioaerosols are urgently needed. In this regard, cold atmospheric pressure plasma (CAP) can represent a suitable option, thanks to its ability to produce reactive species, which can exert antimicrobial action. In this study, results;on the total inactivation of SARS‐CoV‐2 contained in bioaerosols treated using CAP generated in air are reported, demonstrating the possible use of CAP systems for the control of SARS‐CoV‐2 diffusion through bioaerosols. [ FROM AUTHOR] Copyright of Plasma Processes & Polymers is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Persistence of Antibody Responses to the SARS-CoV-2 in Dialysis Patients and Renal Transplant Recipients Recovered from COVID-19.

Nephropathic subjects with impaired immune responses show dramatically high infection rates of coronavirus disease 2019 (COVID-19). This work evaluated the ability to acquire and maintain protective antibodies over time in 26 hemodialysis patients and 21 kidney transplant recipients. The subjects were followed-up through quantitative determination of circulating SARS-CoV-2 S1/S2 IgG and neutralizing antibodies in the 6-month period after clinical and laboratory recovery. A group of 143 healthcare workers with no underlying chronic pathologies or renal diseases recovered from COVID was also evaluated. In both dialysis and transplanted patients, antibody titers reached a zenith around the 3rd month, and then a decline occurred on average between the 270th and 300th day. Immunocompromised patients who lost antibodies around the 6th month were more common than non-renal subjects, although the difference was not significant (38.5% vs. 26.6%). Considering the decay of antibody levels below the positivity threshold (15 AU/mL) as "failure", a progressive loss of immunisation was found in the overall population starting 6 months after recovery. A longer overall antibody persistence was observed in severe forms of COVID-19 (p = 0.0183), but within each group, given the small number of patients, the difference was not significant (dialysis: p = 0.0702; transplant: p = 0.1899). These data suggest that immunocompromised renal patients recovered from COVID-19 have weakened and heterogeneous humoral responses that tend to decay over time. Despite interindividual variability, an association emerged between antibody persistence and clinical severity, similar to the subjects with preserved immune function.

A deletion in the N gene may cause diagnostic escape in SARS-CoV-2 samples.

Five SARS-CoV-2-positive samples showed N-gene drop-out with a RT-PCR multiplex test. WGS found all samples to harbor a deletion in the same region of the N gene, which is likely to impair the efficiency of amplification. This highlights the need for a continued surveillance of viral evolution and diagnostic test performance.

Bacterial infections in critically ill patients with SARS-2-COVID-19 infection: results of a prospective observational multicenter study.

PURPOSE: To investigate the prevalence, incidence and characteristics of bacterial infections and their impact on outcome in critically ill patients infected with COVID-19. METHODS: We conducted a prospective observational study in eight Italian ICUs from February to May 2020; data were collected through an interactive electronic database. Kaplan-Meier analysis (limit product method) was used to identify the occurrence of infections and risk of acquisition. RESULTS: During the study period 248 patients were recruited in the eight participating ICUs. Ninety (36.3%) patients developed at least one episode of secondary infection. An ICU length of stay between 7 and 14 days was characterized by a higher occurrence of infectious complications, with ventilator-associated pneumonia being the most frequent. At least one course of antibiotic therapy was given to 161 (64.9%) patients. Overall ICU and hospital mortality were 33.9% and 42.9%, respectively. Patients developing bacteremia had a higher risk of ICU mortality [45.9% vs. 31.6%, odds ratio 1.8 (95% CI 0.9-3.7), p = 0.069] and hospital mortality [56.8% vs. 40.3%, odds ratio 1.9 (95% CI 1.1-3.9), p = 0.04]. CONCLUSION: In critically ill patients infected with COVID-19 the incidence of bacterial infections is high and associated with worse outcomes. Regular microbiological surveillance and strict infection control measures are mandated.

Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.