Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness. IMPORTANCE: This study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

The effectiveness of booster vaccination of inactivated COVID-19 vaccines against susceptibility, infectiousness, and transmission of omicron BA.2 variant: a retrospective cohort study in Shenzhen, China.

Little studies evaluated the effectiveness of booster vaccination of inactivated COVID-19 vaccines against being infected (susceptibility), infecting others (infectiousness), and spreading the disease from one to another (transmission). Therefore, we conducted a retrospective cohort study to evaluate the effectiveness of booster vaccination of inactivated COVID-19 vaccines against susceptibility, infectiousness, and transmission in Shenzhen during an Omicron BA.2 outbreak period from 1 February to 21 April 2022. The eligible individuals were classified as four sub-cohorts according to the inactivated COVID-19 vaccination status of both the close contacts and their index cases: group 2-2, fully vaccinated close contacts seeded by fully vaccinated index cases (reference group); group 2-3, booster-vaccinated close contacts seeded by fully vaccinated index cases; group 3-2, fully vaccinated close contacts seeded by booster-vaccinated index cases; and group 3-3, booster-vaccinated close contacts seeded by booster-vaccinated index cases. Univariate and multivariate logistic regression analyses were applied to estimate the effectiveness of booster vaccination. The sample sizes of groups 2-2, 2-3, 3-2, and 3-3 were 846, 1,115, 1,210, and 2,417, respectively. We found that booster vaccination had an effectiveness against infectiousness of 44.9% (95% CI: 19.7%, 62.2%) for the adults ≥ 18 years, 62.2% (95% CI: 32.0%, 78.9%) for the female close contacts, and 60.8% (95% CI: 38.5%, 75.1%) for the non-household close contacts. Moreover, booster vaccination had an effectiveness against transmission of 29.0% (95% CI: 3.2%, 47.9%) for the adults ≥ 18 years, 38.9% (95% CI: 3.3%, 61.3%) for the female close contacts, and 45.8% (95% CI: 22.1%, 62.3%) for the non-household close contacts. However, booster vaccination against susceptibility did not provide any protective effect. In summary, this study confirm that booster vaccination of the inactivated COVID-19 vaccines provides low level of protection and moderate level of protection against Omicron BA.2 transmission and infectiousness, respectively. However, booster vaccination does not provide any protection against Omicron BA.2 susceptibility.

Vaccine Effectiveness against SARS-CoV-2 among Household Contacts during Omicron BA.2-Dominant Period, Japan.

We calculated attack rates for household contacts of COVID-19 patients during the SARS-CoV-2 Omicron BA.2-dominant period in Japan. Attack rates among household contacts without recent (<3 months) vaccination was lower for contacts of index patients with complete vaccination than for contacts of index patients without complete vaccination, demonstrating indirect vaccine effectiveness.

A mini review of reinfection with the SARS-CoV-2 Omicron variant.

Background: COVID-19 has caused severe morbidity and mortality worldwide. After the end of the dynamic zero-COVID policy in China in December, 2022, concerns regarding reinfection were raised while little was known due to the lack of surveillance data in this country. Aims: This study reviews the probability, risk factors, and severity of severe acute respiratory syndrome coronavirus 2 Omicron variant reinfection, as well as the interval between infections, risk of onward transmission by reinfected cases, and the role of booster vaccination against reinfection. Sources: References for this review were identified through searches of PubMed and Web of Science up to September 24, 2023. Results: The rate of reinfection ranges from 3.1% to 13.0%. Factors associated with a higher risk of reinfection include being female, having comorbidities, and being unvaccinated. Reinfection with the BA.4 or BA.5 variant occurs approximately 180 days after the initial infection. Reinfections are less clinically severe than primary infections, and there is evidence of lower transmissibility. The debate surrounding the effectiveness and feasibility of booster vaccinations in preventing reinfection continues. Conclusions: The reinfection rate during the Omicron epidemic is significantly higher than in previous epidemic periods. However, the symptoms and infectivity of reinfection were weaker than those of the prior infection. Medical staff and individuals at high risk of reinfection should be vigilant. The efficacy of booster vaccinations in reducing reinfection is currently under debate.

Dynamics of asymptomatic Plasmodium falciparum and Plasmodium vivax infections and their infectiousness to mosquitoes in a low transmission setting of Ethiopia: a longitudinal observational study.

OBJECTIVE: A 15-month longitudinal study was conducted to determine the duration and infectivity of asymptomatic qPCR-detected Plasmodium falciparum and Plasmodium vivax infections in Ethiopia. METHOD: Total parasite and gametocyte kinetics were determined by molecular methods; infectivity to Anopheles arabiensis mosquitoes by repeated membrane feeding assays. Infectivity results were contrasted with passively recruited symptomatic malaria cases. RESULTS: For P. falciparum and P. vivax infections detected at enrolment, median durations of infection were 37 days (95% confidence interval [CI], 15-93) and 60 days (95% CI, 18-213), respectively. P. falciparum and P. vivax parasite densities declined over the course of infections. From 47 feeding assays on 22 asymptomatic P. falciparum infections, 6.4% (3/47) were infectious and these infected 1.8% (29/1579) of mosquitoes. No transmission was observed in feeding assays on asymptomatic P. vivax mono-infections (0/56); one mixed-species infection was highly infectious. Among the symptomatic cases, 4.3% (2/47) of P. falciparum and 73.3% (53/86) of P. vivax patients were infectious to mosquitoes. CONCLUSION: The majority of asymptomatic infections were of short duration and low parasite density. Only a minority of asymptomatic individuals were infectious to mosquitoes. This contrasts with earlier findings and is plausibly due to the low parasite densities in this population.

Is It Possible to Test for Viral Infectiousness?: The Use Case of (SARS-CoV-2).

Identifying and managing individuals with active or chronic disease, implementing appropriate infection control measures, and mitigating the spread of the COVID-19 pandemic highlighted the need for tests of infectiousness. The gold standard for assessing infectiousness has been the recovery of infectious virus in cell culture. Using cycle threshold values, antigen testing, and SARS-CoV-2, replication intermediate strands were used to assess infectiousness, with many limitations. Infectiousness can be influenced by host factors (eg, preexisting immune responses) and virus factors (eg, evolution).

Diagnostic performance of rapid antigen tests for SARS-CoV-2 transmission risk based on cycle threshold values in the emergency department.

BACKGROUND: This study aimed to investigate the diagnostic performance of the rapid antigen test (RAT) for screening patients with cycle threshold (Ct) values of SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) in the emergency department. Previous studies have shown that Ct values could be used as indicators of infectiousness. Therefore, we considered the Ct value an indicator of potential infectiousness. METHODS: This single-center retrospective observational study was conducted between January 1, 2020, and March 31, 2022. Patients who underwent both RT-PCR and RAT for the diagnosis of COVID-19 were included. Patients with negative RT-PCR results were excluded. Patients with Ct values lower than 26 and 30 were considered potentially infectious for COVID-19. RESULT: A total of 386 patients were analyzed. At Ct value cutoffs of 26 and 30, the result of the RAT showed a sensitivity of 82% and 74%, specificity of 84% and 89%, and area under the curve (AUC) of 0.829 and 0.813, respectively, in the receiver operating characteristic curve. However, the NPV was relatively low at 55% and 25%. CONCLUSION: The RAT might be a rapid screening tool for detecting patients with the infectiousness of SARS-CoV-2. However, considering the low NPV, it is challenging to depend only on a negative test result from an antigen test to terminate quarantine. Clinicians should consider additional factors, such as the duration of symptoms and the immunocompromised state, for SARS-CoV-2 transmission.

Association of serum cathelicidin and Vitamin D levels with infectiousness in patients with pulmonary tuberculosis: A prospective cohort study.

Background: The immunomodulatory effects of Vitamin D expand to induce the synthesis of an antimicrobial peptide, cathelicidin. There is evidence showing altered levels of cathelicidin in tuberculosis (TB). It has been suggested that Vitamin D-mediated antimicrobial activity depends on its ability to induce cathelicidin. The present study was designed to assess the alterations in serum anti-microbial peptide cathelicidin and 25-hydroxy Vitamin D levels in patients with newly diagnosed pulmonary TB at different treatment times and to study the association between serum Vitamin D levels and cathelicidin. Methods: Serum 25-hydroxyvitamin D and cathelicidin levels were estimated in 147 patients with newly diagnosed pulmonary TB at different times: at the start of anti-tubercular treatment, end of the intensive phase of treatment, and at the end of treatment. Results: There was a statistically significant difference between the levels of serum 25-hydroxyvitamin D and serum cathelicidin at different treatment periods. However, no significant correlation was found between serum Vitamin D and cathelicidin levels or between serum Vitamin D and cathelicidin levels with infectiousness in patients with pulmonary TB. Conclusion: Serum Vitamin D levels and serum cathelicidin levels were significantly reduced at diagnosis, and there was an incremental increase following treatment. However, there was no correlation between the levels of serum cathelicidin and serum Vitamin D or with the infectiousness of the illness.

Epidemic dynamics with time-varying transmission risk reveal the role of disease stage-dependent infectiousness.

A key characteristic of acute communicable diseases is the infectiousness that varies over time as the infection dynamics evolve within a host, which influences the risk of transmission in different stages of the disease. Despite the evidence of time-varying transmission risk, most dynamic models of epidemics assume a constant transmission rate during the infectious period. Recent work has shown the difference in epidemic dynamics when this assumption is relaxed and different transmission rates are used by discretizing the infectious period into multiple sub-periods. Here, we develop an age-structured model to integrate a continuous time-varying transmission risk, based on an established correlation between the viral dynamics and infectiousness profile. Taking into account the natural history and parameter estimates of COVID-19 caused by the original strain of SARS-CoV-2, we demonstrate the difference in temporal epidemic dynamics when a continuous time-varying transmission probability is used as compared to multiple constant transmission probabilities. Our results show a significant difference between the incidence curves in terms of the magnitude and peak time, even when the reproduction number and total number of infections are the same for continuous and discrete transmission probabilities. Finally, we demonstrate the spurious outcome of preventing an epidemic through the isolation of infectious individuals when constant transmission probabilities are used, highlighting the importance of integrating a continuous time-dependent transmission parameter in dynamic models. These findings suggest a more cautious interpretation of model outcomes, especially those that are intended to evaluate the effectiveness of interventions and inform policy decisions for disease mitigation strategies.

Host infection and disease-induced mortality modify species contributions to the environmental reservoir.

Environmental pathogen reservoirs exist for many globally important diseases and can fuel epidemics, influence pathogen evolution, and increase the threat of host extinction. Species composition can be an important factor that shapes reservoir dynamics and ultimately determines the outcome of a disease outbreak. However, disease-induced mortality can change species communities, indicating that species responsible for environmental reservoir maintenance may change over time. Here we examine the reservoir dynamics of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats. We quantified changes in pathogen shedding, infection prevalence and intensity, host abundance, and the subsequent propagule pressure imposed by each species over time. We find that highly shedding species are important during pathogen invasion, but contribute less over time to environmental contamination as they also suffer the greatest declines. Less infected species remain more abundant, resulting in equivalent or higher propagule pressure. More broadly, we demonstrate that high infection intensity and subsequent mortality during disease progression can reduce the contributions of high-shedding species to long-term pathogen maintenance.

Effect of COVID-19 vaccination on household transmission of SARS-CoV-2 in the Omicron era: The Vaccine Effectiveness, Networking, and Universal Safety (VENUS) study.

OBJECTIVES: To evaluate the effectiveness of vaccination on reducing household transmission of SARS-CoV-2 among common household types in Japan during the Omicron variant wave. METHODS: This retrospective study was conducted using vaccination records, COVID-19 infection data, and resident registry data from two Japanese municipalities. Households that experienced their first COVID-19 case between January and April 2022 were categorized into two groups according to the presence/absence of children aged ≤11 years. We constructed multivariable logistic regression models with generalized estimating equations to calculate the odds ratios (ORs) and 95% confidence intervals for household transmission according to the vaccination statuses of primary cases and household contacts. RESULTS: We analyzed 7326 households with 17,586 contacts. In all households, the OR for household transmission was <0.6 (P <0.001) when the primary case and/or contact were vaccinated. In households with children aged ≤11 years, the OR was 0.71 (P <0.001) when only the contact was vaccinated. In households with all members aged ≥12 years, the OR was <0.5 (P <0.001) when the primary case and/or contact were vaccinated. CONCLUSION: COVID-19 vaccination effectively reduced household transmission in Japan during the Omicron variant wave.

SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains.

Introduction-The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods-We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested via RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results-A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS-CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.

Vaccine effectiveness against transmission of alpha, delta and omicron SARS-COV-2-infection, Belgian contact tracing, 2021-2022.

OBJECTIVES: Vaccine effectiveness against transmission (VET) of SARS-CoV-2-infection can be estimated from secondary attack rates observed during contact tracing. We estimated VET, the vaccine-effect on infectiousness of the index case and susceptibility of the high-risk exposure contact (HREC). METHODS: We fitted RT-PCR-test results from HREC to immunity status (vaccine schedule, prior infection, time since last immunity-conferring event), age, sex, calendar week of sampling, household, background positivity rate and dominant VOC using a multilevel Bayesian regression-model. We included Belgian data collected between January 2021 and January 2022. RESULTS: For primary BNT162b2-vaccination we estimated initial VET at 96% (95%CI 95-97) against Alpha, 87% (95%CI 84-88) against Delta and 31% (95%CI 25-37) against Omicron. Initial VET of booster-vaccination (mRNA primary and booster-vaccination) was 87% (95%CI 86-89) against Delta and 68% (95%CI 65-70) against Omicron. The VET-estimate against Delta and Omicron decreased to 71% (95%CI 64-78) and 55% (95%CI 46-62) respectively, 150-200 days after booster-vaccination. Hybrid immunity, defined as vaccination and documented prior infection, was associated with durable and higher or comparable (by number of antigen exposures) protection against transmission. CONCLUSIONS: While we observed VOC-specific immune-escape, especially by Omicron, and waning over time since immunization, vaccination remained associated with a reduced risk of SARS-CoV-2-transmission.

The effect of variation of individual infectiousness on SARS-CoV-2 transmission in households.

Quantifying variation of individual infectiousness is critical to inform disease control. Previous studies reported substantial heterogeneity in transmission of many infectious diseases including SARS-CoV-2. However, those results are difficult to interpret since the number of contacts is rarely considered in such approaches. Here, we analyze data from 17 SARS-CoV-2 household transmission studies conducted in periods dominated by ancestral strains, in which the number of contacts was known. By fitting individual-based household transmission models to these data, accounting for number of contacts and baseline transmission probabilities, the pooled estimate suggests that the 20% most infectious cases have 3.1-fold (95% confidence interval: 2.2- to 4.2-fold) higher infectiousness than average cases, which is consistent with the observed heterogeneity in viral shedding. Household data can inform the estimation of transmission heterogeneity, which is important for epidemic management.

Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study.

BACKGROUND: Guidelines for SARS-CoV-2 have relied on limited data on duration of viral infectiousness and correlation with COVID-19 symptoms and diagnostic testing. METHODS: We enrolled ambulatory adults with acute SARS-CoV-2 infection and performed serial measurements of COVID-19 symptoms, nasal swab viral RNA, nucleocapsid (N) and spike (S) antigens, and replication-competent SARS-CoV-2 by viral growth in culture. We determined average time from symptom onset to a first negative test result and estimated risk of infectiousness, as defined by positive viral growth in culture. RESULTS: Among 95 adults, median [interquartile range] time from symptom onset to first negative test result was 9 [5] days, 13 [6] days, 11 [4] days, and >19 days for S antigen, N antigen, culture growth, and viral RNA by RT-PCR, respectively. Beyond two weeks, virus growth and N antigen titers were rarely positive, while viral RNA remained detectable among half (26/51) of participants tested 21-30 days after symptom onset. Between 6-10 days from symptom onset, N antigen was strongly associated with culture positivity (relative risk=7.61, 95% CI: 3.01-19.22), whereas neither viral RNA nor symptoms were associated with culture positivity. During the 14 days following symptom onset, the presence of N antigen remained strongly associated (adjusted relative risk=7.66, 95% CI: 3.96-14.82) with culture positivity, regardless of COVID-19 symptoms. CONCLUSIONS: Most adults have replication-competent SARS-CoV-2 for 10-14 after symptom onset. N antigen testing is a strong predictor of viral infectiousness and may be a more suitable biomarker, rather than absence of symptoms or viral RNA, to discontinue isolation within two weeks from symptom onset.

Low SARS-CoV-2 Cq values in healthcare workers with symptomatic COVID-19 infections, regardless of symptom severity, The Netherlands, January to August 2022.

We analysed SARS-CoV-2 PCR Cq values from 3,183 healthcare workers who tested positive between January and August 2022. Median Cq values were lower in symptomatic than in asymptomatic HCW. The difference in Cq values between HCW with mild vs moderate/severe symptoms was statistically significant but negligibly small. To prevent nosocomial infections, all symptomatic HCW should be tested irrespective of symptom severity. This information can support decisions on testing and isolation, in the context of ongoing pressure on healthcare systems.