Longitudinal wastewater surveillance addressed public health priorities during the transition from "dynamic COVID-zero" to "opening up" in China: a population-based study (preprint)

Background Wastewater surveillance provides real-time, cost-effective monitoring of SARS-CoV-2 transmission. We developed the first city-level wastewater warning system in mainland China, located in Shenzhen. Our study aimed to reveal cryptic transmissions under the "dynamic COVID-zero" policy and characterize the dynamics of the infected population and variant prevalence, and then guide the allocation of medical resources during the transition to "opening up" in China. Methods In this population-based study, a total of 1,204 COVID-19 cases were enrolled to evaluate the contribution of Omicron variant-specific faecal shedding rates in wastewater. After that, wastewater samples from up to 334 sites distributed in communities and port areas in two districts of Shenzhen covering 1.74 million people were tested daily to evaluate the sensitivity and specificity of this approach and were validated against daily SARS-CoV-2 screening. After the public health policy was switched to "opening up" in December 7, 2022, we conducted wastewater surveillance at wastewater treatment plants and pump stations covering 3.55 million people to estimate infected populations using model prediction and detect the relative abundance of SARS-CoV-2 lineages using wastewater sequencing. Findings In total, 82.4% of SARS-CoV-2 Omicron cases tested positive for faecal viral RNA within the first four days after the diagnosis, which was far more than the proportion of the ancestral variant. A total of 27,759 wastewater samples were detected from July 26 to November 30 in 2022, showing a sensitivity of 73.8% and a specificity of 99.8%. We further found that wastewater surveillance played roles in providing early warnings and revealing cryptic transmissions in two communities. Based on the above results, we employed a prediction model to monitor the daily number of infected individuals in Shenzhen during the transition to "opening up" in China, with over 80% of the population infected in both Futian District and Nanshan District. Notably, the prediction of the daily number of hospital admission was consistent with the actual number. Further sequencing revealed that the Omicron subvariant BA.5.2.48 accounted for the most abundant SARS-CoV-2 RNA in wastewater, and BF.7.14 and BA.5.2.49 ranked second and third, respectively, which was consistent with the clinical sequencing. Interpretation This study provides a scalable solution for wastewater surveillance of SARS-CoV-2 to provide real-time monitoring of the new variants, infected populations and facilitate the precise prediction of hospital admission. This novel framework could be a One Health system for the surveillance of other infectious and emerging pathogens with faecal shedding and antibiotic resistance genes in the future. Funding Sanming Project of Medicine in Shenzhen, Shenzhen Key Medical Discipline Construction Fund.

Rapid evaluation of neutralizing antibodies in COVID-19 patients (preprint)

The ongoing coronavirus disease 2019 (COVID-19) pandemic calls for a method to rapidly and conveniently evaluate neutralizing antibody (NAb) activity in patients. Here, an up-conversion phosphor technology-based point-of-care testing (UPT-POCT) and a microneutralization assay were employed to detect total antibodies against the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and NAb activity in COVID-19 patients' sera, respectively, in order to determine if UPT-POCT could be used as a surrogate method for rapid evaluation of serum NAb activity in COVID-19 patients. In total, 519 serum samples from 213 recovered and 99 polymerase chain reaction re-positive (RP) COVID-19 patients were used in this report. We found that UPT-POCT reporting values correlated highly with NAb titers from 1:4 to 1:1024, with a correlation coefficient r = 0.9654 (P < 0.001), as well as protection rate against RP (r = 0.9886, P < 0.0001). As a significant point for reducing re-positive rate, UPT-POCT values of 4.380, corresponding to NAb titer of 1:64, may be appropriate as an indicator for evaluating high efficiency of protection. This study demonstrates that the quantitative lateral flow based UPT-POCT, could be used to rapidly evaluate NAb titer, which is of importance for assessing vaccine immunization efficacy, herd immunity, and screening patient plasma for high NAbs.

Prime-boost protein subunit vaccines against SARS-CoV-2 are highly immunogenic in mice and macaques (preprint)

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assessed prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

Viral RNA level, serum antibody responses, and transmission risk in discharged COVID-19 patients with recurrent positive SARS-CoV-2 RNA test results: a population-based observational cohort study (preprint)

SummaryO_ST_ABSBackgroundC_ST_ABSManaging discharged COVID-19 (DC) patients with recurrent positive (RP) SARS-CoV-2 RNA test results is challenging. We aimed to comprehensively characterize the viral RNA level and serum antibody responses in RP-DC patients and evaluate their viral transmission risk. MethodsA population-based observational cohort study was performed on 479 DC patients discharged from February 1 to May 5, 2020 in Shenzhen, China. We conducted RT-qPCR, antibody assays, neutralisation assays, virus isolation, whole genome sequencing (WGS), and epidemiological investigation of close contacts. FindingsOf 479 DC patients, the 93 (19%) RP individuals, including 36 with multiple RP results, were characterised by young age (median age: 34 years, 95% confidence interval [CI]: 29-38 years). The median discharge-to-RP length was 8 days (95% CI: 7-14 days; maximum: 90 days). After readmission, RP-DC patients exhibited mild (28%) or absent (72%) symptoms, with no disease progression. The viral RNA level in RP-DC patients ranged from 1{middle dot}9-5{middle dot}7 log10 copies/mL (median: 3{middle dot}2, 95% CI: 3{middle dot}1-3{middle dot}5). At RP detection, the IgM, IgG, IgA, total antibody, and neutralising antibody (NAb) seropositivity rates in RP-DC patients were 38% (18/48), 98% (47/48), 63% (30/48), 100% (48/48), and 91% (39/43), respectively. Regarding antibody levels, there was no significant difference between RP-DC and non-RP-DC patients. The antibody level remained constant in RP-DC patients pre- and post-RP detection. Virus isolation of nine representative specimens returned negative results. WGS of six specimens yielded only genomic fragments. No clinical symptoms were exhibited by 96 close contacts of 23 RP-DC patients; their viral RNA (96/96) and antibody (20/20) test results were negative. After full recovery, 60% of patients (n=162, 78 no longer RP RP-DC and 84 non-RP-DC) had NAb titres of [≥]1:32. InterpretationRP may occur in DC patients following intermittent and non-stable excretion of low viral RNA levels. RP-DC patients pose a low risk of transmitting SARS-CoV-2. An NAb titre of [≥] 1:32 may provide a reference indicator for evaluating humoral responses in COVID-19 vaccine clinical trials. FundingSanming Project of Medicine in Shenzhen, China National Science and Technology Major Projects Foundation, Special Foundation of Science and Technology Innovation Strategy of Guangdong Province of China, and Shenzhen Committee of Scientific and Technical Innovation grants.

Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection (preprint)

Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic. Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)