SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children … – Nature.com

Combined with clinical data and contact tracing, wastewater-based epidemiology (WBE) enables monitoring of SARS-CoV-2 community transmission, elucidating the onset, decline, and re-emergence of the epidemic15. Compared to the general community wastewater, pediatric dental wastewater can provide a targeted sample of asymptomatic children. This study uniquely detected SARS-CoV-2 RNA in the dental wastewater. The detection of SARS-CoV-2 RNA in 17 samples coincided with COVID-19 emergence among children in Saitama Prefecture. A sample obtained at the end of February 2022 contained the Omicron variant of SARS-CoV-2. Thus, the wastewater of pediatric dental clinics can provide information regarding asymptomatic COVID-19 in children.

Unlike clinical COVID-19 specimens, wastewater samples contain viral RNA at low concentrations. Therefore, we used Ct values as a reference without considering the copy number16. To detect SARS-CoV-2, we used the N1 and N2 primers and the probe set developed by the US Centers for Disease Control and Prevention (CDC), which amplify two regions of the nucleocapsid (N) gene17. In the kit used in this study, CDC N1 and N2 are modified using Cy5 and subjected to PCR in the same tube, thereby doubling the fluorescence intensity and improving assay sensitivity.

In Japan, the age-dependent epidemiological characteristics of COVID-19 varied among epidemic waves18. The sixth wave (Omicron variant) differed significantly from previous waves. The proportion of SARS-CoV-2 infections in individuals aged>70years was highest during the first wave (variant B.1.1)19, with the number of elderly cases gradually decreasing. The number of cases in 2050-year-olds markedly increased in the second to fifth waves (variants B.1.1.284, B.1.1.214, Alpha, and Delta, respectively)18,19. During the sixth wave (Omicron variant)18, the number of cases in individuals aged<19years increased rapidly.

Regarding vaccination in Japan, the mRNA vaccines developed by Pfizer and Moderna were mainly used20. Vaccination using the Pfizer vaccine began in April 2021, while the Moderna vaccine was introduced in May 2021. Initially, the vaccines were available to the elderly, and later the age range for vaccination has been gradually expanded to 12years and older. Vaccination of children aged 511years started in February 2022. Individuals in the age groups that were not vaccinated may have accounted for the increased number of cases.

Reports from other countries have indicated a high prevalence of the Delta and Omicron variants (particularly the latter) among children, implying that the characteristics of each virus variant were the main determinants of the number of cases21,22,23. In this study of children aged<10years in Saitama Prefecture, we observed an indistinct fourth wave that did not fall under the definition of an epidemic wave18. During the fifth and sixth waves, the NWRNC/100,000 population similarly increased and decreased for both preschoolers and schoolers aged<10years (p<0.0001, Wilcoxon matched-pairs test).

Although this study did not detect SARS-CoV-2 RNA during the period of Alpha-variant dominance, detection began in the same week as that of the fifth wave. Meanwhile, the sixth wave began 5weeks before SARS-CoV-2 RNA detection. We think that the SARS-CoV-2 RNA detection resulted from asymptomatic patients visiting the hospital. Generally, SARS-CoV-2 excretion begins 2days before symptom onset and persists for 710days. The incubation period is 1.5days longer in children than in the elderly24, while it is about one day longer for the Delta variant (4.41days; 95% CI, 3.765.05) than for the Omicron variant (3.42days; 95% CI, 2.883.96)24. The prolonged incubation period of the Delta variant could have increased the number of hospital visits by asymptomatic patients. Conversely, a significantly greater number of children were affected by the Omicron variant, including those with asymptomatic infections, compared to the Delta variant, and it is estimated that the number of asymptomatic children was much greater during the Omicron-predominant period than during the Delta-predominant period.

In Japan, COVID-19 vaccination began in April 2021. The vaccination coverage rate (two doses) was approximately 20% during the fifth wave and reached around 75% by December 2021, with over 70% in 1219-year-olds20. While no vaccine was available for children aged<12years at the time, the high community vaccination coverage rates could have contributed to the 5-week delay in SARS-CoV-2 RNA detection following the start of the sixth epidemic wave (Fig.2). The markedly increased NWRNC during the first 5weeks of the sixth wave may have been associated with increased number of asymptomatic patients visiting the clinic.

Vaccine coverage rate (the 2nd shot, %)27, detection period of SARS-CoV-2 RNA in wastewater from a pediatric dental clinic, and cumulative number of weekly reported new cases (NWRNC) per 100,000 population aged under 10years in Saitama Prefecture, Japan. The SARS-CoV-2 RNA detection period was observed in Weeks 2942 of 2021 (the fifth wave, Delta VOC predominant) and Weeks 821 of 2022 (the sixth wave, Omicron VOC predominant).

In this study, SARS-CoV-2 RNA was detected in wastewater until 3weeks after the end of the fifth wave, representing a period of high risk in terms of asymptomatic patients visiting the hospital, since COVID-19 infections in children are typically mild or asymptomatic. Additionally, the viral RNA can be detected for 1014days after symptom resolution. The number of days required to achieve PCR negativity has been reported to be similar between the Delta and Omicron (BA.1) variants25. Long COVID-19 was recognized by the WHO in 202126 and may account for the prolonged period of detection after the fifth wave.

The Omicron variant was detected simultaneously with the initial detection of SARS-CoV-2 RNA during the sixth wave, probably representing the peak of RNA release in this epidemic period. Despite a rapid decline of NWRNC following the peak of the sixth wave, SARS-CoV-2 RNA continued to be detected until the end of the survey period.

In WBE studies, SARS-CoV-2 RNA detection is generally considered a leading indicator of an increase in COVID-19 cases. However, in this study, the Delta and Omicron waves (Weeks 2939 of 2021 and Weeks 321 of 2022, respectively) preceded SARS-CoV-2 RNA detection in dental wastewater (Weeks 2942 of 2021 and Weeks 821 of 2022, respectively). Asymptomatic patients may have visited the clinic as the number of local cases increased. The increase in the number of cases was marginal during the fourth wave (Alpha variant), and SARS-CoV-2 RNA was not detected in wastewater from the pediatric dental clinic. This implies that a threshold number of cases is necessary for SARS-CoV-2 RNA detection in dental wastewater. Receiver operating characteristic (ROC) analysis was used to estimate the cut-off NWRNC/100,000 population value.

The cut-off and risk analysis demonstrated a strong association between SARS-CoV-2 RNA detection in dental wastewater and the NWRNC/100,000 population. The RR, 95% CI, and Fishers exact test results were identical among the three groups (RR, 5.36 [95% CI, 1.7216.67]; Fishers exact test, p=0.0005), although the cut-off values differed among the age groups.

During the SARS-CoV-2 RNA detection period, asymptomatic patients may have undergone dental treatment and been in contact with others in the preschool, school, or home settings. For all age groups, the RR was higher in the SARS-CoV-2 RNA detection period compared to that in SARS-CoV-2 RNA detection. Therefore, physicians and public health managers should be aware of the possibility of asymptomatic COVID-19 children when the NWRNC/100,000 population for those aged<10years reaches12.8 (preschoolers,13.4; schoolers aged<10years,20.2).

SARS-CoV-2 RNA positivity began when the NWRNC among children aged<10years reached12.8/100,000 population (RR, 7.8 [95% CI, 2.6323.21]; Fishers exact test, p<0.0001). A sample obtained in Week 8 of 2022 was found to harbor the Omicron VOC. This method may also be used to detect other viral pathogens in dental wastewater.

This study had several limitations. Since the data were all from Saitama Prefecture, the findings may not be generalizable to other locations. Therefore, further multicenter studies, using our study as a guide, are needed. Because wastewater samples contain viral RNA at low concentrations, a more comprehensive analysis could be conducted by improving the sensitivity of the assay. Additionally, multivariate analyses are required to overcome the effects of potential confounding factors and bias. To this end, efforts to gather more data for multivariate models, conduct several multicenter studies, and sensitivity improvement trials are currently underway.

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SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children ... - Nature.com