图文摘要 | Graphical abstract
导读 | Introduction
废水监测是一种有效监测病毒循环并可以作为早期预警系统的工具，能够同步检测有/无症状疾病的传播并识别特定病毒变体及其循环水平。废水监测已在世界范围内成功应用于甲型流感、呼吸道合胞、脊髓灰质炎、诺如、甲型肝炎等病毒性疾病的监测。季节性病毒在COVID–19控制措施的暂时停止下又卷土重来，其中以甲型流感病毒和乙型流感病毒(IAV/IBV)以及呼吸道合胞病毒(RSV)为主，它们占与SARS–CoV–2共同感染宿主的大多数。这三种病毒的相似症状妨碍了它们的临床诊断。为了将SARS–CoV–2从这些主流季节性呼吸道病毒中区分开，本研究评估、监测和对比西班牙巴塞罗那污水处理厂废水中IAV/IBV、RSV–A/RSV–B和SARS–CoV–2的RNA水平，并测量人类粪便污染的相关指标。
Wastewater–based surveillance (WBS) is an efficient tool to monitor viral circulation and serves as an early warning system, which can detect both symptomatic and asymptomatic transmission and identify specific variants and their circulation levels. WBS has already been successfully applied to monitor viral diseases such as influenza A, respiratory syncytial virus (RSV), poliovirus, norovirus, and hepatitis A around the world. Seasonal viruses have made a comeback after their temporary suppression by COVID–19 control measures. The most relevant are influenza viruses A and B (IAV/IBV), and RSV, which account for majority of the coinfections with SARS–CoV–2. The similar symptomatology of this trio pf viral treats hinders their clinical diagnosis. In response to differentiate SARS–CoV–2 from dominant seasonal respiratory viruses, this study evaluated, monitored, and compared RNA levels of IAV/IBV, RSV–A/RSV–B, and SARS–CoV–2 in wastewater treatment plant (WWTP) wastewater in Barcelona, Spain, as well as measured indicators of human fecal contamination.
一、废水中呼吸道病毒检测
| Detection of respiratory viruses in wastewater
SARS–CoV–2的阳性率显著高于其他病毒。两个污水处理厂的所有废水样品中都有检测到SARS–CoV–2，而流感病毒和RSV阳性检出率<50%，且在污水处理厂1中检出率更高。
The positivity rate of SARS–CoV–2 was significantly greater than that of other target viruses. While SARS-CoV-2 was detected in all samples collected from both WWTPs, influenza virus and RSV were <50% of the total samples, and more frequently in WWTP1 than WWTP2.
图1 所有呼吸道病毒阳性率。采样期间两个污水处理厂废水中呼吸道病毒分析阳性样品的百分比。
Fig. 1 Positivity rate of all respiratory viruses. Percentage of positive samples for the respiratory viruses analyzed in wastewater of both WWTPs during the sampling camping.
二、废水中粪便污染指标的水平
| Levels of indicators of fecal contamination in wastewater
两个污水处理厂废水样品中大肠杆菌和体细胞噬菌体的平均密度水平相当，且在分析样品间与污水处理厂间无显著差异(p>0.05)。所有分析的微生物指标与呼吸道病毒间没有相关性，同时考虑很多监测数值都低于检出限，这意味着它们的实际相关性更低。
Comparable average concentrations of E. coli and somatic coliphages in both WWTPs wastewater samples, and no significant differences were observed among the analyzed samples and between the WWTPs (p>0.05). The microbial indicators analyzed did not correlate with the respiratory viruses, and considering that many values were below the limit of detection, the real correlation should be lower.
表1 取样期间两个WWTPs大肠杆菌和体细胞噬菌体的平均浓度。
Table 1 Average concentrations of E. coli and somatic coliphages during the sampling campaign for both WWTP.
三、废水中呼吸道病毒的水平
| Respiratory virus levels in wastewater
流感和RSV基因滴度在104–105 GC/L范围内，低于相应样品中SARS–CoV–2基因滴度。SARS–CoV–2滴度在冬季略有增加（第13周至第23周）；IAV在2月底到3月初期间达到一个显著的峰值，且在污水处理厂1中更明显，而IBV仅第55周在污水处理厂1中被检测到一次；在两个污水处理厂样品中，两个明显的RSV子高峰在冬季月份被检测到（第8周至20周和第55周至61周）。
The concentrations of the genes influenza and RSV were in the range of 104–105 GC/L, being consistently lower than those of the SARS–CoV–2 genes in the corresponding samples. SARS–CoV–2 concentration increased slightly during the winter season (Week 13 to 23); IAV reached a prominent peak in late February/early March, more obvious in WWTP1, while IBV was only detected once in week 55 in WWTP1; an obvious peak of detection of both subtypes was apparent during the winter months in samples from both WWTPs (weeks 8 to 20 and 55 to 61).
图2 RSV和流感病毒的滴度。点表示病毒的每个采样点的结果，线表示病毒的样品平滑度。底线表示流感病毒(蓝色)和RSV(红色)的检测限。未检出用1/2检测限替换。
Fig. 2 Viral titers of RSV and influenza virus. Dots show the results of each sampling point for each virus and lines show the spline smoothers for each virus. Bottom line indicates the limit of detection (LOD) of Influenza virus (blue) and RSV (red). Non–detects have been replaced with LOD values/2.
图3 RSV、流感病毒和SARS–CoV–2的比较。热图显示结果中每种病毒的丰度。图例显示了以log 10单位表示的最强力颜色对应的最高值。虚线表示每种病毒的检出限。
Fig. 3 Comparison of RSV, influenza virus and SARS–CoV–2. Heatmap showing the abundance of each virus along the study. The legend shows the highest value corresponding to the most intense color expressed in log10 units. Dotted lines indicated the LOD of each virus.
四、废水监测数据与临床记录对比
| Comparison of WBS data with clinical records
废水监测的病毒浓度和相关疾病或症状发生率模式与临床数据相近。流感病毒和SARS–CoV–2的检出与对应的病例数呈显著正相关，而毛细支气管炎与RSV之间没有相关性，这可能是因为用作标记的临床疾病对RSV感染的特异性较低。另一方面，RSV在废水中的检出率高于报告中的临床病例数，这可能是无症状或症状轻微的患者没有去医疗中心导致的。
Similarity was observed in the patterns of viral concentrations and incidence of associated diseases/symptoms between WBS and clinical data. The detections of influenza viruses and SARS-CoV-2 were significantly related to the number of cases, while no correlation was found between bronchiolitis and RSV, perhaps because the clinical disease used as marker is less specific of RSV infections. Another, RSV were detected at a higher rate in wastewater compared to the number of reported clinical cases, which could be attributed to non-attendance at health care centers of asymptomatic individuals or those with mild symptoms.
图4 污水中呼吸道病毒检测结果及公共卫生数据库报告病例数。取样期间两个WWTPs 中SARS–CoV–2、流感病毒和RSV的检测(左y轴log10 GC/L)。两个污水处理厂覆盖地区的初级保健和医院诊断(左y轴log10 报告病例数)。
Fig. 4 Results of respiratory virus detection in wastewater and of number of cases reported in public health databases. Detection of SARS–CoV–2, influenza virus, and RSV during the sampling campaign in both WWTPs (left Y–axis log10 GC/L). Primary care and hospital diagnostics in the area covered by the two WWTPs (right Y-axis log10 number of reported cases).
总结| Conclusion
本研究为巴塞罗那呼吸道病毒的存在和演变提供了重要的依据，揭示了与COVID–19大流行前不同的流行病学模式。废水监测可以独立于临床记录提供人群中病毒动力学的相关信息，但废水流行病学仍需进一步研究以实现作为传染病爆发确认和预测系统的潜力。
This study has provided significant data on the presence and evolution of respiratory viruses in the city of Barcelona, revealing different epidemiological patterns to those before the COVID–19 pandemic. WBS can provide relevant information about viral dynamics in a population independently of clinical records, and the epidemiological study of wastewater needs further research to realize its potential as a system that can confirm and anticipate outbreaks of infectious diseases.
第一作者：Daniel Toribio-Avedillo
university of barcelona
通讯作者：Maite Muniesa
university of barcelona
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