Estimating the prevalence of hepatitis B by wastewater-based epidemiology in 19 cities in China
Graphical abstract
Introduction
Hepatitis B is the liver cell necrosis and inflammation disease caused by the infection with Hepatitis B virus (HBV). Chronic hepatitis B could be developed into liver cirrhosis and liver cancer and ultimately lead to death (Dehesa-violante and Nuñez-Nateras, 2007; McMahon, 2014; Seto et al., 2018; Liu et al., 2018; Koffas and Kennedy, 2019). China has the world's largest burden of HBV infection, the weighted prevalence of hepatitis B surface antigen (HBsAg) adjusted for people was 7.2% in 2006 according to the data from national surveys in China and was estimated to be 6.1% in 2016 (Razavi-Shearer et al., 2018). At present, there are almost 86 million people carried HBsAg in China, which accounts for approximately 25% of the global total. About 32 million suffered from chronic hepatitis B and needed necessary antiviral treatment (Liang et al., 2009; Wang and Jia, 2011; Wang et al., 2019a, Wang et al., 2019b; Liu et al., 2019). To respond to the global health sector strategy to eliminate viral hepatitis as a public health threat by 2030, monitoring the prevalence of HBV in near real-time is required.
Traditional methods of epidemiological survey including cross-sectional study, cohort study and case-control study are mainly based on questionnaire surveys, national census, previous literature, clinical cases, reports and market research which might cost considerable labor, equipment, expense and time, meanwhile, the results are not in real time and might not be completely objective. To obtain more reliable results, wastewater-based epidemiology (WBE) was considered and applied (Xiao et al., 2019). WBE was firstly proposed by Daughton in 2001 (Daughton, 2001) and has now become an important way in monitoring the abuse of illicit drugs worldwide (Zuccato et al., 2005; Thomas et al., 2012; Ort et al., 2014; Du et al., 2015; Du et al., 2017). Recently, WBE was also increasingly used in other aspects such as estimating the abuse or consumption of tobacco, alcohol, caffeine and some pharmaceuticals including antiviral drugs and antibiotics (Castiglioni et al., 2014; Wang et al., 2016; Santos et al., 2009; Feng et al., 2019a, Feng et al., 2019b; Prasse et al., 2010; Nannou et al., 2020), assessing population size (by measuring certain PPCPs) (Senta et al., 2015; Gao et al., 2016; Rico et al., 2017) and evaluating the health status of inhabitants in certain areas (by estimating some endogenous biomarkers) (Ryu et al., 2016; Choi et al., 2018; Sims et al., 2019). It should be noted that the trend and prevalence of some diseases could also be assessed by WBE. Xiao et al. (2019) recently estimated the trend of diabetes mellitus by analyzing metformin in wastewater in Dalian China and obtained reasonable results.
Lamivudine is a nucleoside analogues anti-HBV/anti-HIV pharmaceutical (The therapeutic compounds lead to synthesis inhibition and chain termination of DNA by mimicking physiological nucleosides or inhibit key enzymes involved in RNA synthesis) (Galmarini et al., 2001). In China, the amount of chronic Hepatitis B patients is much larger than HIV/AIDS patients (32 million versus 0.86 million) (Wang et al., 2019a, Wang et al., 2019b; China HIV Country Profile, 2019). Meanwhile, about 9.5%–15% of the HIV-positive patients were HIV-HBV co-infected (Liu, 2015; Xie et al., 2016; Wu, 2019). Besides, according to the Data-center of China Public Health Science, the incidence of Hepatitis B was around 70/lakh/per year in recent years (72.6137/lakh in 2017) with about 1 million cases per year (1 million in 2017) (Hepatitis, n.d), which was much more than the yearly new HIV infections (around 80,000 in 2018) (Lv and Chen, 2019). Hence, lamivudine is mainly used to treat chronic Hepatitis B in China. Lamivudine is a synthetic drug without natural sources and is detectable and stable in wastewater (Nannou et al., 2020); moreover, approximately 70% of the dose is excreted as unchanged drug in urine after administration (U.S. National Library of Medicine). Thus, according to the selection principals of biomarkers in WBE (Daughton, 2012; Chen et al., 2014), lamivudine was selected as a biomarker to estimate the prevalence of HBV.
This research aimed to estimate the prevalence of HBV by WBE with antiviral drug lamivudine as a biomarker. Sewage samples from 92 sewage treatment plants (STPs) in 19 cities in China were measured to obtain the concentration of lamivudine. Meanwhile, the consumption (mg/day/1000inh) and prevalence of lamivudine were evaluated based on it. After considering various parameters, the prevalence of HBV was ultimately estimated.
Section snippets
Chemicals and reagents
High Performance Liquid Chromatography (HPLC) grade methanol (MeOH) and acetonitrile (ACN) were obtained from TEDIA (Ohio, USA) and formic acid from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Ultrapure water was produced by a water purification machine from Nanjing Miaozhiyi Electronic Technology Co., Ltd. (Nanjing, China). Analytical standards lamivudine was purchased from Yuanye Biotechnology Co. Ltd. (Shanghai, China). Isotopic internal standards lamivudine-13C1, D2 was purchased
Method validation
All method validation results of the LC–MS/MS analytical methods are listed below. Calibration curves prepared in wastewater were all linear at the concentration range 2.5–2500 ng/L with the correlation coefficient (r2) of 0.9957 for lamivudine. The limit of quantification (LOQ) was determined to be 2.0 ng/L based on a signal-to-noise ratio (S/N) of 10 and the limit of detection (LOD) was 0.8 ng/L (S/N = 3). Absolute and Relative recoveries of lamivudine were 114.4% ± 3.2% and 94.25% ± 1.80%,
Conclusion
This research is the first research on estimating HBV prevalence by WBE in China. Lamivudine was used as a biomarker for WBE, and the prevalence of HBV was further back-calculated by the estimated lamivudine consumption. The results were consistent with the prevalence reported in other surveys. This demonstrated that the estimation of HBV prevalence by WBE with lamivudine as biomarker was feasible in big cities in Southern China and might assist the national programme on eliminating HBV
CRediT authorship contribution statement
Chenzhi Hou: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. Zhendong Hua: Writing - review & editing. Peng Xu: Writing - review & editing. Hui Xu: Writing - review & editing, Supervision, Project administration. Youmei Wang: Writing - review & editing, Supervision, Project administration. Jun Liao: Formal analysis, Data curation. Bin Di: Conceptualization, Methodology, Resources, Writing - review & editing,
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This research was funded by Key Projects of National Key Research and Development Plan of China no. 2018YFC0807402.
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