The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir.

1. HEV RNA in Surface Water Samples

From 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figure 1 and Figure 2).

In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of the campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 1a).

In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 1b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 1b).

2. HEV RNA in Drinking Water Samples

From 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figure 1 and Figure 2). 

In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 1a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.

In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 1b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December. 

In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 2), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.

Figure 1. Variation in the concentration of hepatitis E virus (HEV) RNA detected in concentrated water sampled in four sampling sites during 2019. (a) River and WTP_R (n = 34). (b) Dam reservoir and WTP_D (n = 20). RT-qPCR results (average values from two independent reactions), in gc/L, indicate estimated genomic copies per liter of sampled water (based on data from Table 1).

Figure 2. Variation in the concentration of HEV RNA detected in concentrated drinking water from the sampling point in the distribution network during 2019 (n = 9). RT-qPCR results (average values of two independent reactions), in gc/L, indicate estimated genomic copies per liter of sampled water (based on data from Table 1). 

3. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination

3.1. River vs WTP_R

In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).

3.2. Dam Reservoir vs WTP_D

In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).

Table 2. Quantification of HEV RNA in concentrated samples from surface water sources and their associated water treatment plants, and evaluation of the treatment efficacy (reduction in RNA copies).