Molecular detection of human adenovirus in urban wastewater in Egypt and among children suffering from acute gastroenteritis

Incidence of enteric viruses in sewage, the efficacy of wastewater treatment plants to remove these viruses, and health effects from their release into the surface water are very important environmental issues in the microbiology field. One of the most pathogenic enteric viruses is adenovirus which can cause a serious disease such as gastroenteritis with low grade fever and mild dehydration in humans. In this study we performed qualitative polymerase chain reaction (PCR) analysis of HAdV on 60 stool samples from children with acute gastroenteritis admitted to Abu-Rish hospital and 96 environmental samples (32 raw sewage, 32 treated sewage, 32 sewage sludge) collected from Zenin wastewater treatment plant (WWTP). HAdV were detected in 17 (28.3%) of stool, 27 (84.4%) of raw sewage, 16 (50%) of treated sewage and 25 (78%) of sludge samples. The viral concentrations were in the range of 2.02 × 10–7.23 × 10, 8.7 × 10–4.3 × 10, 1.22 × 10–3.7 × 10 and 1.48 × 10–1.77 × 10 GC/mL in stool, raw sewage, treated sewage, and sludge, respectively. HAdV was detected throughout the whole year of sample collection. Moreover, our results suggested that males were more susceptible to adenovirus infections than females. The results indicate that the high incidence of HAdV in the treated sewage may cause adverse health effects. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2019.303 s://iwaponline.com/jwh/article-pdf/17/2/287/623563/jwh0170287.pdf Elmahdy M. Elmahdy (corresponding author) Nehal I. Ahmed Mohamed N. F. Shaheen Environmental Virology Laboratory, Department of Water Pollution Research, Environmental Research Division, National Research Center, Dokki, Giza, Egypt E-mail: mahdynrc@yahoo.com EL-Chaimaa B. Mohamed Samah A. Loutfy Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.


INTRODUCTION
Many HAdV types can be shed in high concentrations (ranging from 10 5 to 10 13 viral particles per gram of stool) in the stool of an infected person for months (Wold & Horwitz ). Fecal indicator bacteria are commonly used for water quality measurements, however they can fail to predict the presence of other pathogens such as human enteric viruses that are more environmentally tolerant than the fecal indicator bacteria (Hewitt et al. ). HAdV are more tolerant to chemical/physical parameters and UV irradiation than other water-borne pathogens such as enteric viruses and fecal indicator bacteria (Nwachuku et al. ). Many studies have been conducted to investigate HAdV incidence in clinical and environmental samples, but few have simultaneously studied the communal level incidence of HAdV in fecal, raw wastewater, treated wastewater and sludge samples, which represent different nodes in the fecal-oral route of transmission of enteric pathogens. However, it should be emphasized that this study is not focused on source tracking of HAdV in the fecal and environmental samples. In this study, HAdV was detected in clinical stool samples and wastewaters by conventional polymerase chain reaction (PCR) and then the viral concentrations were determined in some positive samples by real time RT-PCR.

Wastewater and sludge samples
During the period January-December 2017, a total of 64 sewage samples (32 of raw sewage and 32 of treated sewage) and 32 sewage sludge samples from a secondary treatment step were collected from Zenin wastewater treatment plant (WWTP) to cover the seasonality of HAdV occurrence in wastewater and sludge. Eight samples from each study site were collected per season. The capacity of this wastewater treatment plant is 330,000 m 3 /day and the finally treated wastewater is discharged into the Nahia effluent system and then to the River Nile.

Clinical samples
In 2017, a total of 60 stool samples were collected from children suffering gastroenteritis who were admitted to Abulreesh hospital (Sayeda Zeinab, Cairo Governorate). Samples were collected in clean containers then transferred to the laboratory within 3 hours of collection for viral examination.

Concentration of sewage samples
Sewage samples (raw and treated wastewater) were concentrated according to previously published protocol (USEPA ) yielding a mean recovery efficiency of 75%. Briefly, 2.5 mL of 1 M magnesium chloride (Merck-Schuchardt, Germany) was added to a 1 L sample to increase the stability of the viruses during the transportation process (APHA ).
The samples were adjusted to pH 3.5 by 1 N HCL, and then concentrated by filtration using a nitrocellulose membrane filter (0.45 μm pore size, and 142 mm in diameter) on a stainless-steel filter holder. The adsorbed viruses on membrane filter were eluted in 100 mL of 3% beef extract-0.05 M glycine solution, pH 9.5. All samples were reconcentrated using an organic flocculation method (USEPA ). Finally, these samples were stored at À20 C until used.

Concentration of sludge samples
One hundred grams of sludge was dissolved in 300 mL of (10% beef extract, 1.34% Na 2 HPO 4 .7H 2 O and 12% citric acid). The mixture was stirred for 30 min at pH 7 and then centrifuged at 4,500 rpm for 15 min. The supernatant was adjusted to pH 3.5, stirred for 30 min and centrifuged at 4,500 rpm for 15 min, finally the obtained pellet was dissolved in 0.15 M Na 2 HPO 4 and stored at -20 C until analysis (USEPA ).

Concentration of clinical stool samples
Approximately 0.2 g of stool samples were weighed, diluted by 1 mL PBS (20% wt/v), and vortexed for 30 s. Samples were clarified by centrifugation at 7,000 rpm, 4 C for 10 min, and then the supernatant was taken and archived at -80 C until use. The HAdV thermal cycling protocol consisted of four steps: step 1: 95 C for 10 min for DNA denaturation; step 2: three temperature cycles, repeated 40 times, at 95 C for 30 s, 50 C for 30 s, and 72 C for 30 s; step 3: final extension at 72 C for 7 min; step 4: melting curve 95 C for 15 s, 60 C for 1 min, and 95 C for 15 s and finally cooling at 4 C forever. The genome copies for adenovirus was determined by comparison with a standard curve constructed using tenfold serial dilution (10 -1 to 10 -6 copies/mL) by cloning the PCR amplicon into a plasmid (pBR22 Invitrogen USA).

Physicochemical characteristics of raw treated wastewater
The physico-chemical parameters such as pH, total chemical oxygen demand (COD tot ), soluble chemical oxygen demand (COD sol ), biochemical oxygen demand (BOD tot ), total suspended solids (TSS), total Kjeldahl nitrogen (TKN), ammonia, nitrite-nitrogen (NO 2 -N), nitrate-nitrogen (NO 3 -N), and total phosphorus (TP) of the sewage samples were analyzed. All these parameters were characterized using standard methods (APHA ).

Statistical analyses
To evaluate the possible correlation between viral detection in samples from each point of collection, a Pearson correlation and linear regression test were performed using GraphPad Prism 5.0 (USA); data were considered statistically significant at P 0.05.

Seasonal variation of HAdV in sewage
We analyzed the occurrence rates of HAdV in sewage samples throughout the year of sample collection. A total of 16 sewage samples (eight raw sewage and eight treated effluents) were collected each season. HAdV was detected throughout the whole year with a peak in winter 100% (16/16), followed by autumn 68.7% (11/16), spring 62.5% (10/16), and summer 37.5% (6/16), as shown in Figure 3.

DISCUSSION
Diarrheal illnesses remain a major public health concern and represent the third leading cause of death, globally. In Phosphorus mg/L 1.9 ± 0.7 1.1 ± 0.5  In the present study, we studied the incidence of HAdV have been conducted to investigate the incidence of HAdV in clinical and community water samples simultaneously.
In the present study, HAdV was detected in 67% (43/ of Tyume River in South Africa with a viral load ranging between 6.54 × 10 3 and 8.49 × 10 4 GC/L (Sibanda & Okoh ). This was lower than our result because of the highest viral load are known to be present in wastewater and stool.
In another study, HAdV was detected in final effluent of wastewater samples at concentrations ranged from 1.10 × 10 4 to 2.37 × 10 5 GC/L (Osuolale & Okoh ) but our results showed that the viral load of HAdV in the treated effluent ranged from 1.22 × 10 4 to 3.7 × 10 6 GC/ml, such a difference could be attributed to the efficiency of the wastewater treatment plant.
In conclusion, the presence of HAdV in final treated effluent is a huge public health concern. The levels of viral contamination in treated effluent and sewage sludge of the WWTP must be considered by the regulatory and local authorities for improving the efficiency of conventional WWTP for the removal of pathogens.