ENVIRONMENTAL BIOTECHNOLOGY Efficiency of hepatitis A virus removal in six sewage treatment plants from central Tunisia Imen Ouardani 1,2 & Carmen F. Manso 2,3 & Mahjoub Aouni 1 & Jesús L. Romalde 2 Received: 26 May 2015 /Revised: 27 July 2015 /Accepted: 30 July 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract The efficiency of six Tunisian sewage treatment plants (STP) for the removal of hepatitis A virus (HAV) from wastewater was analysed in order to evaluate the potential risk for human health linked to reuse or discharge of treated waste- water into the environment. The STP utilize different biolog- ical wastewater treatments including primary treatment, which involves the physical removal of organic and inorganic solids, and secondary treatment that involves different processes, such as activated sludge or lagoon. Quantitative real-time re- verse transcription polymerase chain reaction (RT-PCR) and conventional RT-PCR were used for the analysis of the 325 wastewater samples (163 raw and 162 treated) obtained. Results revealed highest contamination in west-central of Tunisia in raw wastewater with 62.96 % of samples positive for HAV and predominance during winter and autumn, where- as east-central region showed 50.62 % of positive samples with high prevalence from winter through summer. The quan- titative analysis revealed a range between 4.29 × 10 1 and 1.24 × 10 5 RNA copies/mL in treated wastewater, showing clearly the inefficiency for total removal of HAV regardless of the treatment method used. The vast majority of HAV se- quences belonged to the sub-genotype IA, except one that was assigned to sub-genotype IB. Keywords Hepatitis A . Wastewater . Sewage treatment plants . qRT-PCR . Genotyping Introduction Infections by human enteric viruses, including viral hepatitis, represent an important human health problem worldwide. Hepatitis A virus (HAV), classified within the Hepatovirus genus of the Picornaviridae family, is the most significant pathogen associated with waterborne and foodborne out- breaks of hepatitis in humans worldwide. It is a positive single-stranded RNA non-enveloped virus, and its genome consists of a 7.5-kilobase (kb) RNA molecule coding for a single open reading frame (Pintó et al. 2012). The virion cap- sid is composed of the structural proteins VP1, VP2, VP3, and possibly VP4, encoded in the P1 region of the genome (Costafreda et al. 2012; Hollinger and Emerson 2007). HAV have been classified into four human (I, II, III and VII) and three simian (IV, V and VI) genotypes (Costa-Mattioli et al. 2006). Genotypes I and III have been further divided into sub- genotypes A and B (Byun et al. 2001). Viral contamination of wastewater has been frequently re- ported as a primary source of hepatitis outbreaks. Considering the low infectious dose of this virus, only a small amount present in the contaminated water can be sufficient to infect a human host. The non-enveloped HAV particles are stable, especially when associated with organic matter, which signif- icantly contribute to its resistance to low pH and temperature and its environmental persistence for extended periods Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6902-9) contains supplementary material, which is available to authorized users. * Jesús L. Romalde jesus.romalde@usc.es 1 Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99ES27, Faculté de Pharmacie de Monastir, Université de Monastir, Avenue Avicenne, 5000 Monastir, Tunisia 2 Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain 3 Present address: Public Health England, London, UK Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6902-9