Adenovirus and rotavirus recovery from a treated effluent through an optimized skimmed-milk flocculation method.

Sewage treatment may be insufficient for the complete removal of enteric viruses, such as human adenoviruses (HAdV) and group A rotavirus (RVA). The differences in the efficiency of the treatment methodologies used may interfere with the detection of these viruses. The objective of this study was to optimize a skimmed-milk flocculation technique for the recovery of HAdV and RVA in the samples of treated effluent. The treated effluent collected at the wastewater treatment plant (WWTP) was processed via four protocols including modifications in the initial centrifugation step and the final concentration of skimmed-milk. The viral load and recovery rate were determined by quantitative PCR TaqMan® System. The highest recovery rates of HAdV, RVA, and bacteriophage PP7 (internal control process) were obtained when the concentration of skimmed-milk was doubled and no centrifugation step was used for the sample clarification. The optimized protocol was assessed in a field study conducted with 24 treated effluent samples collected bi-monthly during 2015. HAdV and RVA were detected in 50.0% (12/24) and 33.3% (08/24) of the samples tested, respectively, throughout the year, without seasonal variation (p > 0.05). This study corroborates the use of the organic flocculation method for virus recovery in environmental samples with the adaptation of the protocols to different aquatic matrices.

Optimization of the skimmed-milk flocculation method for recovery of adenovirus from sludge.

Return of treated sludge to the environment poses concerns and has stimulated the development of studies on viral monitoring in this matrix, in order to assess its potential risks for public health. Human adenovirus (HAdV) has been identified as a putative viral marker of faecal contamination due to its stability and resistance to the sewage treatment process. The aim of this study was to optimize the organic flocculation procedure in order to establish an appropriate methodology for HAdV recovery from sewage sludge samples. Four protocols (A-D) have been proposed, with changes in the initial sample dilution, in the stirring time and in the final concentration of skimmed-milk. A single sludge sample was obtained in Wastewater Treatment Plant (WWTP) and divided into aliquots. In each protocol, three aliquots were inoculated with HAdV and bacteriophage PP7 and a non-inoculated one was used as negative control. Viral load and recovery rate were determined by quantitative PCR. HAdV recovery rate varied between the protocols tested (p=0.016) and the best result was obtained through the protocol C. In order to confirm this result a field study with activated, thickened and digested sludge samples was carried out. Different types of sludge were obtained in two WWTPs and processed using protocol C. HAdV was detected in all samples, with a similar or higher viral load than those obtained with other concentration techniques already applied to sludge. Protocol C proved to be really efficient, with the advantage of showing low cost and practicability in routine laboratories.

The role of human adenoviruses type 41 in acute diarrheal disease in Minas Gerais after rotavirus vaccination.

Human adenovirus species F (HAdV-F) type 40 and 41 are commonly associated with acute diarrheal disease (ADD) across the world. Despite being the largest state in southeastern Brazil and having the second largest number of inhabitants, there is no information in the State of Minas Gerais regarding the role of HAdV-F in the etiology of ADD. This study was performed to determine the prevalence, to verify the epidemiological aspects of infection, and to characterize the strains of human adenoviruses (HAdV) detected. A total of 377 diarrheal fecal samples were obtained between January 2007 and August 2011 from inpatient and outpatient children of age ranging from 0 to 12 years. All samples were previously tested for rotavirus, norovirus, and astrovirus, and 314 of 377 were negative. The viral DNA was extracted, amplified using the polymerase chain reaction and the HAdV-positive samples were sequenced and phylogenetically analyzed. Statistical analyses were performed using the Chi-square test (p<0.05), considering two conditions: the total of samples tested (377) and the total of negative samples for the remaining viruses tested (314). The overall prevalence of HAdV was 12.47% (47/377); and in 76.60% (36/47) of the positive samples, this virus was the only infectious agent detected. The phylogenetic analysis of partial sequences of 32 positive samples revealed that they all clustered with the HAdV-F type 41. The statistical analysis showed that there was no correlation between the onset of the HAdV infection and the origin of the samples (inpatients or outpatients) in the two conditions tested: the total of samples tested (p=0.598) and the total of negative samples for the remaining viruses tested (p=0.614). There was a significant association in the occurrence of infection in children aged 0-12 months for the condition 1 (p=0.030) as well as condition 2 (p=0.019). The occurrence of infections due to HAdV did not coincide with a pattern of seasonal distribution. These data indicate the significant involvement of HAdV-F type 41 in the etiology of ADD in Minas Gerais, which demonstrates the importance of other viral agents in the development of the disease after the introduction of rotavirus vaccine immunization.

Relationship between viral detection and turbidity in a watershed contaminated with group A rotavirus.

Enteric viruses are present in aquatic environments due to contamination by raw sewage, even in the absence of fecal coliforms, which are considered to be significant indicators when it comes to microbial water quality assessment. This study investigated the presence of group A rotavirus (RVA) in surface water from a river basin in Minas Gerais, Brazil, assessing the influence of the urbanization, the rainfall, and the microbiological and physico-chemical parameters regarding water quality. Forty-eight surface water samples collected in urbanized and non-urbanized areas, both in dry and rainy periods, were obtained throughout the study. The viral particles were concentrated by adsorption-elution in a negatively charged membrane. Fecal coliforms, as well as physico-chemical water parameters were determined at each point in all collections. The RVA was detected in 62.5 % (30/48) of the water samples through a real-time PCR assay. All the sequenced RVA strains belonged to genotype I1. The RVA was detected in 50.0 % (11/22) of the water samples regarded as being acceptable by current microbiological standards. The presence of the RVA and the viral load were influenced by the collection area (p < 0.05). It was also observed a significant association between the RVA and detecting the turbidity of water (p < 0.05). The collected data showed a high level of contamination in this watershed by the discharge of raw sewage, highlighting the need for urgent measures to improve water quality, ensuring the safe use of this matrix.