Evaluation of methods used to concentrate and detect hepatitis A virus in water samples

Villar, Lívia Melo; Paula, Vanessa Salete de; Diniz-Mendes, Leonardo; Lampe, Elisabeth; Gaspar, Ana

doi:10.1016/j.jviromet.2006.06.008

Cited by 46 publications

(47 citation statements)

References 32 publications

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“…Hence, we compared the performance of three relatively simple virus concentration methods to determine their efficiency in recovering two viral MST markers (HAdVs and HPyVs) in tap and river water samples spiked with raw sewage. The strategy of spiking with sewage rather than with cultured viruses (27,41,42) was used to better mimic a natural scenario that includes viruses in various states of intactness and disruption. Little has been published on the recovery efficiency of these MST viral markers through the processes of concentration, nucleic acid extraction, and purification.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Ahmed

Harwood

Gyawali

et al. 2015

Appl Environ Microbiol

126

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dPathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl 2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ϳ10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg 2؉) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters. Discharges from sewage treatment plants (STPs), storm water drains, improperly designed septic systems, and fecal contamination from livestock and wildlife are known to degrade environmental water quality in terms of elevating fecal indicator bacteria and possibly pathogen concentrations (1-5). Fecal indicator bacteria such as Escherichia coli and Enterococcus spp. have been widely used as an indirect measure of microbial risk associated with environmental waters. However, identifying the health risks associated with enteric viruses and protozoa by monitoring fecal indicator bacteria has been questioned (5-8).The risk of infectious disease associated with recreational water use may well be of viral etiology (9). The transmission of these viruses occurs via the fecal-oral route, nasal mucosa or the conjunctiva and the infected individual may shed up to 10 11 viral particles/gram of feces (10). Some of these viruses are more res...

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Section: Discussionmentioning

confidence: 99%

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Ahmed

Harwood

Gyawali

et al. 2015

Appl Environ Microbiol

126

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“…The real-time PCR technique is an efficient tool in detecting HAV in environmental samples because it combines PCR amplification with the use of a probe to confirm the identity of the PCR product (De Paula et al, 2007). Results of a study carried out by Villar et al (2006) to evaluate methods used to concentrate and detect HAV in water samples also confirmed that compared to qualitative PCR, real-time PCR detects low concentrations of genome per millilitre and is more suitable than qualitative PCR for the detection of HAV RNA in environmental samples.…”

Section: Discussionmentioning

confidence: 70%

Real-time PCR quantitative assessment of hepatitis A virus, rotaviruses and enteroviruses in the Tyume River located in the Eastern Cape Province, South Africa

Sibanda¹,

Okoh²

2013

WSA

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We applied real-time RT-PCR (reverse transcription-polymerase chain reaction) to assess the incidence of hepatitis A virus, rotaviruses and enteroviruses in the Tyume River, an important water resource in the impoverished Eastern Cape Province of South Africa. Detection of noroviruses was done using conventional semi-nested RT-PCR. Water samples were collected once monthly from 6 sampling sites over a 12-month period starting in August 2010 and ending in July 2011. Hepatitis A virus was detected in 13% of the samples in concentrations ranging between 1.67×10 3 genome copies/ℓ and 1.64×10 4 genome copies/ℓ while rotaviruses were detected in 4% of the samples with concentrations ranging from 9×10 1 genome copies/ℓ to 5.64×10 3 genome copies/ℓ. Enteroviruses were not detected in any of the samples, while noroviruses were detected in 4% of the samples. All hepatitis A and rotaviruses positive samples were from the upstream sections of Tyume River while noroviruses were detected in samples from downstream sections only. Statistical analysis showed that occurrence of the viruses in Tyume River was sporadic. Risk analysis showed that hepatitis A virus posed greater risk than rotaviruses for both recreational and domestic water uses. Because of the low infectious dose of enteric viruses, the detection of even low concentrations of hepatitis A virus, rotaviruses and noroviruses in surface water poses a significant risk to public health.

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“…La detección de partículas virales en las muestras ambientales depende en gran medida de la eficiencia de la técnica utilizada para establecer la concentración de partículas virales, como son la floculación con leche descremada, la ultrafiltración, la liofilización y la ultracentrifugación, entre otras (33)(34)(35)(37)(38)(39)47). En el 2013, Calgua, et al (38), publicaron un estudio en el que se registraron diferencias en el rendimiento de la técnica al medir el porcentaje de recuperación viral (38,47).…”

Section: Discussionunclassified

“…En el 2013, Calgua, et al (38), publicaron un estudio en el que se registraron diferencias en el rendimiento de la técnica al medir el porcentaje de recuperación viral (38,47). La técnica de floculación con leche descremada demostró ser más eficiente y conveniente en cuanto a su costo-efectividad y el tiempo de procesamiento de la muestra (38).…”

Section: Discussionunclassified

Evidencia de circulación del virus de la hepatitis A, subgenotipo IA, en muestras ambientales en Antioquia, Colombia

et al. 2016

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Introducción. El virus de la hepatitis A (HAV) es un importante patógeno que se transmite por vía fecal-oral. La epidemiología de la infección está directamente relacionada con el acceso de la población al agua potable y con la infraestructura de alcantarillado. Objetivo. Determinar la presencia del HAV e identificar el genotipo en muestras de agua de abastecimiento y agua residual en ocho municipios, un corregimiento y una vereda del departamento de Antioquia, noroccidente de Colombia. Materiales y métodos. Se hicieron tres muestreos seriados de diciembre de 2012 a abril de 2014 en la fuente principal de abastecimiento de los acueductos y en el principal vertimiento de aguas residuales de cada municipio. Las muestras se concentraron por filtración y ultrafiltración tangencial, y por las técnicas de polietilenglicol y floculación con leche descremada, respectivamente. A partir del ARN total de cada muestra, se amplificaron la región VP3-VP1 para la detección del genoma viral y la región VP1-2B para la genotipificación. Resultados. El genoma del HAV se detectó en las fuentes de agua de abastecimiento de Puerto Berrío, Frontino y Nutibara, y en las muestras de aguas residuales provenientes de los municipios de Arboletes, Zaragoza y Venecia. Mediante el análisis de las secuencias se identificó el subgenotipo IA del virus. Conclusión. Este estudio permitió detectar la presencia del HAV en 6,6 % de las muestras de agua de abastecimiento y en 13,3 % de las muestras de agua residual de los municipios en estudio. Se reporta por primera vez la circulación del subgenotipo IA en muestras ambientales en Antioquia.

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Evaluation of methods used to concentrate and detect hepatitis A virus in water samples

Cited by 46 publications

References 32 publications

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Real-time PCR quantitative assessment of hepatitis A virus, rotaviruses and enteroviruses in the Tyume River located in the Eastern Cape Province, South Africa

Evidencia de circulación del virus de la hepatitis A, subgenotipo IA, en muestras ambientales en Antioquia, Colombia

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