Detecting enteral viruses from

Transcription

1 Detecting enteral viruses from water with real time PCR National Institute of Environmental Health, Hungary M. Balazs, M. Vargha, A. Kern 17 th of December 2013 Sorea Regia Hotel, Bratislava, Slovak Republic

2 Presentation Introduction The background of the method development The main steps of the method The factors inluencing the detection Results Conclusions

3 Introduction The demand for clean water and water quality requirements are increasing The consumption of not adequately treated water can cause (usually gastrointestinal) diseases in humans, or even outbreaks. Emerging pathogens in drinking water: enteric viruses, protozoa, novel bacteria High virulence low levels can be infective Usually difficult to detect need to improve Not indicated by fecal indicators Sporadic water related disease case are difficult to identify in surveillance (aspecific symptoms)

4 The background of the method development The process consists three main steps: 1. Virus concentration 2. Nucleic acid extraction 3. Detection by quantitative real-time polymerase chain reaction (qpcr) method Enteric virus concentration and the method was developed in project Virobathe and in project Viroclime funded by EU 3 alternative methods were tested for virus concentration The optimization of nucleic acid extraction was also performed with comparison of two different extraction kits The adjustment of taxon specific PCR methods and sequence analyses to detection of human viruses (HAdV, NoV) from the water concentrates

5 First step: Virus concentration The concentration step is essential for the detection The titer of viruses is too low without this step. Concentration of viruses was performed: By absorption-elution method on two different absorbents: cellulose nitrate membrane filter and glass wool or with direct flocculation glass wool membrane filter direct flocculation

6 The comparison of virus concentration methods Tens of samples were processed by both membraneand by glass wool concentration methods in order to compare the virus binding efficiency Glass wool seemed to be more effective for virus concentration But the most effective, reliable and practicable method is the direct flocculation (less work) A comparison of the absorptionelution and flocculation methods Surface water 10L Treated wastewater 1L Untreated wastewater 40mL Samples Glass wool Direct flocc. untreated /L HAdV untreated /L HAdV35-12 untreated - 12

7 Second step: Nucleic acid extraction Viral nucleic acids were extracted from sample concentrates Two different extraction kits: magnetic silica bead kit NucliSens minimagtm (Biomérieux, France) and QiaAmp Mini Viral RNA QiaAmp proved to be more efficient Silica spin column based nucleic acid extraction from 140 µl concentrate It is used according to manufacturer s instructions. The final volume of the purified nucleic acid was 80 µl. Nucleic acid extract was stored at -20 C until further analysis.

8 Third step: quantitative PCR The quantity of virus genome copies were determined by quantitative real-time PCR (qpcr) Three-step dilution series for conventional PCR (concentrated, 1:10, 1:100) Our qpcr methods are set to detect For example: Human adenoviruses (dsdna virus) Caliciviridae (+ssrna virus) for 2 human genus: Norovirus, Picornaviridae (+ssrna virus) - Enteroviruses (EV) Porcine adenovirus (PAdV), Bovine Polyomavirus (BPdV) JC polyomavirus (JCV), Hepatitis A virus The detection method based on the presence of the viral nucleic acid a positive test does not mean that the detected virus is infectious!

9 Factors influencing the detection The concentration method (better efficiency) Suspended solids sediment during centrifugation may hinder nucleic acid extraction Certain materials in the water (such as metal ions or organic acids, detergents) can be concentrated during the process and can interfere with the molecular detection through the inhibition of the polymerase enzyme Samples are analyzed in more dilutions (usually 3) to overcome the effect

10 The complete method Approx. 10 L surface water 50 ml sewage Adjusting to ph 3.5 direct flocculation centrifuging Purification of nucleic acid Determination of amount of virus with virus-specific qpcr

11 Results I: Presence of human viruses in surface water samples Humán vírusok előfordulási gyakorisága a felszíni vízmintákban 100% 80% 60% 40% 20% 0% Humán adenovírus Norovírus GII Norovírus GII Szolnok Vízmű Szolnok Szennyvízbefolyó után Vezseny Kompátkelő Tiszakécske Szabadstrand Positive sampless %

12 Results II: Virus genome copy numbers in wastewater samples. 1,00E+08 lg gen nom copies/l 1,00E+07 1,00E+06 1,00E+05 1,00E+04 1,00E+03 Adenovírus Norovírus GI Norovírus GII 1,00E+02 1,00E+01 1,00E+00 Szolnok, Tisztított szennyvíz Szolnok, Nyers szennyvíz Sampling sites

13 Results III: Calicivirus outbreak, Miskolc (June 2010) 3673 reported patients, infection through the consumption of contaminated drinking water 3 of the 6 samples were source water (raw water) and positive for adenovirus 1 of them was from the water network and was also positive for calicivirus 3 source water (raw water) samples and two mains drinking water samples were positive for adenovirus The presence of virus was confirmed As a result of high rainfall in this karstic ground the surface and drinking waters were infected by viruses

14 Summary - Conclusions Water is the main transport medium for many human pathogens: bacteria, protozoa and enteric viruses. Studies also confirm, that human pathogen viruses are present in water bodies. Virus detection is not a part of the water quality monitoring scheme, mainly due to the absence of routine analysis methods This elaborated method is applicable for the detection of several viruses from different water bodies: Drinking water, pool water Surface water (including Seawater) Sewage water Therefore it is adequate for the detection of outbreaks which are often with unknown etiology

15 Thank you for your attention For further information please contact Anita Kern, Mihaly Kadar, Katalin Szomor, György Berencsi, Beatrix Kapusinszky and Marta Vargha 2013 Detection of enteric viruses in Hungarian surface waters: first steps towards environmental surveillance Journal of Water and Health