Emerging Waterborne Infection: Contributing Factors, Agents, and Detection Tools

2. Contributing Factors Increased numbers of immunocompromised people People in institutional settings Rural urbanization Inadequate sanitation and detection Antibiotic resistance Change in agricultural practices

3. Breakdown of Public Health Measures Pathogens remain in reservoir hosts, the environment or in small pockets of infection leaving them to take advantage of breakdowns in preventative measures. Presence of pathogens in water is only evident when a large number of people become ill and methods of routine monitoring are lacking. Largest U.S. waterborne disease outbreak was in Milwaukee in 1993 Over 400,000 affected and hundreds killed Due to nonfunctioning filtration plant and lack of monitoring

4. Water treatment management Filtration- microsporidia too small Flocculation- reduces bioloads, however, contaminants and biofilms often persist Disinfection- insufficient contact time, resistant cyst, oocyst and microsporidia Remediation of wastes using microbes Detection recognize threat level to utilize efficient sanitizing options

5. Challenges to detection Stressed VBNC- depth & duration: EHEC is suspect in water but not detected Cyst, coccoid- H. Pylori not isolated with traditional methods Indicator species show no correlation with level of infection Many viruses have no detection method Other debris present in water

6. Pathogenic Bacteria Helicobacter pylori VBNC allows H. pylori to survive in sterile distilled water for up to two weeks Enterohemoragic Escherichia coli Methodological problems prevent EHEC from being isolated in drinking water but there is evidence of infection via recreational water, well water, public water and pools Campylobacter presence does not correlate with level of fecal contamination and coliform tests fail VBNC

7. Aeromonas Biofilms protected from drinking water Mycobacterium Isolated from all parts of drinking water facilities Several species are opportunistic pathogens M. avium is common in patients with HIV likely transmitted in water Yersinia enterocolitica Although few outbreaks reported, the method of infection in usually unknown

8. Traditional and nucleic acid based techniques.  Detection for enteric bacteria

9. Culturing: Bacterial Population density vs. Species richness Membrane filtration VBNC Resuscitation techniques Subculturing: differential and selective medias Metabolic and phenotypic analysis

10. Enzyme immunoassays Qualitative and quantitative Single tests or batch wells Problems: cross reactivity may result from changes that result from treatment

11. Nucleic acid techniques Gene probes Immunomagnetic beads for capture, concentration and purification PCR amplification- popular, rapid and sensitive. FISH- generally need to be unstressed, low bioload, suspended solid interference Benefits- Can detect VBNC. Indirect enrichment dilutes dead cells and inhibitors

12. Parasitic Protozoa Cryptosporidium Most common drinking water contaminant 1987 in Carrollton, GA 13,000 cases and 1993 in Milwaukee, WI 403,000 Cysts resist chlorination and filtering reduces by 2 to 3 orders of magnitude Infectious dose is between 10-100 cysts Giardia presents same problems Microsporidia survives filtration Cyclospora indentified in 1990

13. Traditional and nucleic acid based techniques Detection of protozoa

14. Detection Filtration, membrane filtration (bacteria)-immuno-concentration, Culturing: enrichment method, differential and selective media (bacteria) Enzyme Assaying PCR Probe hybridization

15. Filtration and microscopy for Protozoa Cellulose acetate filters Microscopy-direct & indirect Percol sucrose Immunoflourescent staining examined under UV microscope, recorded by size and shape. Label monoclonal antibodies CaCO3 precipitation ELISA

16. FITC, DAPI, DIC

18. Nucleic acid based Detect low levels in large volumes PCR, multiplex can simultaneously detect Cryptosporidium and Giardia Restriction Fragment Length Polymorphism (RFLP) Limitations: Enzyme inhibition, quantification, viability excitation RT-PCR assays for viability based on enzyme expression Immunomagnetic separation,cell culture and PCR

19. Viruses Under reporting of outbreaks and limited detection has resulted in severe underestimations in viral importance Infectious risk 10 to 10,000 times higher than bacteria Norwalk and rotaviruses isolated in chlorinated drinking water and in biofilms No method of detection for pesti-, corona-, toro- or picobirnaviruses and little information about aquatic survival

20. Viruses Microporous filter, beef extract eluent Precipitation with propylene glycol (PEG) Culture – Most probable endpoints and plaques Handling methods determine success ELISA and nucleic acid techniques

21. Nucleic acid –based detection PCR, RT-PCR Need good extraction techniques Antibody-antigen complexes Integrated cell culture-RT-PCR, nested PCR

22. Detecting a threat: Signs and symptoms More than 50% of waterborne outbreaks remain undetected Pathogen presence is usually identified after outbreak Recognize risk of equipment failures Employ monitoring surveillance and diagnostics

23. Emergence Changes in who needs clean water Agriculture: Livestock, plants, aquaculture, etc.. Pathogen survival potentials Contamination from wildlife immigration and trade Virulence expression

24. Qualify, quantify, viability and virulence Can we relate detection to pathogen infectivity? Multiple testing approach: subculturing, biochemical, metabolic, phenotyipic, immunologic, nucleic acid-based

25. Combining techniques offers quantity and viability Flourescent antibody with tetrazolium dye reduction (Presence, enumeration and viability) PCR with RFLP can distinguish between Cryptosporidium oocycsts Immunomagnetic capturing, separation with PCR and hybridization. Culture prior to to PCR detection

26. Treatment and detection Value of detection and cost: benefit analysis Relate detection with infectivity potential, while making it possible to detect, culture, amplify. Stress, detect, culture amplify… Effected by where, how, when you sample Noncultivable pathogens

27. Water treatmentIs complete eradication of pathogen threat sustainable?Control vs. Management