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Contributing Factors. Increased numbers of immunocompromised peoplePeople in institutional settingsRural urbanizationInadequate sanitation and detection Antibiotic resistanceChange in agricultural practices. Breakdown of Public Health Measures. Pathogens remain in reservoir hosts, the environme
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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