Water treatment Is complete eradication of pathogen threat possible Control vs' Management

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Emerging Waterborne Infection Contributing
Factors, Agents, and Detection Tools
By Theron and Cloete Presented by Hewitt and
Meador
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Contributing Factors

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

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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

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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

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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

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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

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• 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

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Traditional and nucleic acid based techniques.

• Detection for enteric bacteria

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Culturing Bacterial

• Population density vs. Species richness
• Membrane filtration
• VBNC Resuscitation techniques
• Subculturing differential and selective medias
• Metabolic and phenotypic analysis

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Enzyme immunoassays

• Qualitative and quantitative
• Single tests or batch wells
• Problems cross reactivity may result from
  changes that result from treatment

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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

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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

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Traditional and nucleic acid based techniques

• Detection of protozoa

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Detection

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

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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

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FITC, DAPI, DIC
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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

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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

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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

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Nucleic acid based detection

• PCR, RT-PCR
• Need good extraction techniques
• Antibody-antigen complexes
• Integrated cell culture-RT-PCR, nested PCR

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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

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Emergence

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

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Qualify, quantify, viability and virulence

• Can we relate detection to pathogen infectivity?
• Multiple testing approach
• subculturing, biochemical, metabolic,
  phenotyipic, immunologic, nucleic acid-based

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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

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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

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