Providing Safe Drinking water

USGS Workshop
Bangkok

Muhammad Naseem Khan

Scientific Officer
PCSIR
Road Map
• Background
• Methods
• WQMP overview
 Pathogens in Water and
Public Health Microbiology
Important concepts
• Viruses, bacteria, and protozoa are
very different types of organisms.
• Many fecal-oral pathogens that are
important to public health are
waterborne.
• Non-pathogenic bacteria are used
to test for the presence of
pathogenic viruses, bacteria, and
protozoa .
 Waterborne disease
• Caused by drinking or accidentally ingesting
water contaminated with pathogens.
• World-wide: 4 billion episodes of diarrhea
result in about 1.5 million deaths each year,
mostly children. Waterborne bacterial
infections account for approximately half of
these episodes and deaths.
 Waterborne pathogens—a
public health concern
• Potential sources of pathogens: • Pathogens ingested from:
– Human, animal wastes, – drinking water,
– sewage and sewage sludge, – recreational water,
– septic tanks, – contaminated fish or shellfish.
– landfills
 Types of waterborne
pathogens

Viruses Protozoa
(tiny, non- (larger,
living) complex cells)
Bacteria
(medium size,
Simple cells)
 Drinking-water outbreaks in the US
in 2005 and 2006—Cause of acute
gastrointestinal illnesses
Surface
Water

Mixed
Unident.

Viral

Parasitic
Ground
Bacterial Water

Etiologic Agent Source

Source and cause of
gastroenteritis associated with
recreational water, 1997 – 2006
Factors affecting incidence of
waterborne Illness in the US
• Increasingly greater threat to public
health
– Increase in population
– Aging water-treatment systems
– Aging population
– Mismanagement of animal wastes
Waterborne Diseases of
Specific Concern
• Afghanistan
– bacterial and protozoal diarrhea, hepatitis A,
and typhoid fever
• Pakistan
– bacterial diarrhea, hepatitis A and E, and
typhoid fever
• Thailand
– bacterial diarrhea and hepatitis A
• Similar to U.S., but at higher rates, with
more mortality in young
Factors affecting incidence of
waterborne Illness in
developing nations
• Consistent threat to public health
– Poor sanitation
– Inadequate or lacking infrastructure
• Especially in rural or dispersed populations
– Mismanagement of animal wastes from
agriculture
• Similar to U.S.
 Viruses
• DNA or RNA with a protein coat
• No nucleus
• Cannot metabolize or reproduce without a host
• 0.02-0.3 micron

Photo Credit: Maria-Lucia Rácz Rotavirus - 0.07 µm

 Fecal Viral Pathogens—
Examples of Illnesses
Pathogen Disease or symptom
Interferes with liver function, fever, jaundice,
Hepatitis A & E
diarrhea
Adenoviruses Respiratory & eye infections, diarrhea
acute-onset vomiting, watery non-bloody
Norovirus
diarrhea with abdominal cramps
Echoviruses Infants:Liver failure, myocarditis, menegitis
Polio, ranging from gastroenteritis to
Poliovirus
irreversible paralysis
Reovirus Gastroenteritis
Most common cause of severe diarrhea in
Rotavirus
small children
Bacteria
• Independently metabolizing and reproducing
organisms. Bacteria
• No true nucleus E. coli - 4 µm long
• Generally 0.2 to 5 micron
 Bacteria—Examples of
Illnesses
Pathogen
Salmonella typhi and sp.
Shigella sp.
Escherichia coli O157:H7
Campylobacter jejuni and sp.
Legionella pneumoniae
Helicobacter pylori
Vibrio cholerae and sp.
Yersinia spp.
Aeromonas hydrophila
Disease or Symptom
Typhoid fever
Shigellosis: dysentery
can lead to kidney failure
gastroenteritis
fever, pneumonia
Gastritis, ulcers
cholera
Yersiniosis: diarrhea
pneumonia
 Protozoa
• Unicellular members of
the Animal kingdom
• true nucleus
• 2 to 200 microns

Giardia lamblia -- 12 to 15 µm
 Protozoa—Examples of Illnesses
Pathogen Disease or Symptom
Cryptosporidium parvum Cryptosporidiosis, diarrhea

Giardia lamblia Diarrhea

Blindness or brain damage in
Toxoplasma gondii
children infected in-utero

Microsporidia Acute diarrhea (esp. those with HIV)

Cyclospora Persistent diarrhea

Primary amebic meningo-

Naegleria fowleri encephalitis (headache, fever, and
vomiting)
Summary
• Similar diseases world wide in developing
and developed nations
– Difference is in intensity and water use
– Due to:
• Infrastructure differences
• Population age distribution differences
• Health Care differences
• All nations can potentially benefit from
having well designed and implemented
microbial water quality monitoring, for
drinking and recreational water
Colilert Method for Total
Coliforms
& E.coli
 Colitag Method

• Commercially available enzyme –

substrate liquid broth medium.
• Simultaneously detection of total
coliforms & E.coli
• Available in MPN or
Presence/absence format.
BACTERIAL INDICATORS

E. coli cells (from USEPA web site)

Use Aseptic Technique

• Aseptic means “sterile”

• Minimizes sample
contamination
• Assume you are
surrounded by
contaminating
organisms; disinfect
your work area before
Dr. Harold Sears, U of S. Carolina
and after use
• Use aseptic techniques
during sample bacteria on a pin
collection, sample
Defined substrate
technology (DST)
sterile DI water

mixing bottle

sample
pre-measured
media
quantitray
 How does DST work?

• Nutrient medium for

coliforms
– ortho-nitrophenyl-β-D-
galactopyranoside (ONPG)
and
– 4-methylumbelliferyl-β-D-
glucuronide (MUG)
• Total coliform enzyme
metabolizes ONPG to form
a yellow product.
• E. coli enzyme metabolizes
MUG to form a fluorescent
product.
 DST Steps

2. Seal, incubate

3. Count the
positive cells
1. Mix media with
sample; pour into
Quantitray
Preparing a sample for
incubation
Total coliforms and E. coli:
Colilert Quantitray
• Under ambient
light, total
coliform
colonies are
yellow
• The same tray
under UV light;
total coliform
colonies that
are E. coli
fluoresce
Determining a final count
• Compare color
intensity to the
“comparator” tray
• Count the number Blank Comparator Positive
of large and small test

wells that are

positive
• Look up the most
probable number
on the IDEXX table
How does DST work?

• Nutrient medium
for enterococci
– 4-
methylumbelliferyl-
β-D-glucoside
• Enterococci
enzyme
metabolizes the
reagent to form a
fluorescent product
Enterococci: Defined Substrate
Technology
• Enterococci
produce Blue
fluorescence
under UV light
• Marine samples
must be diluted
to reduce false
positives
Coliphages
 Viral Indicators:
Coliphages
• Coliphages are viruses that infect
coliform bacteria
• Recommended by USEPA for use
as a GW fecal indicator.
• Indicator for the transport and
survival of viruses in the
environment
MS2 phage capsids
– Smaller than bacteria
– No metabolic needs
Viral Indicators:
Coliphages
• Two main groups of coliphage:
– Male-specific - Infect bacteria by
attaching to hairlike projections “F pili”
– Somatic coliphage - Infect bacteria by
attaching to outer cell wall
• Found in high numbers in sewage;
low incidence in human feces
• Good indicator of sewage pollution
 Coliphage analyses

• Simple and inexpensive

• Requires a trained analyst in the laboratory
• Coliform host (E. coli) exposed to water sample
– Presence-absence test (USEPA 1601): host added
to sample. Test for coliphage after enrichment.
– Single agar-layer test (USEPA 1602): sample
mixed with liquid agar containing a host. Coliphage
detected directly from sample.
• Plaques form where coliphage were present and
killed surrounding cells
Flow Chart of Presence/absence(1601) Method
 Coliphage presence-absence
test

Positive Negative
control control

Test
sample

USEPA Method 1601

Flow Chart of Quantitative (1602) Method
 Coliphage quantification
by single-layer agar
method

plaques

USEPA Method 1602 From Madigan and others, 2000

IMS/ATP RAPID METHOD
IMS/ATP METHOD
• Method initially developed by
researchers Lee & Deininger (2004)
• IMS/ATP method has been
developed for E.coli & Enterococci.
• It is based on antigen-antibody
reaction.
IMS/ATP Method Overview
– Capture by immunomagnetic separation (IMS)
• Uses antibody-coated magnetic beads which bind
to antigens present on the surface of cells
– Detection by adenosine triphosphate (ATP)
luminescence
• Energy molecule in all cells
• Detects viable organisms

Results are reported M

in relative light units A
(RLUs) G
N
E
T
 Method
Flow
Add beads

30 min @ 18 rpm

Sample + diluent Large magnet

Wash with PBS

Add LL

Luminometer Small magnet

Add SRA
Wash with PBS
Add BRA
Advantages
• IMS/ATP is a rapid method for the
detection of E. coli and Enterococci
– Detects viable cells so directly related to
health risk.
– Can analyze 1 sample in less than 1 hr;
6-8 samples in less than 2 hrs
– Low startup and per sample cost
– Equipment is portable, can be used in
the field
Water Quality Monitoring
Program
Goal

• To provide Safe Drinking Water in

Sind Province
Objectives

• Identify the source of water supply

• Identification of Sources of Contamination
( Industrial/Hospital/Environmental )
• Identifying the type of contamination ( Physical /
Chemical / Microbial )
• Identifying Seasonal/periodic variation of
contaminations
• Identifying hydrological boundaries of
contamination
• Analysis of Monitoring Data
• Planning and Designing for remedial measures
• Physical works / Implementation of Remedial
measure
Beneficial Organization

• Concern implementing Agency

• Stakeholder and Consumer
• Academic institute
• Capacity / Capability / Resources of
Concern organization
• Need to be Analyses by
sharing information
• Who will Lead
• SIDA
Responsibilities / Role of
concern organization
• Sampling ----- SIDA, PCRWR,
PCSIR, KU
• Testing ------ PCRWR, KU, PCSIR
• Analysis of DATA----- KU and
PCRWR
• Coordination during implementation by
Lead Agency
• Cooperation with lead Agency
• Analysis and Sharing and Distribution of
collected data
• Will be assign base on Capacity /
capability / resources of concern
organization
• PCRWR has data warehouse --- Role
data management
• Advisory / Steering Committee for WQMP
Geographical Boudries
Sindh—All barrages Kotri Barrage,
Guddo Barrage, Sakhar Barrage
Time frame

Duration ---- 5 years

Frequency --- 10 to 20 samples/ year / Site
Surface Water ( More or Equal to
above frequency )
Ground water ( Less then above
frequency )
Volume ------
Bacteriological – 500ml
Virology -- 10000 ml (10 liter )
Chemical ---- 1000 ml
Mediums

Surface water
River
Canals
Sub canals
Drains
Ground water
Wells
Treated water
Houses
Hospital
Educational institutes
Hydrants
Filter water
Pipe water
Store Water
PROGRAM DESIGN

• Hydrological information majorly provided

by SEDA
• Previous Studies would collected from
PCRWR and other organization if have any
information
• Steering committee design WQMP
• All parameter would be tested
• Major parameter on the basis of site
requirements
Data interpretation

• Storage--- Data warehouse in

PCRWR
• Its must full fill the Objective
• Comparison – PSQCA
• Group of experts – Analysis and
trend
• Also determine whether
objectives achieved or not