Mark Lebeck1, Troy McCarthy1, Nancy Hall, BS, MT (ASCP)2, Kevin L. Russell, MD, MTM

1
Mark Lebeck1, Troy McCarthy1, Nancy Hall, BS, MT
(ASCP)2, Kevin L. Russell, MD, MTMH3, Dean D.
Erdman, DrPH4 , Gregory C. Gray, MD, MPH1
1Center for Emerging Infectious Diseases,
Department of Epidemiology, University of Iowa
College of Public Health, Iowa City,
IA. 2Environmental Microbiology, University of
Iowa Hygienic Laboratory, Oakdale Campus,
Coralville, IA. 3Navy Respiratory Disease
Laboratory, Naval Health Research Center, San
Diego, CA. 4Respiratory and Enteric Viruses
Branch, Centers for Disease Control and
Prevention, Atlanta, GA.

• Why study adenovirus as an indicator of surface
  water contamination?
• Adenoviruses are prevalent in polluted water
  sources.
• Adenoviruses have resistance to ultraviolet light
  inactivation and long survival rates in the
  natural environment.
• Adenoviruses are now recognized as an emerging
  indicator of fecal coliform/E.coli contamination
  and public health concern in surface water.

• Five of 33 water samples screened (15.2) were
  positive for adenovirus.
• See representative gel (figure 1).
• Human adenovirus types 1, 2, 3, 4, and 35 were
  detected by sequence typing (table 1).

Background - Because of their prevalence in
polluted water sources, their resistance to
ultraviolet light inactivation and long survival
rates in the natural environment, adenoviruses
are now recognized as an emerging indicator of
fecal coliform/E.coli contamination and public
health concern in surface water. As their
occurrence and pathogenicity from water have not
been well studied, we are developing molecular
techniques to determine the prevalence of
adenovirus and to identify the specific serotypes
associated with surface water contamination. Metho
ds - A panel of 33 ambient Iowa surface water
samples, previously studied with traditional
coliform detection techniques, was provided to us
by The University of Iowa Hygienic Laboratory.
Specimens were syringe filtered and DNA was
extracted. Nested PCR was performed per the
Centers for Disease Control and Preventions
adenovirus hexon gene sequence typing protocol.
Adenovirus-positive PCR products were sequenced.
A panel of 5 adenovirus-negative and 5
adenovirus-positive water samples was sent to an
outside laboratory for blinded validation
studies. Results - Five of 33 water samples
screened were positive for adenovirus. Human
adenovirus types 1, 2, 3, 4, and 35 were detected
by sequence typing. Blinded validation studies
yielded high agreement (Kappa0.90, p-value
lt0.05). Conclusion - We have shown that
adenovirus contamination can be detected in
surface water samples. The presence and
variability suggest our molecular techniques to
be a promising method for detection of adenovirus
in surface water. Its strength over traditional
fecal coliform/E.coli detection methods is its
potential to determine the origin of
contamination. This method has yet to be
validated for zoonotic adenoviruses.

• Examine other regions of the adenovirus genome.
• Validate this method for animal adenoviruses.
• Optimizing sample storage and preparation with
  larger sample size.
• Correlation study comparing coliform/E.coli
  assays with adenovirus presence, type and source.

• We have shown that it is possible to detect
  adenovirus in surface water samples and to
  determine the adenovirus type and species without
  collecting large volumes of water.
• Our validation showed a 100 agreement for
  negative samples and 80 agreement for positive
  samples. Our methodology may be more sensitive
  than the methodology used by the independent
  laboratory. To determine if differences are
  caused by sensitivity levels or false positives
  more validation studies need to be performed.
• This study would be strengthened by a larger
  sample size using a wider, more diverse
  collection of surface water samples.
• To better distinguish adenovirus species and
  type, it would be advantageous to examine other
  regions of the adenovirus genome. In addition to
  hexon gene sequencing, we plan to adapt similar
  sequencing data for the fiber gene and another
  hypervariable region of the hexon gene.
• Currently, this method has only detected human
  adenoviruses. To better determine the source of
  water contamination, we plan to expand this
  protocol to include animal adenoviruses .

Table 1
Figure 1

• 33 ambient Iowa surface water samples were
  provided to us by The University of Iowa Hygienic
  Laboratory.
• Water samples were Millex syringe-driven
  filtered.
• DNA was extraction with QIAGEN kit.
• Nested polymerase chain reaction was then
  performed per the Centers for Disease Control and
  Preventions adenovirus hexon gene sequence
  typing protocol (figure 2).
• Adenovirus-positive PCR products were then
  purified by the QIAquick PCR Purification Kit.
• Samples were submitted to the University of
  Iowas DNA Core Facility for sequence typing.
• The wild type sequences were compared to the CEID
  Adenovirus sequence library that we created by
  sequencing known adenoviruses using this
  technique and NCBI.
• A panel of 5 adenovirus-negative and 5
  adenovirus-positive water samples was sent to
  Respiratory Disease Laboratory, DoD Center for
  Deployment Health Research, Naval Health Research
  Center, San Diego, CA for blinded validation
  studies.

• In contrast to most methods of adenovirus
  detection, this method did not require large
  volumes of water or an expensive water apparatus.
  
• This hexon gene sequencing technique holds great
  promise to determine the source of water
  contamination (e.g. human, swine, poultry, etc.)
  and to identify the specific adenovirus type
  (e.g. human Adv41).

Blinded validation studies yielded high agreement
(Kappa0.90, p-value lt0.05)
Figure 2
Collaborators - Howard Lehmkuhl, PhD, Kevin
Knudson, PhDCEID Staff - Whitney Baker, Ana
Capuano, Mark Lebeck, Ghazi Kayali, Troy
McCarthy, Sharon Setterquist