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An Updated Review on Medical Detection of Dog

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  • Techno India Univerity

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For centuries, our sense of smell has been used as a diagnostic tool in the practice of medicine, be it for recognising gas gangrene on the battle field or diabetic ketoacidosis in the emergency room. In recent decades, many scent detection studies have been performed with human, animal and electronic noses. The ability of humans to diagnose disease by smelling has only rarely been the subject of quantitative studies. Scent detection by animals, on the other hand, has been addressed in several diagnostic studies, which all suggest similar or even superior accuracy compared with standard diagnostic methods. Examples include, amongst many others, the use of dogs for the detection of lung cancer in breath samples, or rats for Mycobacterium tuberculosis detection in sputum. Studies using different types of electronic noses in conditions such as pulmonary disease and cancer have also shown promising results with high overall sensitivity and specificity. However, results of different types of noses are not easily general sable and independent confirmation studies are generally lacking, which should be a focus for future research. Scent detection by animals and electronic noses holds promise for the future and should receive higher priority in terms of research effort and funding.
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Asian J. Pharm. Ana. 2016; Vol. 6: Issue 1 [AJPAna.]
47
ISSN- 22315667 (Print) www.asianpharmaonline.org
ISSN- 22315675 (Online)
REVIEW ARTICLE
An Updated Review on Medical Detection of Dog
Somsubhra Ghosh*, Arnab Jana, Beduin Mahanti
Department of Pharmaceutical Analysis, Bharat Technology, Uluberia, Howrah, W. B-711316, India.
*Corresponding Author E-mail: som_subhra_ghosh@yahoo.co.in
ABSTRACT:
For centuries, our sense of smell has been used as a diagnostic tool in the practice of medicine, be it for
recognising gas gangrene on the battle field or diabetic ketoacidosis in the emergency room. In recent decades,
many scent detection studies have been performed with human, animal and electronic noses. The ability of
humans to diagnose disease by smelling has only rarely been the subject of quantitative studies. Scent detection
by animals, on the other hand, has been addressed in several diagnostic studies, which all suggest similar or even
superior accuracy compared with standard diagnostic methods. Examples include, amongst many others, the use
of dogs for the detection of lung cancer in breath samples, or rats for Mycobacterium tuberculosis detection in
sputum. Studies using different types of electronic noses in conditions such as pulmonary disease and cancer
have also shown promising results with high overall sensitivity and specificity. However, results of different
types of noses are not easily general sable and independent confirmation studies are generally lacking, which
should be a focus for future research. Scent detection by animals and electronic noses holds promise for the
future and should receive higher priority in terms of research effort and funding.
KEYWORDS: Cancer, Trained Dog, Blood Sugar, Medical detection, Sence.
INTRODUCTION:
Every year, across the world, billions of dollars are
invested into curing and preventing cancer; the world’s
leading cause of death. Advanced screening and
detection methods are developed and new drugs and
technologies are trialled to help fight the many forms of
this disease. Yet still, the number of deaths caused by
cancer continues to grow. Current figures show that
approximately 8 million people die from cancer each
year and the World Health Organisation predicts that this
figure will increase 80% by 2030 (Centers for disease
Contol and Prevention, 2014). Early detection may be
man’s greatest from of defence. However, this is often
difficult as screening techniques can be expensive and in
some cases are not without their own health risks.
Received on 09.02.2016 Accepted on 28.02.2016
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 6(1): January- March, 2016; Page 47-52
DOI: 10.5958/2231-5675.2016.00008.9
In order to encourage people to seek regular care, less
invasive, and simpler methods would be advantageous,
especially if they can prove to be more sensitive and
accurate in detecting early stages of cancer. This demand
has led to many alternative methods and technologies
being explored.The theory that dogs may be able to
detect human cancer using their superior olfactory
system is one such alternative that has been widely
researched in recent years. A dog’s sense of smell is said
to be anywhere from 1000 to 10,000 times more
sensitive than humans. Research has already shown that
a dog can sense when an epileptic person is going to
have a seizure or when a diabetic’s blood glucose level
drops.
This is due to the dog’s ability to smell pheromones or
chemical changes taking place in the body. Extensive
training and tests have been carried out to discover if
dogs can also be trained to identify and detect human
cancers. Findings from these studies have prompted
scientists to ask ‘what is it exactly that the dog can
smell?’ ‘Does cancer have distinguishing odour
Asian J. Pharm. Ana. 2016; Vol. 6: Issue 1 [AJPAna.]
48
signature made up of Volatile Organic Compounds
(VOCs) which are emitted into the air?’ And if this
chemical compound could be isolated and replicated
then might it be possible to develop an electronic nose
‘which could potentially mimic the dog’s olfactory
sense?’ It is hoped to explore these ideas and attempt to
answer the questions posed. The scientific basis of the
ability of dogs to detect the odour of cancer is believed
to be linked to volatile organic compounds produced by
malignant cells. It has been established that during
tumour growth changes occur in some of the cellular
proteins, leading to peroxidation of the cell membrane
components, which then produces volatile organic
compounds that can be detected in the headspace of the
cells. There is strong indication from a number of studies
that these cancer volatiles are excreted in urine or
exhaled on the breath at an early stage in the disease
process [1].
Brief history:
The first clinically robust investigation of cancer
detection by dogs was reported by Willis and colleagues
(British Medical Journal 2004). During this study, dogs
were trained by individuals from Medical Detection
Dogs (aka Cancer and Bio-detection Dogs) to detect
bladder cancer (transitional cell carcinoma) by smelling
urine samples from patients and healthy controls. Six
dogs of varying breeds were used, none had been trained
for previous scent work. Overall diagnostic accuracy was
41% (compared with the 14% success rate expected from
chance alone). However there was considerable variation
in the success rate between dogs, the best dog achieved
56%, but two dogs had no success at all.
The charity, in collaboration with Willis et al, published
a further study in the journal Cancer Biomark 2011,
‘Volatile organic compounds as biomarkers of bladder
cancer: Sensitivity and specificity using trained sniffer
dogs’. Specificity ranged from 92% for urine samples
obtained from young, healthy volunteers, decreasing to
56% for samples taken from older patients with non-
cancerous urological disease.
Supporting evidence has been published around the
world including a study by Cornu and colleagues,
‘Olfactory Detection of Prostate Cancer by Dogs
Sniffing Urine: A Step Forward in Early Diagnosis’,
indicated the possibilities of canine cancer detection
(sensitivity 91%, specificity 91%). A second study
published online in 2011 by Sonoda and colleagues
investigated colorectal cancer screening using faecal
samples, and demonstrated equally promising results
(sensitivity 97%, specificity 99%). Ehmann et al
(European Respiratory Journal, 2012) carried out the
first published study showing that sniffer dogs can
reliably detect lung cancer from a breath sample
(sensitivity 71% specificity 93%) [2].
A recent study from Italy (Taverna et al, Journal of
Urology 2014), reported the diagnostic accuracy of dogs
trained to recognize specific volatile organic compounds
of prostate cancer in urine samples (sensitivity 98
100%; specificity 9899%) [3].
Procedure:
1. Detection of changes in Blood sugar level:-
Dogs have been shown to respond when their owners’
sugars are low or high, but as yet we cannot be sure as to
what they are actually responding. Odour cues are the
most plausible explanation especially as dogs show
“alert” behaviours when their owners are asleep and
presumably emitting few behavioural cues (although
changes in breathing rate may occur).
In addition, owners frequently report their dogs
responding when they are in another room and
behavioural cues therefore implausible.
It is likely that dogs detect changes in the chemical
composition of their owners’ sweat, or breath (including
products of ketosis), using their acute sense of smell.
This is supported by the fact that MDD are increasingly
training new dogs using remote odour samples collected
from clients during times of hypoglycaemia, before they
introduce dog and owner. Unlike the training of seizure
alert dogs, MDD do not intentionally train dogs to
respond to behavioural cues.
However, once placed some dogs may learn to utilise
additional predictive cues as well as odour, including
subtle changes in their owners’ mood or behaviour (e.g.
trembling, becoming disorientated). Research is now
required to determine the precise cues used and to
identify any odour signature involved.
This study is the first to examine the effectiveness of
trained glycaemia alert dogs and has demonstrated that
most clients are willing and able to collect data, although
some improvement in recording methods is
recommended. Although based mainly on owner-
recorded data, multiple findings point consistently to the
potential value of trained alert dogs, but for conclusive
proof, longitudinal studies are now required, examining
matched clients pre- and post-dog allocation [4].
Such studies can never be truly randomised, as the
population willing to use a dog as an intervention will by
necessity be self-selected. However, comparison of
waiting list applicants to those who have acquired a
trained dog, will help to determine the full value of this
intervention.
Asian J. Pharm. Ana. 2016; Vol. 6: Issue 1 [AJPAna.]
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2. Cancer detection:-
Fig no 1:- Shows Dog detecting Cancer
In Cancer detection some patients gave a urine sample at
their first visit to the thyroid clinic before they went on
to have a biopsy of suspicious thyroid nodules and
surgery.
The surgical pathology result was diagnosed as cancer in
some patients and benign thyroid disease in rest of the
patients.
These urine samples were presented, by a gloved dog
handler, one at a time to Frankie to sniff. Neither the dog
handler nor the study coordinator, who recorded the
dog's responses after the handler announced them, knew
the cancer status of those subjected patients with urine
samples.
The handler interspersed some urine samples that had a
known cancer status so he could reward the dog for
correct answers: alerting to a cancer sample by lying
down, and turning away from a benign sample to alert
the absence of cancer [5].
Training of the dogs:
Six dogs of varying breeds and ages completed a seven
month period of training. All were familiar with
obedience commands, but none had been previously
trained for search or scent discrimination tasks. We
made no attempt to include dogs with a particular
suitability for scent discrimination.
The training objective was to enable the dogs to
discriminate between urine from patients with bladder
cancer and urine from diseased and healthy people, using
samples entirely new to them, so as to preclude simple
memory recognition of participants' unique odour
signatures. Dogs were trained to detect (“alert to” or
“indicate”) one urine sample from a patient with bladder
cancer placed among six control specimens. We selected
this task format (of being able to select one urine from
seven) with reference to data on dogs' behaviour.
Training was by operant conditioning, using the clicker
training method; the dogs were taught to indicate the
appropriate sample by lying beside it. Early recognition
of the tumor scent was achieved by using search and find
games, which were gradually replaced by discrimination
phases of increasing complexity. Urine from patients
with bladder cancer was presented sequentially against
water, diluted urine from healthy people, undiluted urine
from healthy controls, urine (containing blood) from
menstruating women, and urine from patients with non-
malignant active or recent urological disease or other
disease. Samples were not pooled at any stage.
Participant selection:
Patients presenting with new or recurrent transitional cell
carcinoma of the bladder gave urine before surgical
intervention.
Then male controls aged over 50 only if recent prostate
histology had been negative for cancer was included.
And then patients with premalignant urological disease
or a history of urological carcinoma was excluded.
A history of other malignancy was acceptable providing
the patient was now considered disease-free. All other
past or current medical conditions were permissible and
made no exclusions on the basis of drugs, menstrual
cycle, ethnicity, diet, alcohol consumption, smoking
habits, exposure to chemicals, or findings on urinalysis.
After all details of all of these factors for each
participant, in case needed to consider their influence on
the composition and odour of the urine at any stage was
recorded.
Analysis and processing of urine samples:-
After urinalysis refrigeration of fresh urine specimens
within 45 minutes and froze them 2-32 hours later as 0.5
ml aliquots in glass vials.
Then stored them at -40°C for up to five months. For
presentation to the dogs, samples were defrosted and
pipetted on to filter paper in Petri dishes (58 x 15 mm)
and used either immediately in a wet state or within four
weeks after overnight air drying and storage at room
temperature[4].
Dog training:
A Belgian Malinois shepherd was trained by a
professional and dedicated team of two people from the
beginning to the end of the detection process. The first
objective was to teach the dog to discriminate between
urine from individuals with PCa and urine from controls.
The dog was trained by the clicker training method (a
kind of operant conditioning). The dog was given his
ball as a reward for alerting to a cancer urine. The dog
was taught to sit in front of the sample of urine
recognized as cancer.
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Patients and samples:
Urine samples were obtained from Caucasian patients
recruited in tertiary reference who had given written
consent for analysis of their urine for detection,
including genetic analysis. All men included were
referred to an urologist because they had an elevated
PSA level or abnormal findings on Digital Rectal
Examination (DRE).
Data collected at the visit were age, height, weight, PSA
level, and DRE data. Urine was collected during the first
consultation after DRE.
Then all patients underwent prostate biopsies according
to a standard procedure (12 cores) and were classified as
cases or controls after pathologic examination of the
specimens. Patients were not selected in case of history
of urothelial carcinoma or other malignant disease. There
were no exclusion criteria regarding other medical
history, alcohol consumption, drugs, food, tobacco
consumption, or other habits.
Study design:
The double-blind testing phase consisted of consecutive
runs. For each run, the dog was presented six samples
(five controls and one cancer). During each run, the
cancer urine was one of the selected cancer samples and
the 5 control urines were samples randomly selected
among controls. Samples were anonymised and
numbered so that people conducting the test were not
able to discriminate cancer from control samples. The
samples were frozen at 4 8C from the time of urine
collection to the time of testing. Each urine sample was
slowly heated to 37 8C with the same material
immediately before examination in a dedicated area
outside the testing room.
During each run, the dog had to scent successively the
six samples that were hidden in boxes. Each box had a
hole so that the dog could not access the urine itself, but
only its odor. After a mean time of 30 sec, the dog had to
sit in front of a box to designate the cancer sample. In
case of success (dog sitting in front of PCa urine
sample), the result was classified as a true positive and
the controls as true negatives, and the next cancer sample
was tested. In case of mistake (dog sitting in front of
control urine sample), the control sample was classified
as false positive and the cancer sample as a false
negative. The false-positive sample was excluded from
the pool of controls used for the future runs, and the
cancer sample was retested in association with other
controls. A new prostate biopsy was proposed to the
patient who provided the false-positive sample [6].
3. Seizure detection:-
Over the last decade a new kind of service animal has
emerged. Seizure alert dogs warn people with epilepsy
of an oncoming attack minutessometimes hours
before it occurs. This allows the person time to take
seizure blocking medication, get to a safe place, or call
for assistance.
How dogs detect an oncoming seizure in a human is a
mystery. Some trainers and researchers think they detect
subtle changes in human behaviour or scent before an
episode occurs. There are no scientific studies, however,
to prove these theories. Trainers also believe the
behaviour is not breed, age or gender specific in dogs.
Dogs can be trained to stay with the person during a
seizure. When selecting a potential seizure alert dog to
work with, she performs a trainability test [7].
Understanding dog's senses
A big part of understanding a dog is understanding its
senses and accepting that they are indeed different than
humans. Both humans and dogs have the same three
senses: sight, hearing and smelling, however while most
humans communicate by hearing, seeing, and then
smelling, dogs primarily communicate by smelling,
seeing and lastly hearing. Dogs also have a universal
sense which humans do not have, where they can feel the
energy (emotions) of the other beings around them. The
statistics below will vary slightly with different types of
breeds, for example a sight hound may have slightly
better vision and a coonhound type of dog may have a
slightly better sense of smell than other types.
Properties of each of senses
The Nose: A dog interprets the world predominantly by
smell, whereas a human interprets it by sight. As a
human I cannot even imagine what that would be like to
get most of my information from what I smell. This is
why a blind or deaf dog can get along just fine if allowed
to be a dog, given the proper leadership and exercise and
their sensory whiskers are not cut off when they are
groomed. While a dog's brain is only one-tenth the size
of a human brain, the part that controls smell is 40 times
larger than in humans. A dog’s sense of smell is about
1,000 to 10,000,000 times more sensitive than a human’s
(depending on the breed). A human has about 5 million
scent glands, compared to a dog, who has anywhere
from 125 million to 300 million (depending on the
breed).
Ever wonder why your dog's nose is wet? The mucus on
a dog's nose actually helps it smell by capturing scent
particles. When a dog’s nose is dry they may lick it to
aid them in scent.
When dogs smell something they are not just registering
a smell, they get an entire story. They can smell
pheromone, which is not only found in the urine and
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fecal, but on the skin and fur. From this they can tell a
lot about another dog or human including if they are
male or female, what they ate, where they have been,
what they have touched, if they are ready to mate, if they
have recently given birth, or had a false pregnancy, and
what mood they are in. They have even been known to
smell cancer on people, alerting them to it and saving
their lives. This means when your dog smells another
person, tree that another dog has peed on, pant leg that
another dog has rubbed up against, or chair that someone
has sat in, they are actually reading a story, not just
smelling an interesting scent. While a human will smell
something like spaghetti sauce as one smell, a dog smells
each individual ingredient. Unlike humans, dogs can
move their nostrils independently, allowing them to
know what direction a smell is coming from.
A dog can both sniff and breathe. These are two different
functions. Breathing is for air, but when they sniff with
short breaths they actually save some scent that does not
get exhaled. When a dog is overheated and actively
panting, its sense of smell is reduced by as much as 40
percent as it uses the air to cool itself rather than for
smelling. Puppies have heat sensors in their noses to help
find their mother during the time when their eyes and
ears are closed. These sensors disappear by the time they
are adults.
The Eyes:
Since dogs do not have a spoken language, their thoughts
are more like a sequence of images, much like a child
before it learns to speak.
A common question among humans is, "Are dogs colour
blind?" The answer is no, not exactly, meaning they do
not only see in shades of only black and white. Studies
have shown that dogs see in colours of various shades of
blue and yellow. For example, a rainbow to a dog would
be as follows: dark blue, light blue, light gray, light
yellow, dark brownish yellow, and dark gray.
Purple and blue are both seen as shades of blue.
Greenish-blue is viewed as a shade of gray. Red is seen
as a black or dark gray. Orange, yellow and green all are
seen to a dog as various shades of yellow. This means
that, to a dog, bright orange toys are the same yellowish
shade as the green grass. If you want your dog to clearly
see his toys in the green grass you are better off giving
the dog blue toys; if you have orange, yellow or green
toys, the dog will be able to find them with his nose.
Dogs can see best at dusk and dawn. Their low-light
vision is much better than a human’s, but their overall
vision is not better. While a human’s vision is considered
perfect at 20/20, a dog's vision is on average 20/75. Dogs
cannot see as well at a distance as a human with normal
eyes. Humans can also see things close up better than a
dog can. On average, a human can see something clearly
as close as 7 cm away, compared to a dog that sees
things burry if they are closer than 33 cm away. Dogs
can recognize objects better when they are moving and
sometimes overlook the same object when it is still.
Dogs see images on a TV screen, but most likely also see
a rapidly flickering light, almost like a strobe light, in the
picture; a human’s flicker resolution ability is about 55
Hz and a dog's is about 75 Hz.
Puppies are born deaf and cannot hear until they are
about 21 days old. Their eyes are also closed. During this
time they rely solely on scent to interpret their world. By
the time their sense of hearing is completely developed
they can hear about 4 times the distance of a human who
has normal hearing. Dogs can hear higher pitched sounds
that humans cannot hear. They often bark at vacuums
because they hear a very loud annoying pitch to their
motors.
Dogs detect sounds in the frequency range of
approximately 67 - 45,000 Hz (varies with different
breeds), compared to humans with the approximate
range of 64 - 23,000 Hz. As humans and dogs get older
they both lose the ability to hear certain frequencies.
Dogs have 18 or more muscles in their ears allowing
them to be mobile, whereas a human has only 6 and can
only move their ears slightly, if at all. Dogs with perked
ears can usually hear better than dogs with hanging ears,
especially if they can move their ears in the direction of
the sound.
Animals can feel energy. If you break it down to simple
science, the kinetic energy a dog detects might simply be
a frequency. Light, sound and heat are all frequencies.
This energy is a universal animal language. Have you
ever been watching a group of wild animals out in the
yard, perhaps a squirrel, rabbit and a deer all eating
peacefully? Clearly these animals are not speaking
words to one another asking if they all come in peace;
somehow they all know that they are not going to harm
one another. Or perhaps you know a dog that other dogs
do not tend to like, or a cat that likes one dog but not
another. Or perhaps you know of a person who dogs are
prone to bark at. Dogs can sense fear. It is believe they
can smell the pheromone and perhaps they can even feel
it radiating from a being. Some dogs can tell a few
minutes before a human is about to have a seizure even
before the person knows. When I was a kid growing up I
had a Lab mix who loved everyone. There was not a
single person he didn't like, except for my uncle. When
my uncle would come around he would bark at him. I
later discovered that many dogs tended to bark at my
uncle and as I got older I realized my uncle was a very
Asian J. Pharm. Ana. 2016; Vol. 6: Issue 1 [AJPAna.]
52
tense, nervous person. Another example was a time
when my husband and I were driving down the road with
our two dogs in a van that did not have any windows in
the back. The dogs were sleeping on the van floor.
Suddenly our Pit Bull stood up and started growling. I
was in the passenger seat and didn’t see or hear
anything. My husband, on the other hand, was amazed.
He had just passed a cop and for a split second thought
he may have been speeding and at the exact moment he
felt a chill of fear run down his spine, his dog had
popped up from his curled up sleep and growled, not at
us but toward the walls of the moving van. The dog had
felt his fear and was jumping up in protection mode.
Dogs interpret human emotionssuch as worry,
anxiety, fear, anger, pity and nervousness, as weaknesses
and they do not listen to these emotions. Dogs listen best
to someone who is calm but firm in their approach. They
use their sense of energy to determine who should be the
leader of their pack. The being with the strongest and
most stable energy is the one they look to, be it
themselves or another being around them. While you can
hide your emotions from another human, you cannot
hide them from a dog [8].
Advantages
1. As compared to other conventional detection
method, this method is much more accurate.
2. Less time consuming method.
3. Sensitivity is much better.
4. Future scope is very promising.
Disadvantages
1. More expensive.
2. Need a well trained dog.
3. Need a perfect trainer to trained the dogs .
4. Needs more research.
5. Low awareness.
Applications of Medical Detection of Dogs
Medical Alert Assistance Dogs (Shirley).
The most prominent application of MDD is lies in
the field of cancer detection. Various type of cancer like
breast cancer, bladder cancer, prostate cancer, ovarian
cancer, lungs cancer can be detected by this advance
screening and early detection method of MDD.
Assists individual in reducing hyperglycaemic
associated conditions.
Future Scope:
Through the research & development of electronic
systems (E noses) is introduced recently that will assist
in the early detection of cancer through cheap non-
invasive tests. In the short term the cancer dogs can
provide second line screening for cancers that are
currently very difficult to diagnose reliably, such as
prostate cancer. Trained dog can be used to assist an
individual with severe narcolepsy. Plans for the future
include the training of dogs to detect substances that
result in severe allergic response in atopic clients.
CONCLUSION:
Approach to training was vindicated by the results
achieved when the dogs were formally evaluated.
Despite the fact, that do not use dogs without proved
scenting abilities, and despite the inclusion of age
matched diseased controls statistically significant
success rate was achieved by scientists. Great deal of
learning was achieved during the study, and it was
certain that improvements in the success rate can be
achieved by modifications to the training regimen.
Suitable reward mechanisms for the trainers those who
are blinded to the samples, so as not to confuse the dogs.
Also, for this approach in cancer detection to have more
clinical relevance, need to train the dogs for respond to
more than one positive sample at a time, and to have a
signal for “no positive sample present.” Future of
medical detection is very bright if new & latest
techniques are incorporated time to time. Some other
types of detection may be possible in future if proper
research is done on this project.
REFERENCE:
1. Available at: http://medicaldetectiondogs.org.uk, collected on
21/10/2015.
2. Ehmann R, Boedeker E, Friedrich U, Sagert J, Dippon J, Friedel
G, Walles T, Canine scent detection in the diagnosis of lung
cancer: revisiting a puzzling phenomenon, Eur Respir J., 2012
Mar; 39 (3): 669-76.
3. Gianluigi Taverna, Lorenzo Tidu, Fabio Grizzi, Valter Torri,
Alberto Mandressi, Paolo Sardella, Giuseppe La Torre,
Giampiero Cocciolone, Mauro Seveso, Guido Giusti, Rodolfo
Hurle, Armando Santoro, Pierpaolo Graziotti, Presented at annual
meeting of American Urological Association, Orlando, Florida,
May 16-21, 2014.
4. Available at: http://news-medical.net, collected on 7/11/2015.
5. Willis CM, Church SM, Guest CM, Cook WA, McCarthy N,
Bransbury AJ, Olfactory detection of human bladder cancer by
dogs: a proof of principle study. BMJ 329:712-4, 2004.
6. Jean-Nicolas Cornu, Geraldine Cancel-Tassin, Valeria Ondet,
Caroline Giradet, Olivier Cussevot; Olfactory detection of
Prostate cancer by dogs sniffing urine : A Step Forward In Early
Diagnosis, European Association Of Urology, Elsevier B.V, 198,
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collected on 7/11/2015.
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