SEM2: URINARY TRACT INFECTION

TRADITIONAL AND EMERGING TECHNOLOGIES FOR DIAGNOSIS

Ms. Ms. Julie Ann Mercado | Week 11

URINARY TRACT INFECTION Traditional Methods of Diagnosis

What is urinary tract infection?
● UTIs are common infections that happen when a Physical Examination
bacteria often from the skin, or the rectum enter the COLOR
urethra and infect the urinary tract ● Pink/Red/Brown: Presence of Blood
● The infections can affect several parts of the urinary ● Yellow: Microscopically, RBC is seen
tract but the most common type is what we call the CLARITY
bladder infection also known as cystitis ● Turbid: Presence of Sediments
● While we also have what we call the kidney infection ○ Leukocytes
or the pyelonephritis ○ WBC
○ Less common but more serious rather than ○ Bacteria
bladder infection because the bladder is on the ○ Red blood cells
lower part which Odor
is closer to the ● We don't report it, but it is good to take note what kind of
infection or bacterial infection is giving
bacteria. If it ● Foul ammonia like odor: This is because of the
goes up to the bacterial multiplication causing the breakdown of urea
kidney, the to ammonia
infection will be
a lot more Chemical Examination
serious Specific Test for Diagnosis
○ Usually women NITRITE
are more ● The nitrite test is positive for those bacteria that can
affected since reduce nitrate to nitrite. And this organism are mostly
females and their urethra are shorter and gram-negative organism for example:
closer to the rectum so it makes it easier for ○ Enterobacteria family
the bacteria to enter the urinary tract ● Gram-positive organism they are negative for this test
especially those do not produce nitrite
Symptoms ○ Enterococcus
1. Dysuria: ○ Staphylococcus
● Defined as the sensation of pain or burning or ● It also detects bacteria at a concentration of >105
stinging or itching of the urethra which can be cfu/mL (colony forming unit per ml) or according to
experienced during urination Strasenger it is 100 000 bacteria per ml
2. Frequent urination ● Positive: Uniform pink
3. Bloody urine ● Negative: Yellow
4. Pressure or
cramping in the
groin or the
lower abdomen Note:
5. Fever Drawback: Gram-positive organisms are negative
6. Chills
7. Lower back pain LEUKOCYTE ESTERASE
or pain in the ● Detects an enzyme produced by leukocytes whose level
side of your increases in the urine during infection. However, it is
back not recommend to just base the diagnosis on this test
8. Nausea or vomiting because levels may not be high enough to be
detected early in an infection for the leukocyte esterase
Traditional Diagnosis ● Cells that possesses the esterase enzymes
As a Medtech, to aid in the diagnosis of a disease, we are ○ Neutrophil
required to have a specimen to test ○ Eosinophil
Midstream Clean Catch: The specimen of choice of voided ○ Basophils
urine samples ○ Monocytes

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 ○ Histiocytes Antimicrobial Susceptibility Test
○ Trichomonas ● Resitance→Burden: The rise in antibiotic resistance
● Cells that do not possess this esterase enzyme we among neuropathogens is a growing concern and adds
have the: to the economic burden of healthcare. This is due to the
○ Lymphocytes inappropriate prescriptions and use
○ RBC or erythrocytes ● Nitrofurantoin: One of the first antibiotics used for UTIs
○ Bacteria and is now prescribed as the first line of defense. This
○ Renal tissue cells is effective against:
● Positive: Violet ○ E. coli
● Negative: Yellow ○ Enterococci
● But many europathogens are intrinsically resistant to
nitrofurantoin such as:
○ Proteus
○ Pseudomonas
Microscopic Examination ○ Providencia
● Detect white blood cells and bacteria ○ Acinetobacter species
● Pyuria and bacteriuria: are helpful for UTI diagnosis ● ESBL-producing bacteria (Extended Spectrum
when symptoms are present. However, if the person is betalactomies): Especially the Enterobacteriaceiae are
asymptomatic and there's a presence of bacteria, it may resistant to most antibiotics except:
be result of contamination ○ Carbapenems
● Sample of UTI infection under a microscope: - It is the third line of drugs given to
○ Bacteria: Malilikot sa slides those who have infection. That's why
○ PUS cells: Nagpapadumi sa urine and most it is worrying for the CDC when they
commonly seen in UTI because they are dirty report that there are a carbapenem
in appearance resistant acinetobacter and
○ RBC: small like donuts carbapenem resistant
enterobacteriaceiae and that causes a
threat
The knowledge of antibiotic susceptibility of an infection in
addition to the identification of the pathogen is essential to
provide an appropriate therapy. TIhe standard method of
determining antibiotic susceptibility testing is through the:
● Phenotypic bacterial growth in the presence of
Antibiotics:
○ The strain is classified as either:
- Resistant
- Susceptible
- Intermediate
Urine Culture ○ The conventional manual methods of
● This is considered as the most diagnostic test determining the phenotypic AST includes:
● Escherichia coli or E coli: The most common cause - Broth dilution
of UTI across all age group - Disk diffusion
● Other common uropathogens are: - Gradient diffusion
○ Enterobacteriaceae family ○ Involve a manual sample preparation steps
○ Proteus and additional time of:
○ Saphylococcus - 16 to 24 hours from culture isolation
○ Enterococcus species ➔ 18 to 30 hours of urine
● Group B streptococcus: they are frequently isolated culture add more the hours
from pregnant women that we will be needing for
● Chromogenic agar: Utilize this for culturing of the urine the anti-microbial
for UTI UTIs susceptibility testing (ganon
● 18 to 30 hours: takes for the test result (foreign article) siya katagal)
● 24 to 48 hours: 24 hours for the initial result and 48
hours for the final result (based on lab practice in PH) ● Newer high-troughput instrument → reduced time of
● Biochemical testing: if positive, It will undergo 10-16 hours:
biochemical testing to identify the organism ○ That is why there this instruments that are now
● Antimicrobial Testing: Last employed by several large Laboratories that
enables the automated sample preparation
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 increased sensitivity and reduced time of 10
to 16 hours.
○ So this commercially available and FDA
approved instruments include the:
- Microscan walkaway (Beckman
Coulter)
- Phoenix automated microscopy
system (Beckton-Dickinson)
- Vitek-2 (bioMerieux)
New Emerging and Diagnostic Technologies
Note:
A timely diagnosis of UTI is essential to prevent the
inappropriate antibiotic treatments and to promote
antibiotic stewardship. The pathogen and its antibiotic
susceptibility should be identified within a few hours of
sample collection. Such that the correct therapy can be
started within the same day that the patient visits the doctor.
So a new diagnostic technologies have the potential to
promote both patient care and management as well as
antibiotic stewardship by delivering rapid results. Because
as we all know urine culture is the most time consuming
step in the current laboratory diagnosis therefore new
technologies need to be able to work directly on urine samples
without the need for enrichment or bacterial isolations
Screening Methods
Manual → Automated instruments
● The method aims to improve the laboratory workflow
and reduce the cost by screening samples to quickly
identify and remove negative samples, while clearly not
missing any positive samples.
● The example of this are the:
○ Flow cytometry
○ Light scattering instruments
○ Urine sediment analyzers
so they are a useful screening tools with high
predictive value
- It means that the test is more sensitive. So
less likely that an individual that tested
negative will have the disease
Flow Cytometers
● This can analyze urine by sorting and counting cells
● They can distinguish between:
○ Bacteria
○ Yeast
○ White blood cell
○ Red blood cells
○ Epithelial cells
Bacterioscan 216 Dx
● This is an FDA approved semi-automated system that
uses light scattering profiles in the urine sample to
detect bacteria in three hours
Automated urine analyzers
● It has a high throughput chemical urinalysis by
assaying the:
○ Protein
○ Glucose
○ Nitrites
○ Leukocyte esterase
among other parameters
Take it as this point when we are doing the chemical analysis
of urine, we are utilizing a dipstick and the reading of the result
in that dipstick is based on the medtech which are prone to
errors. So, if the machine is the one reading the test, that it is
certainly correct
Automated urine sediment analyzers
● It can microscopically analyze the urine sediment for:
○ Leukocytes
○ Erythrocytes
○ Epithelial cells
○ Bacteria
○ Yeast
● Sometimes they are also integrated with microscopic
analyzers or flow cytometers to provide a complete
automated urine analysis. And this system can flag
samples for further analysis such as urine culture
Monoclonal antibody-based immunoassay
● It has a potential as a point of care dipstick assay
● Currently this has been approved for veterinarian use
● Currently being evaluated to be used on human
samples
Identification Methods
So after the screening let us now move on with the identification
method and some of which also has the ability for antimicrobial
susceptibility testing
Matrix Assisted Laser Desorption Ionization Time of Flight
(MALDI-TOF) Mass Spectroscopy (MS)
So the malditovit has two phases:
1. Ionization
● When we say ionization, the samples are fixed in a
crystalline matrix and are bombarded by a laser
● The sample molecules vaporize into the vacuum while
being ionized at the same time without fragmenting or
decompassing
2. Time of Flight:
● The time of flight this is a mass spectrometry method
that separates ions by their mass to charge ratio and
determines that mass to charge ratio by the time it takes
for the ion to reach a detector
● What is new about this is that, this method generates
characteristic mass spectral fingerprints which are
compared with the large library of mass spectra
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 ● As the spectral fingerprints are unique signatures for
each microorganism, accurate microbial identification at
the genus and species level is done using bioinformatics
pattern profiling
(Ibig sabihin is merong specific data na naka store. For example
yung mga libro, naka store yn sa library, parang ganon din yung
data ng mga bacteria na very specific for that specific kind of
bacteria that is why madali ang pag i’identify. Kase kapag
pinasok mo yung specimen, tapos na read niya, tapos maiidentify
niya na yung bacteria kaya mabilis yung process)
● Maldi-tof are now being used increasingly in clinical
laboratories for their unambiguous species level
identification of bacteria and yeast
● Advantage of this method it is a:
○ Cost friendly: there are savings on technical
labor and reagent cost
○ Rapid Turn-around-time for results: The
delivering of the results are very rapid
○ Accurate results
● Disadvantage is that it has a:
○ High cost: it is expensive when you bought the
machine but then it becomes cost friendly
machine when you use it because it saves on
technical labor at the same time reagent cost
○ Main disadvantage: It requires a pure culture
of bacteria and is typically used with isolated
bacteria colonies following the standard urine
culture (need mo padin gumawa ng culture)
○ The usage of urine sample directly is not
advised because the urine matrix which are
specifically the:
- pH, eectrolyte concentration and
cellular composition
varies from human to human. At the same time
it varies from same person at different times.
○ It has also the inability to process polymicrobial
urine samples
Uses of MALDI-TOF
It can be used in combination of the screening tools. For
example is the:
● Flow cytometry + MALDI-TOF:
○ Accuracy when they tested this one is 74% to
94%
● Urinalysis + MALDI-TOF:
○ When they tested this there is an increased
sensitivity and specificity for pathogen
identification
○ And the result are within just an hour
● It can detect Carbapenemase-producing Producing
Enterobacteriaceiae using the Imipenem as a marker
● This method provides identification in less than four
hours (>4 hours) using urine
Multiplex PCR
With the inability of the MALDI-TOF to process polymicrobial
urine samples, comes the multiplex PCR. As a PCR an
extracted DNA will be utilized for the next process
● Simultaneous detection of multiple Targets in a
Single Reaction
○ With the different pairs of primers for each
Target that's why it is called Multiplex PCR
● This Multiplex PCR offers a:
○ Cost effective
○ Rapid approach to pathogen identification
○ Quantification of the pathogen DNA is
necessary for diagnosing UTIs, since it is
necessary to distinguish between
contamination and infection
● Multiplex PCR delivers a substantial time saving over
the standard urine culture the PCR panel delivered AST
result within just three (3) hour
● The study concluded that such panels could help in
getting appropriate and immediate antibiotic therapy
Microfluid platforms for AST
● This is a miniaturized microfluidic devices which are
now becoming attractive for the emerging diagnostic
tools for antibiotic susceptibility testing
● The portability confers great potential as a point of care
devices because of their small size
● We can also use minimal amount of sample as well as
volume
● Cost-affective and amenable to automation,
multiplexing, high-throughput processing, and
single-cell analysis
○ Because there is a small confined spaces
within the microphobic device, it will allow the
detection of bacterial cell division and growth in
far less time than conventional phenotypic AST
assays
● This can be used for pathogen identification and AST
or it can also be used for AST only
● Results are within 3-6 hours (30 minutes)
● By manipulating a small volumes of samples in an
integrated microchannel, A microfluidic lab on a chip
device is able to combine various steps of bacterial
analysis together. For example it includes:
○ Single bacterial culture
○ Cell lysis
○ Nucleic acid purification
○ Sequence amplifiation and
○ Target detection
(So ibig sabihin, within the chip na ginagamit for this test,
nagagawa na niya lahat ng pwedeng gawin sa ibang machine na
matagalan)
Here in the center, is the antibiotic well. This will be the antibiotic
to be placed. And then your microfluidic channel containing the
bacteria surround the antibiotic well. And after some time it will
be check for some growth
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For example here:
● After 1-5 hours → growth →
after three 3 hours → growth is
more prominent = Resistant to
the antibiotic
● If it remains the same →
Susceptible

Real-Time Microscopy System

● It utilize the Brightfield Optical Microscopic Imaging
System which can monitor the bacterial growth and cell
division in real time directly in the urine either with the
presence or absence of antibiotic thus, it can deliver
AST results much faster than any standard phenotypic
methods and that is usually within three 3 hours time
frame
● Bacterial cells may be imaged while immobilized or
while freely moving. So Imaging of a freely moving
cells can give advantage of reduced sample preparation
This is a screenshot of an article published Under World Health
Organization regarding the anti-microbial resistance and here we
can see that this is a global health and development threat at the
same time it is one of the top 10 Global Public Health threats
facing Humanity. This is due to the misuse and overuse of
anti-microbials

Biosensors
● They are small and cost effective and they deliver
rapid results
● They have the potential to be integrated into a point of
care devices
● Several biosensors are with Electrochemical,
Mechanical or Optical transducers that have been
explored for the detection of UTI
○ When we say transducers, these are the
sensors.
Summary
● Development of new testing methods to provide a faster
way of diagnosis especially right now that there are a lot
of antimicrobial resistant cases
● When we provide a faster way of diagnosis, this can
improve the patient care and management

Sequence-Based Diagnostics
● This is highly sensitive and are able to identify
pathogens at a species level
○ (since sequence-base by sequence tinitake
para basahin or makilala ang organism)
● Detect difficult organism
○ when we say difficult organism these are the
organisms that are difficult to cultivate such as:
- slow growing anaerobic
- fastidious organisms
● They can give you a comprehensive picture of the
bacterial eukaryotic and viral communities in the
urinary tract
○ Again because it is sequence based, so based
on its sequence to properly identify the
organism
○ But the problem is it's hard to interpret the
sequencing result and training to interpret this
result is a must
● There should be a caution within using a urine
sample
○ because urine sample has a lot of
contaminants and there are a lot of organism
inside the urine sample that why caution is
important and reading of the result