Leukocyte Presence in Ukweli Urinary Tract Infection Test Strips
Rhea Sullivan, Aracelis Velez, David Fanelli
April 3rd, 2015
Introduction
Ukweli test strips are primarily used to test for urinary tract infections (UTIs), and
diabetes in developing countries such as Kenya and Sierra Leone. These countries are often
home to citizens who are not well informed on health and hygiene practices, so some diseases
may be more common in these areas. The use of these test strips makes the presence of disease
or infection easy to understand for anyone. A color change translates a complex, chemical
reaction with an abnormal health result into a simple method of understanding.1
Leukocytes are another name for white blood cells, which are colorless cells that circulate
in the blood and body fluids and are involved in counteracting foreign substances and disease.
The normal white cell count is usually between 4 and 11 x 109 cells per liter. They make up
approximately 1% of the total blood volume in a healthy adult.2 An increase in the number of
leukocytes over the upper limits is called leukocytosis, and a decrease below the lower limit is
called leukopenia. Studies from Tehran University have concluded that an elevated leukocyte
count, even within the normal range was associated with the presence of a UTI. 3White blood
cells in urine are detected by measuring the amount of leukocyte esterase, an enzyme produced
by white blood cells. Positive leukocyte esterase tests are usually an indicator of a UTI.
The main ingredients in the strip test are pyrrole amino acid ester and diazonium salt.
Leukocyte esterase in urine catalyzes hydrolysis of diazonium salt, resulting in an azocoupling
reaction that forms an Azo dye. Azo dye is an aromatic compound that contains a double bond.
The double bond nature of the compound allows the dye to absorb certain wavelengths, which
are complementary to those of purple wavelengths. As a result, the test strip changes from beige
to purple, purple indicating an infection. Healthy urine will cause no color change and will result
as negative for urinary tract infections.4
The reaction is as shown:
Ar ( diazonium salt on pad) – N+ = N+ Ar’ (aromatic compound)--> Ar-N=N-Ar (dye)
Figure 1: Diazonium salt hydrolyzed to form Azo dye
Middle Section
Environmental Effects
While the leukocyte esterase dipsticks are generally reliable methods of early UTI, it is
important to note that there are certain environmental effects that could alter the sensitivity of the
dipstick or otherwise skew the results so as to lead to an unreliable diagnosis. Specifically,
special care should be taken with regards to temperature, humidity, and UV light (Ultraviolet
light) when handling and using the leukocyte esterase dipsticks.
The constantly high temperatures of Kenya and Sierra Leone could pose various
problems for the dipsticks, mostly regarding the stability of the chemical components on the
dipsticks themselves. Sierra Leone, on average, has a range of relatively high temperatures from
26 - 36˚C. Kenya’s average temperatures can vary depending on the exact location, as seen
below.5 However, it should be noted that these are day time temperatures, and the temperature
becomes substantially cooler during the night.1
Table 1. Temperature Ranges of Kenya Based on Location5
Area
Temperature Range (˚C)
Central Highlands and Rift Valley
21 - 26
Western Kenya
27 - 29
Northern and Eastern Kenya
30 - 39
Coastal Belt
28 - 31
As mentioned previously, certain components of the dipstick have temperature
sensitivities. Specifically, leukocytes boil at 256˚F (124˚C), which is not necessarily a huge
problem because the temperatures will not reach such high values. However, at 37˚C, leukocytes
will begin to disintegrate relatively rapidly in hypotonic alkaline solution, meaning the cells will
die.6 If the cells die, then leukocyte esterase will not be active and the test strip will subsequently
produce a false-negative result. Nevertheless, while it is important to refrain from using and
storing the test strips in high temperatures, care must be taken to keep them cool at temperatures
below 10˚C. At 5 - 10˚C, the diazonium salt is relatively unstable and can spontaneously
decompose by loss of a nitrogen to produce a carbocation (see diagram below). This would
interfere with the reaction and as a result produce unreliable results. 6
Figure 2: Formation of unstable phenol under high temperatures6
Humidity is another factor that could cause problems for the UTI test strip if not handled
properly. The average humidity of Kenya (specifically Nairobi) and Sierra Leone vary depending
on the season. The Dry Seasons are characterized by less rainfall and hence lower humidity
compared to the Wet Seasons. In Nairobi, Kenya, the Dry Season typically has a humidity
averaging less than 36%, while the Wet Season has a humidity typically greater than 97%. In
Sierra Leone, the humidity is typically around 50% during the Dry Season and 80% during the
Wet Season. Unfortunately, the test strips are in fact moisture sensitive.5 Figure 3 demonstrates a
reaction involving excess humidity and diazonium salt. The test strip involves a hydrolysis
reaction with the diazonium salt, so extra moisture could potentially cause the diazonium salt
bond to be cleaved prematurely, leading to the formation of a phenol (shown below.
Figure 3: Effect of excess humidity on diazonium salt
Furthermore, if a test strip has been compromised by added moisture, any tests done with
it will take longer to process or may be completely invalidated. This is because the formation of
a phenol will hinder the test strip to recognize presence of diazonium salt bonding, and therefore
it will not indicate a color change. No specific data could be found that characterizes “moisturecompromised” test strips, so it is advised that routine test experiments be done with known
samples to determine what range of humidity the test strips still produce reliable results.6
Finally, exposure to UV light could produce problems for the leukocyte esterase dipstick
that could lead to unreliable results. The diazonium salt is sensitive to UV light, and when
exposed to enough of it, the salt will completely breakdown. This results from UV light exciting
the electrons in the diazonium salt bond with such intense energy that the compound’s orbital
order will be destroyed. The diazonium salt is a key component in the reaction for the dipstick,
so its loss would render the dipstick useless.7
Chemical Effects
There are many components of urine that vary between the diseased and “normal” state.
However, these, for the most part, do not alter the sensitivity of the dipstick or otherwise alter the
results. It should be noted that the dipstick is testing for an elevated amount of leukocyte
esterase, and the typical concentration of leukocytes is between 4 x 109 and 11 x 109 cells per
liter (~1% of total blood volume in a healthy adult). 7As such, a lack of leukocytes would impact
the dipstick because there is simply not enough to detect. Nevertheless, exposure to certain
chemicals and reactants are of concern for the accuracy of the leukocyte tests.
Certain oxidizing agents can cause problems with the dipstick, and there are in fact a few
powerful oxidizing agents that have been found in urine. These include peroxide, oxychloride,
and ferric ions. These oxidizing agents could potentially increase the pH of urine by hydrolyzing
water. The increased pH falls outside of the normal pH range (~6.05) that presumably alters the
activity of leukocyte esterase. It should be noted that the exact mechanism by which this occurs
is unclear. It is assumed that the high pH denatures the enzyme leukocyte esterase, but the exact
implications require further experimentation with leukocyte esterase. In diseased urine
(specifically diabetic urine), there is usually a more acidic pH, so increasing the pH would
suggest that there is no disease, ultimately leading to a false-negative result. There are also
certain substances that a person might have in their urine that could potentially cause problems
with the dipstick test. 8Ascorbic acid (Vitamin C), which a person acquires from eating fruits and
vegetables, can be present in sufficient amounts so that it is metabolized to form oxalate ions that
are noticeable in urine. The increased amount of oxalate ions can lead to false-positive results
because the oxalate ion is most commonly associated with the calcium oxalate compound.
Calcium oxalate crystals can be found in urine and are the main component of kidney stones. As
such, high concentrations of the oxalate ion would suggest that there is a kidney stone which
may in turn suggest that there is a urinary tract infection, leading to the false positive.7
Furthermore, high levels of glucose and proteins (which can be present due to diabetes, for
instance), although usually negligible, can cause false-negative results in the dipstick. Proteins
and glucose act as solutes, and when present in high amounts they form a hypertonic solution
around the cell that causes crenation (shrinking) of the cell. The crenation prevents the
granulocytes from releasing esterase, consequently leading to a decreased amount of azo dye and
false-negative result. In women, a particular concern is contamination from vaginal discharge,
which may increase the number of leukocytes in the urine. As a result, there would be more
leukocyte esterase and subsequently the test would show up positive.7
Chlorine bleach and other types of detergents can cause false-positive results in the
leukocyte esterase dipstick due to their properties of being strong oxidizing agents. As such, it is
imperative that the storage containers for the urine are properly tended to and do not contain
excess amounts of detergents.9 During the leukocyte esterase reaction, the pyrrole either reacts
with the azo dye or is oxidized to produce an indigo dye that reacts with the diazonium salt to
produce the violet dye. As a result, an increase in the number of oxidizing agents would increase
the amount of colored dye that is produced and hence lead to a false-positive result.7
Figure 4: Formation of false-positive (top) and false-negative results on test strip7
Another important chemical used in storage is formaldehyde, which can lead to a falsenegative result. Formaldehyde is commonly used for fixation of cells. Thus, formaldehyde in the
container would cause the cells to die and not produce any leukocyte esterase, ultimately causing
a false-negative result. Common antibiotics such as tetracycline, cephalexin, and cephalothin
can also cause false-negative results in the dipstick. This is due to their excretion through the
kidney. When they are excreted through the kidney, antibiotics can decrease the activity of
leukocyte esterase, and as a result they lead to less azo dye being produced, which would suggest
that there is no infection.9 Also, it is important to note that dipsticks should not be exposed to air
for too long because this too can cause problems with the dipstick. This is presumably due to the
presence of oxygen in the air, which is a strong oxidizing agent. As such, oxygen has a similar
effect as other oxidizing agents such as chlorine.8
Storage and Maintenance
Specific procedures for collecting and analyzing the urine samples are detailed so as to
minimize the risk of producing unreliable results. First and foremost, the patient SHOULD NOT
urinate directly on the test strip; it should be collected in a sterile container.10 When collecting
the sample, the collection should begin midstream of the urination. This is because the initial
stream is not a reliable aliquot of the urine; concentrations of various urine substances may be
present in seemingly higher or lower amounts. Depending on the test strip, the reaction can take
between 1 - 5 seconds (for newer test strips) or after 2 minutes (for older test strips). The specific
amount of urine needed depends on the test-strip being used.10 Below are sample calculations for
two different types of Walgreens and an Azo Test Strip.
**According to the US National Library of Medicine, the average male and female
urinates 21mL/sec. Since in question two we established that the most up to date urine strips can
react in 5 seconds, we can assume that it will take 105mL to create a visual response.
Walgreens Brand:
21mL/sec x 5 sec = 105mL of urine needed to react.
AZO Products:
21mL/sec x 120sec = 2520mL of urine to react.
Furthermore, storage should be restricted to cool temperatures, but NOT freezing temperatures,
and as mentioned before moisture should be less than 2%. In order to ensure that moisture is kept
at a minimum, a desiccant such as silica gel or activated alumina should suffice. 11
Conclusion
In addition to all the factors earlier discussed such as environmental elements, effects of
the chemical, storage, and maintenance, there are other factors that need to be considered to
make the Ukweli strips more potent in the community for which they will be used. To maximize
the effect of test trips in the communities, we should consider the prevalence of UTIs in the
region. How applicable will these test strips be in the community? Is there actually a dire need
for these user-friendly test strips?
Malnutrition, poor hygiene, and low socio-economic status are associated with urinary
tract infections and these factors are rife in rural settings. In 2014, there was a study that
addressed the prevalence of UTIs in Kenyan communities. According to the study, there was a
40% chance of acquiring a UTI within the community.12 This percentage overwhelmingly
supports the movement towards creating a more hygiene/health informed Kenyan people. By
bringing in user-friendly test strips such as the Ukweli brand, education and accessibility to
health reform can be established in the region. Because the color indicators are so simple to
understand, Ukweli can create a sustainable way to monitor leukocyte levels.
Chemistry really works. It can and is changing people’s lives halfway around the world.
By studying reactions including a double bond, aromatic compound structure, pyrrole amino acid
ester and diazonium salt, we have the power to break down the walls of complex science, and
harness its beneficial ability to bring to rural communities. A complex hydrolysis of diazonium
salt can be delivered simply in the form of a test strip, and truly make a difference, long term, in
people’s lives around the world.
References
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