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Dodge Kemmer (05362584)
Parasites and Pestilence, Winter 2010
Dr. Scott Smith
The Current State of Molecular Diagnostics for Parasitic Protozoa
Parasitic protozoa affect millions of people worldwide and account for hundreds of
thousands of deaths annually. These diseases are most common in places of poor
sanitation, and so affect a disproportionate amount of poverty-stricken communities and
countries, in which baseline healthcare is virtually non-existent. This makes it imperative
that these communities have access to cheap and easy diagnostic tests and treatment to
prevent further deaths and cases caused by these diseases.
In addition to amebiasis (represented later by Entamoeba histolytica), which infects
1% of the worlds population and causes 100,000 deaths per year, the other eight protozoan
parasites discussed herein (malaria’s 500 million infected and 2.5million deaths per year is
excluded) account for at least 25 million infections and about 250,000 deaths per year.i As
Lynne Garcia points out, diagnostics for these types of infections (referring specifically to
Giardia, E. histolytica, and Cryptosporidium) must not only “be acceptable in terms of
sensitivity and specificity but they must provide clinically relevant, cost-effective, rapid
results, particularly in a potential waterborne outbreak situation.”ii Most of these parasites
do have dependable, easy, and affordable diagnostics available, which can be facilitated in
prescribing acceptable treatment. These include Trichomonas vaginalis, Trypanosoma cruzi
(Chagas’ disease), Entamoeba histolytica, Toxoplasma gondii, Giardia, and Balantidium coli.
These protozoa and their respective available diagnostics are explored first.
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However, two of the most fatal non-malarial protozoa infections—Leishmanaisis
and African Trypanosomaisis (also known as African sleeping sickness)—do not have
acceptable diagnostic tests available. The toxicity and monetary cost of treatment for these
diseases makes it imperative that diagnoses are accurate. As Marleen Boelaert observes,
“starting a course of anti-leishmanial treatment solely on the basis of clinical suspicion is
not acceptable.”iii And as Pere Simarro, et al, suggests, referring to African
Trypanosomaisis, “The desired characteristics of a new test [include] being ‘ready for use,’
stable at room temperature, and affordable by national health systems. The new test should
provide an uncontroversial diagnosis of both forms of the disease and require minimum
training and equipment to allow its execution by any health worker.”iv Below, I will explore
the current state of diagnostics and their drawbacks for these two diseases, as well as
present the roadblocks in the availability of more effective diagnostic tools.
Trichomonas vaginalis is a sexually transmitted disease affecting between 2 and 3
million American women annually. Trophozites are transmitted during intercourse and
are found in vaginal and urethral discharge during infection. T. vaginalis is easily
diagnosed by the observation of trophozites in wet film preparations or Pap smearsv.
However, a quantitative buffy coat (QBC) tube test has been shown to be slightly more
effective than a wet smear, and found to be 100% sensitive and 92.3% specific.vi
A QBC diagnostic involves introducing a blood sample to a fluorescent dye-coated
tube and then centrifuging. The buffy coat, or thin layer between the plasma and the red
blood cells, which contains white blood cells and platelets, will fluoresce under an
ultraviolet light if T. vaginalis is present.vii
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Chagas’ disease—or American trypanosomaisis—is caused by the Trypanosoma
cruzi species and is found in the Americas. It is unique among trypanosomes as it can dwell
not only in the blood but also in cardiac muscle and other tissuesviii It is transmitted by the
reduvvid bug through its feces, and most commonly affects young children. ix Chagas’ can
be diagnosed by indirect fluorescent antibody (IFA), ELISAx and other commercially
available tests, such as that developed by InBiOS, the “Trypanosoma Detect™ for Chagas
Disease.”xixii
In an IFA assay, known antigen from the parasite is fixed on a slide, to which serum
from a patient is added. If antibodies from the serum recognize the antigen, they will bind.
After the rest of the serum is washed off, a secondary, fluorescently labeled antibody
(usually an antibody to IgG or IgM) is added and detection is seen by fluorescence under a
UV light.xiii
The Chagas’ ELISA uses T. cruzi coated plates to which sample serum is added. If
specific antibodies are present, they will bind to the T. cruzi antigen. After the superfluous
serum is washed, a secondary, enzyme-linked antibody specific for the test antibody
(human Ig) is added. Finally, solution that reacts with the linked enzyme is added, and
antibody that is bound to sample antibodies that recognized T. cruzi will produce a color
change, indicating a positive result.
The InBiOS test requires only adding a sample of blood and buffer solution to their
strip and waiting ten minutes, and has “excellent sensitivity and specificity.”xiv
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Entamoeba histolytica is a lumen-dwelling parasite that feeds on red blood cells and
can cause ulcers, which can in turn cause amoebic dysentery. It infects between 1% and
5% of the worlds population, most often in areas of poor sanitation.xv E. histolytica can be
diagnosed by ELISA and by indirect hemagglutinin (IHA), as well as by assays such as the
Triage parasite panel, an enzyme immunoassay (EIA) that can also detect Giardia lamblia
and Cryptosporidium parvum (discussed later).xvi
An IHA assay uses antigen-coated sheep erythrocytes subjected to test serum. If
antibodies to the antigen are present, they will bind and cause agglutination of the sheep
cells. It is mostly a qualitative assay.xvii
Generally EIA involves the binding of antibody to antigen and an enzyme-linked
visualization method. In the Triage parasite panel EIA, antigens specific for E. histolytica,
Giardia, and Cryptosporidium and fixed on a membrane in a test device. Diluted test sample
is added to the device along with enzyme conjugate and wash solution. Control and
positive lines appear in the window.xviii
Toxoplasmosis is transmitted to humans by ingestion of Toxoplasma gondii oocysts
found in cat feces or trophozites and cysts in infected meat. Most infections are
asymptomatic except in the very young and elderly, in whom symptoms can be severe and
include blindness, with complete recovery being rare.xix Many diagnostics for T gondii
exist, including multiple antibody tests and histologic diagnoses, as well as PCR,xx including
IHA and IFA (mentioned above).xxi
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Cryptosporidium parvum is usually associated with contaminated water and can be
avoided by filtering or boiling surface water, but outbreaks still occur in the US. Diagnosis
methods include observing oocytes in stool specimens, the duodenal string test, or (as
mentioned above), the Triage parasite panel.xxii xxiii
The duodenal string test is utilized when no sample is found in the stool; it involves
swallowing a string that picks up a bile sample to be later used in microscopy.
Giardia is the most common protozoa found in the US; it attaches to the small
intestine using a sucking disk. As Giardia lambia does not consistently appear in stool of
infected individuals, samples from a span of a few days must be observed when using this
technique.xxiv Duodenal fluid obtained by a string test, outlined above, may also be
examined for “one of the most easily recognized intestinal protozoa.”xxv Balantidium coli is
rare worldwide and even more rare in the United States. It can be observed with
microscopy by the organism’s appearance and shape.xxvi
Human African Trypanosomasis (HAT), the African cousin of Chagas’ disease, is
caused by the protozoa Trypanosoma brucei gambiense or T.b. rhodesiense and is
transmitted by tsetse fly bites. It initially causes ulcers and lymph node swelling, followed
by dissemination marked by fever and fatigue, and finally, during the invasion period,
disrupted sleep patterns, meningoenchephalitis, and death if not treated. The protozoa
itself is pleomorphic, with physical characteristics that can be anywhere between a slender
shape of 30um with flagellum to a shorter, rounder form lacking flagellum. This, and the
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fact that T.b bruci, rhodeniese, and gambiense are morphologically indistinguishable make
microscopy a daunting diagnosis.xxvii
Clinical manifestations include elevated IgM levels (caused by the antigenic
variability of the protozoa—mentioned later), but this can be observed in non-infectious
individuals, so are not conclusive. However, normal IgM levels exempt a patient from a
positive diagnosis.xxviii Further diagnosis involves observation of trypanosomes in blood,
spinal fluid, or lymph, but this is not wholly effectivexxix A misdiagnosis of malaria (which is
often co-infective) is common, and if treated as such, can be determined successful, leaving
the ‘cured’ patient with an unnoticed case of Trypanosomiasis.xxx Because of this, as well
as the fatality ratio of the protozoa and the late stage treatment, PG Kennedy states the
“pressing need for a quick, simple, cheap and reliable diagnostic test to diagnose Human
African trypanosomiasis in the field.”xxxi
One of the currently used diagnostic tests is the card agglutination test for
trypanosomiasis (CATT), which uses a sample of patient blood or serum to detect
antibodies to the parasite. Although the CATT test is “quick and easy to perform,”xxxii
“CATT-positive results are not sufficiently sensitive and specific to establish a definitive
diagnosis, and therefore parasitological tests must be performed to confirm the presence of
parasites in seropositive individuals;”xxxiii tests which are not easily performed in the field
and insufficiently sensitive.xxxiv
A unique characteristic of the T.b. protozoa, which accounts for patients’ high IgM
titers, as well as insufficient reliability of the CATT test, is the antigenic variation in their
surface proteins, known as variant surface glycoproteins (VSGs).xxxv Being on the surface of
the organism, it is the only antigen that the human immune system can bind and react to,
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which makes the VSG an effective defense to all immune effectors.xxxvi Additionally, there
are as many as 1000 unique VSGs, any number of which can be present in a single infected
individual.xxxvii This makes quick and easy diagnostics difficult to develop because one
would have to be able to test for all VSGs, and, more importantly, be sensitive enough to
detect the antibody produced as a response to only a fraction of the trypanosomes present
in the infected individual.
In 2007, the WHO, after observing a 69% reduction in new cases of T. gambiense and
21% reduction of T. rhodesiense, concluded that elimination of the disease was
possible.xxxviiiAs a result, it has partnered with the Foundation for Innovative New
Diagnostics, which is currently working on multiple diagnostic techniques for
Tryptomiasis. These include fluorescent microscopy, which detects malaria as well, but
many of the drawbacks of microscopy mentioned above still pertain.xxxix Additionally, an
antigen detection test, using a variety of Trypanosoma high-affinity antibody probes to
detect the antigen is being developed. Finally, a loop-mediated isothermal amplification
(LAMP) of DNA, which is more feasible than PCR because of its isothermal nature, ability to
analyze large numbers of samples at one, and color- or fluorescence- based positive
identification techniques, has shown high sensitivity and specificity to Trypanosoma.xl
The second protozoan disease lacking an acceptable diagnostic test is
Leishmanaisis. Leishamnaisis, caused by species of Leishmania comes in three different
forms: cutaneous, occurring usually on the face, mucocutaneous, affecting the nose and
mouth of the patient, and visceral, which is the most lethal, boasting an 11% case fatality
ratio.xli The former two are easily diagnosed by oriental sores characteristic to that type.
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Diagnosis of visceral leishmaniasis, however, is not as easy: clinical features are similar to
those of malaria, typhoid, and tuberculosis, which can all co-infect, and the protozoan is
often sequestered in the spleen, lymph nodes, or bone marrow.xlii Boelaert claims, “several
antibody-detection tests have been developed for field diagnosis of VL, but…none [are]
sufficiently specific for acute VL disease to be used as a stand-alone test.”xliii An effective
and accessible diagnostic is important given the fatality of the visceral form of the disease
as well as unavailability of a vaccine. The ability of a diagnostic to distinguish “the
detection of infection from the diagnosis of VL disease” is also important,xliv as a patient can
be infected but show no signs of the disease and therefore not require any treatment.
Today, diagnosis depends on the demonstration of the parasite either by splenic or
liver puncture, both of which are often unavailable in endemic areas and are invasive,
hence not “quick and easy.”xlv Buffy coat films and multiple serological tests prove effective
at times, alongside clinical case definition, but more conclusive tests are still needed.xlvixlvii
Factors minimizing the effectiveness of these tests include the inability to detect the
difference between a new case, a relapse, an asymptomatic case, and a cured individual,xlviii
as well as the lack of protozoan in the blood available to make blood tests feasible.
The development of “recombinant K39 (rK39)” ELISA rapid diagnostic test
provedxlixl promising, yet the format achieving 100% sensitivity and 98% specificity is no
longer commercially available.li Currently available options include aspiration for
detection of the parasite, which achieves only variable sensitivity; direct microscopy, which
reports between 50 and 85% sensitivity for lymph node or bone marrow samples; and the
serological test DAT, which has had some favorable results but requires at least eight hours
of incubation.lii Additionally, an indirect immunoflourescence test (IFAT) has been shown
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to discriminate between the acute and remission phases.liii As the parasite triggers IgA,
IgM, and IgG antibody production,liv the development of an antibody-detection kit seems
feasible. However, due to the sequestration of the parasite mentioned above, observing it
in a blood sample using mounted antigen-specific antibodies would be difficult.
Most effective molecular diagnostic techniques, used on the first six diseases
discussed, take advantage of the ability of the human immune system to produce
antibodies specific to that given disease (those that aren’t—like the QBC test—rely on the
parasites’ presence in the blood.) These antibodies can either be detected directly through
a blood sample, or used as a probe to detect the antigen in a sample from a patient.
However, in African Trypanosomaisis and Leishmanaisis, either there is not sufficient
antibody produced in response to the parasite, or it is not present in the blood or tissue
from which a sample can easily be obtained. New and innovative techniques for diagnoses
of these protozoan parasites need to be developed to reduce the cost of lives and money
these diseases command.
Medical Parasitology, 14
Garcia, et al
iii Boelaert, el al
iv Simarro, et al
v Medical Parasitology 56
vi Hammouda, et al
vii “Buffy Coat”
viii Medical Parasitology 117
ix Medical Parasitology 118
x Malan, et al
xi Medical Parasitology 122
xii Inbios.com
i
ii
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Medical Parasitology 430
Inbios.com
xv Medical Parasitology 22-23
xvi Garcia, et al
xvii Medical Parasitology 431
xviii Garcia, et al
xix Medical Parasitology 143
xx Palo Alto Medical Foundation
xxi Medical Parasitology 430-1
xxii Medical Parasitology 68
xxiii Garcia, et al
xxiv Medical Parasitology 50
xxv Ibid 49
xxvi Ibid 62
xxvii Ibid 110
xxviii Ibid 112-3
xxix Ibid 112
xxx Kennedy, PG
xxxi Ibid
xxxii Ibid
xxxiii Simarro, et al
xxxiv Ibid
xxxv Medical Parasitology 112
xxxvi Barry, JD, and McCulloh, R
xxxvii Medical Parasitology 113
xxxviii Simarro, et al
xxxix Find: Sleeping Sickness
xl Ibid
xli [Class notes]
xlii Sundar, Shyam, and Rai, M.
xliii Boelaert, et al
xliv Ibid
xlv Medical Parasitology 137
xlvi Ibid 137
xlvii Boelaert, et al
xlviii Ibid
xlix Ritmeijer, et al
l Zijlstra, et al
li Boelaert, et al
lii Ibid
liii Millesimo, et al
liv Medical Parasitology 138
xiii
xiv
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