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.

            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 smears[v].  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]

 

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 tissues[viii]  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), ELISA[x] and other commercially available tests, such as that developed by InBiOS, the “Trypanosoma Detect™ for Chagas Disease.”[xi][xii]

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]

 

            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]

 

            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 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 effective[xxix]  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, 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.[xxxviii]As 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. 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.[xlvi][xlvii] 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 proved[xlix][l] 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 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.

 



[i] Medical Parasitology, 14

[ii] 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 

[xiii] Medical Parasitology 430

[xiv] 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

 

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“Buffy Coat.”  Wikipedia. 24 February 2010. http://en.wikipedia.org/wiki/Buffy_coatJohn, David T., and Petri, Jr., William A. Medical Parasitology. Philadelphia: Elsiver Inc, 2006.

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