Monday, 11 December 2017 14:37

Xenodiagnosis: Using a Pest as a Test

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Animals have long added color to the physician’s otherwise rather unvarying armamentarium of pills and elixirs. Leeches, famously used for bloodletting in the medieval and early modern eras, have found a role in modern medicine, where they aid in increasing blood flow and relieving venous congestion following reconstructive microsurgery. Maggots are effective at debriding necrotic tissue in non-healing wounds, and ant mandibles have reportedly been used to suture wounds. In a somewhat cuddlier application of animals to human health, visits from therapy dogs have been shown to reduce post-surgical pain and distress associated with venipuncture in children and to decrease pain and improve satisfaction with hospitalization in adults after joint replacements.

In the realm of diagnostics, animals have not been nearly so prominent, but they have not been absent; African giant pouched rats, for example, have been trained to detect Mycobacterium tuberculosis in human sputum by scent. While xenodiagnosis, or the inoculation of vectors or non-human hosts to confirm a suspected infection, has now has been replaced by simpler and more rapid in vitro assays for most applications, it was used historically to identify elusive infectious pathogens. In a xenodiagnostic test, a specimen from a person with a suspected infection is inoculated into a vector (often by allowing an uninfected insect to take a blood meal from the patient), and the vector is monitored for evidence of infection. The method was most useful for diseases in which the organism burden in the human host is too low to detect reliably by the conventional laboratory tests that were available at the time, but reaches levels that are higher or otherwise more easily detectable in a vector or other non-human host.

A True Bug’s Kiss

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Historically, one of the most well-known applications of xenodiagnosis was in Chagas disease, or American trypanosomiasis, a zoonotic infection found primarily in Mexico and Central and South America. This infection, caused by the protozoan parasite Trypanosoma cruzi, is acquired when a triatomine bug [Fig. 1] takes a blood meal and defecates into the wound created during the blood meal. (Also known as “kissing bugs,” triatomines are members of the order Hemiptera, and thus classified as “true bugs”).  T. cruzi trypomastigotes from the insect’s feces enter the wound created by the bite and invade local cells, where they transform into amastigotes, multiply, and ultimately differentiate again into trypomastigotes, which enter the host’s circulation and invade a variety of sites, including the heart and the gut, where they transform back into amastigotes. During the period of acute infection, high-level parasitemia is present, and trypomastigotes can generally be detected in blood smears or films by microscopy [Fig. 2] or PCR, but this phase of illness is often asymptomatic or presents as a mild illness for which a diagnostic workup is not usually undertaken. By contrast, manifestations of chronic Chagas disease occur when parasitemia has decreased or resolved, making a definitive diagnosis at this stage very challenging. These manifestations, which occur in 20-30% of infected patients, constitute the most severe forms of the disease and include myocarditis, impaired esophageal motility, and colonic dilation (megacolon). Diagnosis of asymptomatic chronic Chagas disease is particularly challenging, as both parasitemia and classic clinical findings are absent, yet this is a period during which treatment with antitrypanosomal drugs, especially in children, can reduce the risk of progression to myocarditis and gastrointestinal disease.

2017.12.11 XenoDx 2The standard method for diagnosis of chronic Chagas disease is serology. However, antibody tests for T. cruzi suffer from limitations in both sensitivity and specificity, and different tests use different methods and different trypanosomal antigens. As a result, international organizations such as the Pan American Health Organization recommend the use of at least two different antibody tests to make the diagnosis. However, some patients with molecular and clinical evidence of Chagas disease may still have negative results on multiple serological tests. PCR tests with sufficient sensitivity to reliably detect T. cruzi in blood during the chronic phase of illness are being developed but are not yet widely available.

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Given the limitations of cutting-edge modern techniques in diagnosing chronic Chagas disease, it is not surprising that the diagnostic process was even more problematic in earlier decades, but investigators found an unlikely ally in the T. cruzi vector itself: the triatomine bug. Microscopic identification of parasites in insects that have fed on humans with chronic Chagas infection is possible because a high burden of trypomastigotes develops in the insects’ feces following a blood meal, even from a human host with a low blood parasite level. In a typical xenodiagnostic procedure, boxes containing 10 laboratory-reared, uninfected, and unfed (i.e., hungry) bugs were applied to a patient’s arms and legs for 30 minutes, allowing the insects to take a blood meal [Fig 3.]. The bugs were kept in the box for around 30 days, at which point their feces were harvested, pooled, and examined microscopically for evidence of trypanosomes. If results at this point were negative, fecal inspection could be repeated after another 30 days. Other procedures describe “squashing” or homogenizing entire bugs prior to microscopic examination.

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At first the vector used in these procedures was the third instar, or developmental stage, of Triatoma infestans, the primary vector of T. cruzi [Fig. 4]. However, further investigations determined that the first instar of a different triatomine species, Dipetalogaster maximus, was equivalent in sensitivity to the third instar of T. infestans. This smaller bug had certain advantages over the third instar of T. infestans, whose large size, as a 1979 paper noted, “resulted in poor patient acceptance.” Further contributing to patient acceptance of D. maximus may be the painlessness of its bite. The bite is so painless, in fact, that D. maximus has been investigated as a stress-free means for obtaining blood samples from zoo animals for chemistry and hematology testing. The painless bite seems to be the result of both the local anesthetic effect of the bug’s saliva and of its very fine proboscis, whose diameter, at 0.02 mm, is 32 times smaller than the outer diameter of the 21-gauge needle (the size typically used to draw blood from human patients) that was used as a comparator method in this study. (No such stress-free procedure has apparently been developed for the bug itself, from whose abdomen the ingested blood in the study was extracting using a 21-gauge needle). In a similar procedure, D. maximus nymphs were used to extract blood from incubating birds in the so-called “blood-sucking bug in a hollow egg” method.
Unfortunately, the sensitivity of xenodiagnosis for Chagas disease is not optimal: in patients with three positive IgG serological tests, only about 50% were positive by xenodiagnosis in the studies described above. (These studies did not, however, investigate patients with suspected Chagas disease but negative serologies to see whether any might have positive results by xenodiagnosis.)

Other Applications of Xenodiagnosis

While T. cruzi remains the most well-known infection identified by xenodiagnosis, there have been attempts to apply similar approaches to other pathogens. Perhaps not surprisingly, one of these is African trypanosomiasis, or African sleeping sickness, which is caused by two subspecies of Trypanosoma brucei (T. b. gambiense and T. b. rhodesiense) and transmitted by the bite of the tsetse fly. As in T. cruzi, patients infected with T. b. gambiense may have low-level parasitemia that is difficult to detect microscopically. In one study, investigators infected two cows and a pig with T. b. gambiense and allowed tsetse flies to feed on them. Flies were dissected 2-5 days after the blood meal, and their midguts were examined for trypomastigotes, which were identified even when the mammalian host had very low levels of microscopically detectable parasitemia. Xenodiagnostic methods have also been described for leishmaniasis, a protozoan parasitic infection spread by the bite of the sandfly, and, in the 1950s, for trichinosis; in the latter case, albino rats were fed post-mortem human muscle tissue to identify Trichinella spiralis roundworms.

A xenodiagnostic method for the apicomplexan parasite Babesia microti involves not only the vector (Ixodes ticks), but also a non-human host (hamsters). In this technique, blood obtained by standard phlebotomy from a patient with suspected babesiosis but negative peripheral blood smears is injected intraperitoneally into a Syrian hamster. Smears of the hamster’s blood are obtained at weekly intervals and examined for evidence of parasites. Because of the availability of sensitive PCR methods, however, xenodiagnosis is not currently used in the diagnosis of babesiosis.

A Future for Xenodiagnosis?

Xenodiagnostics have been largely supplanted by molecular and serological techniques, but they continue to have a certain appeal resulting from their unusual methods and the possibility of detecting very low levels of infection through natural amplification. For example, while there is widespread scientific consensus that the lingering symptoms that sometimes follow completion of treatment for Lyme disease do not represent persistence of Borrelia burgdorferi spirochetes in tissue, an ongoing clinical trial is currently investigating whether spirochetes can be detected in ticks that have been allowed to feed on patients with such symptoms. It’s unlikely, for many reasons, that xenodiagnosis will become a component of clinical testing for Lyme disease, but if the idea of having a laboratory-approved, disease-free arthropod vector feast on your blood to further scientific progress is appealing to you, this trial may be one of your last chances to have the experience.

Last modified on Monday, 11 December 2017 15:00
Thea Brennan-Krohn

Thea Brennan-Krohn is a diplomate of the American Board of Medical Microbiology, having completed a CPEP fellowship at Beth Israel Deaconess Medical Center (BIDMC). She is an attending in Pediatric Infectious Diseases at Boston Children's Hospital and a post-doctoral fellow in the laboratory of James Kirby at BIDMC, where her research focuses on antimicrobial synergy testing for multidrug-resistant Gram negative pathogens. You can follow her on Twitter at @Thea_BK.