Chapter 49: Pathogenesis and Diagnosis of Parasitic Infection

The pathogenesis of both protozoan and helminthic disease is highly variable. Many factors contribute to this variability and included among them may be factors such as parasite size, induced injury, reproductive potential, nutritional requirements (including metabolites or toxins produced), niche selection (often influenced by individual life cycles and migration patterns through the host), and last, but not the least, immunologic consequences of infection.

Parasite size may or may not be a predictor of pathogenesis. Many of the parasitic Protozoa, including those that cause malaria (Plasmodium), African sleeping sickness (Trypanosoma brucei subspecies), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania), are among the most pathogenic. The giant cestode, Diphyllobothrium latum, can reach sizes exceeding 10 m, yet produces a pernicious anemia due to vitamin B12 competition with the host in less than 1% of the infected individuals. Ascaris lumbricoides, which can grow up to a foot in length can cause severe intestinal blockage if enough worms are present. The larval hydatid cyst of the tapeworm Echinococcus granulosus can achieve considerable size if given long enough to grow and can put tremendous pressure on organs it may be found within.

Parasite-induced injury frequently results from parasite invasion of host tissues. Hookworms, Strongyloides and Trichuris, repeatedly probe the intestinal or colon lining, promoting and inducing extensive, immunologically mediated inflammatory responses. In these cases, worm burden determines the extent of the pathogenesis. The egg laying of schistosome parasites determines the pathology of this infection as many eggs get trapped in tissues in their attempt to leave the host. The result is extensive inflammation and eventual fibrosis of affected tissues.

The reproductive potential of parasites varies considerably. Protozoa generally have very short generational times. In large part, this is due to the asexual nature of their reproduction for much of their life cycles. Rates vary from several hours (African trypanosomes) to several days (malaria). This can place tremendous pressure on host resources with attendant consequences. Helminthes, however, are usually incapable of reproducing within their definitive hosts, so overall worm burden becomes a greater determinant of pathogenesis. This, in turn, will depend on how many eggs, or larvae, initiated the infection. An exception is encountered in Trichinella, in which fertile females residing within the intestinal lining give birth to larvae that migrate to the musculature.

Nutritional requirements among parasites vary tremendously, although most tend to be facultative anaerobes. All Trypanosoma spp. metabolize carbohydrates from their host, but the metabolites are fermentation-like end products of pyruvate that can affect endothelial linings within the host. Malaria parasites of the genus Plasmodium ultimately have rather synchronous infections and produce byproducts of metabolism, including insoluble hemozoin, which when released from infected cells trigger a rise in proinflammatory cytokines that cause fever and impair the functioning of macrophages. Hookworms, because of their voracious appetite and wasteful digestive methods, deplete the iron in the host, resulting in severe anemia if the worm burden is great enough. Hookworms, and many of their allies, also produce powerful enzymes to help predigest what they take in. These enzymes help produce inflammatory responses. Entamoeba histolytica and Trichomonas vaginalis produce enzymes that help mediate contact-dependent cytotoxicity reactions. In the case of E histolytica, this helps the parasite establish extraintestinal sites of infection. In one very interesting case, the death of filarial parasites or their larvae in a definitive host also releases mutualistic endosymbionts. These are felt to contribute to inflammatory responses seen in such infections as those caused by Onchocerca volvulus and resulting in river blindness.

Where the parasite resides, or migrates during establishment in the host, can also be a strong determinant of pathogenesis. Many helminth parasites undergo an obligatory migration through the blood stream that brings them in contact with lung tissue. This required migration often results in Loeffler syndrome that is manifest as an eosinophilic inflammatory response. Larval stages of Taenia solium are frequently encountered in brain tissue, resulting in neurocysticercosis. Parasites such as Toxocara canis may be unable to complete full development in humans, but larvae try to migrate through tissues, causing visceral larval migrans. Many more examples will be expanded upon in chapters that follow.

Finally, there can be numerous immunologic consequences of infection that help promote pathogenesis. Antigen, antibody, and complement complexes combine to cause excessive anemia and glomerulonephritis in African trypanosomiasis. Allergic reactions play a major role in the cutaneous reactions to invading hookworm, Strongyloides, and schistosome larvae (ground itch, swimmers’ itch). Transient pneumonias induced by the pulmonary migration of Ascaris and other nematode larvae (Loeffler syndrome), nocturnal paroxysms of asthma in some patients with filariasis (tropical pulmonary eosinophilia), and the shock, asthma, and urticaria that follow rupture of a hydatid cyst all are immunologically mediated. The latter frequently results in anaphylaxis. Cardiac damage in Chagas disease is thought, at least in part, to reflect immune-induced inflammatory responses, or perhaps autoimmune-related phenomena. Immune complex diseases are seen in schistosomiasis (Katayama syndrome) and malaria (nephrosis). The granulomatous reaction to schistosomal eggs is the result and antibody-dependent, cell-mediated cytotoxic (ADCC) responses. The entire clinicopathologic spectrum of manifestations arising from leishmanial infections appears to be caused by differences in the ability of cell-mediated immune responses to function properly.

The large size, complex structure, varied metabolic activity, and synthetic prowess of most parasites provide their human host with an intense antigenic challenge. Generally, the resulting immunologic response is vigorous, but its role in modulating the parasitic invasion differs significantly from that in viral and bacterial infections. It is apparent from the chronic course and frequent recurrences typical of many parasitic diseases that complete acquired resistance resulting in sterile immunity is often absent. Immunity does, however, frequently serve to moderate the intensity of the infection and its associated clinical manifestations. In fact, clinical recovery and resistance to reinfection in some instances require the persistence of viable organisms at low concentration within the body of the host (premunition = infection immunity). An excellent example of this is seen in patients infected with Toxoplasma gondii.

All those immune responses generally exercised against the more primitive viral and bacterial microorganisms, including innate responses, driven by the complement system, dendritic cells and natural killer cells, and adaptive (acquired) responses, driven by antibodies, cytokines (lymphokines), cytotoxic T lymphocytes, activated macrophages, memory cells, and ADCC mechanisms, have been shown to play a part in modulating parasitic infection.

Innate immune responses are usually immediate, less specific and evolutionarily considered older than adaptive responses. Innate responses often depend on pattern recognition molecules leading to the destruction of bound organisms by complement activation and phagocytosis. Receptor engagement and activation is often critical to further involvement by adaptive responses. One example of innate responses manifest against parasite infections including those seen against malaria. The innate immune response to malaria involves multiple mechanisms, but rarely results in clearance of the parasite. Like other protozoan parasites, Plasmodium falciparum induces the production of IFN-γ by NK cells and subsequent phagocytosis of free parasites by macrophages. NK cells themselves can also lyse parasite-infected erythrocytes. Complicating the picture, improper activation of innate immune mechanisms during malaria may contribute to the disease. For instance, activation of the complement system is a very common finding in human malaria, but excessive complement activation appears to be associated with increased risk of cerebral malaria and severe malarial anemia in children. Likewise, iron sequestration mediated by hepcidin, another innate immune response against malaria, may also worsen anemia by decreasing erythropoiesis.

Overall, parasites are well adapted to resisting host innate defenses. Adaptive responses, therefore, are critical in attempts by the host to control such infections and include both humoral and cell-mediated responses. As already noted, they are usually not perfect.

Antibodies are one line of defense against parasites. They play roles in opsonization, neutralization, complement activation, and ADCC adaptive responses. Antibodies are largely responsible for eliminating populations of trypanosomes from infected individuals. Interestingly, these antibodies are not formed as a result of classical immune stimulation, but because the antigenic signal coming from the trypanosomes consists of T-independent type antigens that can directly stimulate B cells to form antibody. In this case, the antibody is not the classical IgG, but IgM. Although this is useful in eliminating the dominant population of trypanosomes present, another wave of parasites arises because of antigenic variation. Antibodies, if present in high enough concentration, can neutralize sporozoites and merozoites of malaria, thereby preventing them from invading their target host cells, hepatocytes, and red blood cells. Antibody generation against malarial sporozoites using attenuated and recombinant vaccines is currently undergoing multiple pilot clinical trials in developing countries where malaria is endemic. Complement activation does not usually result in direct parasite killing. In fact, many protozoan parasites have evolved mechanisms to avoid complement-mediated killing. Instead, complement appears to play a role in cell-mediated and especially ADCC responses against parasites.

On invasion of tissue, many helminths, and the schistosomes in particular, stimulate the production of IgE, the Fc portion of which binds to mast cells and basophils. Interaction of the antibody with parasitic antigen triggers the release of histamine and other mediators from the attached cells. These may injure the worm directly or, by increasing vascular permeability and stimulating the release of chemotactic factors, may lead to the accumulation of other cells and IgE antibodies capable of initiating antibody-dependent, cell-mediated destruction of the parasite. This is augmented by complement. The specific killer cell involved is often the eosinophil. These cells attach by their Fc receptor site to IgE antibody-coated parasites and degranulate, releasing a major basic protein that is directly toxic to the worm.

Cellular immunity is likewise important as an adaptive response. It is a hallmark of cutaneous Leishmania tropica infections. Skin lesion biopsies show the presence of lymphocytes and macrophages working in synergy to contain parasites. Activated macrophages are quite capable of destroying engulfed leishmanial parasites. However, defects in this type of cooperation can be seen in leishmanial infections that result in mucocutaneal leishmaniasis. Lesions containing these parasites contain plenty of macrophages, but few or no lymphocytes.

Cytotoxic T cells, or CD8+ T cells, play a very important part in response to many protozoan infections. These cells not only produce INF-γ, but can also produce TNF-α, and recently have been show to produce IL-17. Collectively, all the cytokines have been shown to have varying roles for protective responses in toxoplasmosis, malaria, Chagas disease, and leishmaniasis.

Many cellular responses also work in consort with antibody responses to assist in modulating parasitic infections. An excellent example of this is seen in the case of many nematode infections such as Trichinella, Ancylostoma, Necator, and Strongyloides, where intimate association with intestinal tissue is an integral part of the life cycle. Such interactions lead to inflammatory responses that are the result of antigen signaling through the Peyer patches, movement of cells to mesenteric lymph nodes, and clonal expansion of both T and B cells that migrate back to the intestinal epithelium to promote inflammatory responses that depend on both antibody and cell-mediated constituents. The whole idea of inflammation in this instance is to produce an environment inhospitable for the worms, or to induce worm expulsion as in the case of Trichinella.

Many parasites are capable of evading host immune responses. The strategies used vary considerably and allow the parasite to successfully propagate and spread to other hosts. If immune responses were completely successful in eliminating parasites, parasites would no longer be a problem. However, if all parasites killed their hosts, transmission would be interrupted. What good is a dead host to a parasite? The techniques by which parasites have been shown to evade the consequences of the host’s specific adaptive responses are numerous. Included among them are seclusion within immunologically protected areas of the body, continual alteration of surface antigens (antigenic variation), molecular mimicry, and active evasion or suppression of the host’s effector mechanisms. Several Protozoa are shielded from the host defenses by virtue of their intracellular location. Some have even found ways to avoid or survive the normally lethal environment of the macrophage, a first-line defense cell normally intent on destroying pathogens it encounters. Trypanosoma cruzi, for example, escapes from phagosomes into the cytoplasm early during host infection. Toxoplasma gondii inhibits the fusion of phagosomes with lysosomes, thus preventing phagolysosome formation. Leishmania species, capable of neither of these feats, are resistant to the action of lysosomal enzymes and survive in macrophage phagolysosomes. Once macrophages have been activated to sufficient levels; however, the tables are somewhat turned on these parasites.

In the examples given above, T cruzi and T gondii have alternate mechanisms for escaping host responses. They do so by becoming intracellular in cell types not normally involved in immune responsiveness. The gut lumen is perhaps the largest immunologic sanctuary within the body, because, unless the integrity of the intestinal mucosa is breached by injury or inflammation, this barrier protects lumen-dwelling parasites, many of which are surrounded by a protective tegument, or cuticle, from most of the effective humoral and cellular immune mechanisms of the host, allowing survival and the opportunity to reproduce.

Most immune effector mechanisms are directed against the surface antigens of the parasite, and alteration of these antigens may blunt the immunologic attack. Many parasites undergo developmental changes within their hosts that are generally accompanied by alterations in surface antigens. Immune responses directed at an early developmental stage may be totally ineffective against a later stage of the same parasite. Such stage-specific immunity is very evident in malaria because different life cycle stages express different antigens and even give rise to different types of responses. The issue of stage-specific immunity in malaria is further compounded by species-specific immunity. No wonder we still do not have a totally effective vaccine against this disease. Even more intriguing is the ability of some parasites to vary the antigenic characteristics of a single developmental stage. The trypanosomes that cause African sleeping sickness circulate in the bloodstream coated with a thick glycoprotein surface coat. The development of humoral antibody to this coating results in the elimination of parasites from the blood expressing the dominant surface coat. However, within this dominant population of parasites are a few that have undergone antigenic variation and produced a new variant surface glycoprotein coat. This less dominant population gives rise to the next dominant population and this process repeats itself over and over. Over 1000 variant types can arise via this process. The process is genetically and not immunologically driven. The expression of individual genes from this large genetic repertoire is controlled by the sequential transfer of a duplicate copy of each gene to an area of the parasite genome responsible for gene expression. Continued antigenic variation, unfortunately, causes host immunosuppression with attendant consequences.

Several protozoan and helminthic pathogens are thought to be capable of neutralizing antibody-mediated attack by shedding and, later, regenerating specific surface antigens. Adult schistosomes, in addition, may immunologically hide from the host by masking themselves with host blood group antigens and immunoglobulins and through a process known as molecular mimicry by which they produce substances that are transported to their tegument that mimic substances naturally found within the host.

Several parasites can destroy or inactivate immunologic mediators. Tapeworm larvae produce anticomplementary chemicals, and T cruzi splits the Fc component of attached antibodies, rendering it incapable of activating complement. Several Protozoa, most notably T brucei species that are responsible for African sleeping sickness, induce polyclonal B-cell activation leading to the production of nonspecific immunoglobulins and eventual exhaustion of the antibody-producing capacity of the host. This and other Protozoa can produce nonspecific suppression of both cellular and humoral effector mechanisms, also enhancing the host’s susceptibility to a variety of unrelated secondary infections. Patients with disseminated leishmaniasis display a specific inability to mount a cellular immune response to parasitic antigens in the absence of evidence of generalized immunosuppression.

Finally, the thick, tough cuticle of many adult helminths renders them impervious to immune effector mechanisms designed to deal with the less robust microbes.

Diagnosing parasitic infections can test the limits of the best physicians and diagnostic laboratories. Many of these infections are not frequently encountered in most of the industrialized world, as they are elsewhere, and many laboratories do not routinely handle requests to diagnose such infections. In addition, personnel may be poorly trained to adequately diagnose these infections.

The continuous arrival of travelers and immigrants from endemic areas, and the fact that parasitic infections may at times be life threatening, necessitates consideration of these diseases in differential diagnoses. Unfortunately, the clinical manifestations of parasitic infections are highly varied, often mimic other disease conditions, and are seldom sufficiently characteristic to raise this possibility in the clinician’s mind. It is incumbent upon the physician to ask questions related to travel history, food and liquid intake, activities, exposure to biting insects, etc, to raise the possibility that the individual might have acquired a parasitic disease.

Once considered, an appropriate diagnostic test must be ordered. Typically, diagnosis rests on the demonstration and morphologic identification of the parasite or its progeny in the stool, urine, sputum, blood, or tissues of the human host.

A routine blood differential may raise the specter of such an infection. Eosinophilia has been recognized as an important clue to the diagnosis of parasitic disease. However, this phenomenon is characteristic only of helminthic infection, and even in these cases it is frequently variable. Eosinophilia, which presumably reflects an immunologic response to the complex foreign proteins possessed by worms, is most marked during tissue migration. Once migration ceases, the eosinophilia may decrease or disappear entirely.

In intestinal infections, an O&P, or ova and parasite examination may suffice. This may involve concentration procedures such as floatation or sedimentation, followed by a wet mount or stained smear, or both, of the stool sample. Some parasites, such as Giardia, however, may be passed in the feces intermittently or in fluctuating numbers and repeated specimens are needed to confirm infection. Occasionally, specimens other than stool must be examined. In the case of small bowel infections, such as giardiasis and strongyloidiasis, aspirates of the duodenum or a small bowel biopsy may be required to establish the diagnosis. Similarly, the recovery of large bowel parasites such as E histolytica and Schistosoma mansoni may require proctoscopy or sigmoidoscopy, with aspiration or biopsy of suspect lesions. Eggs of pinworms (Enterobius) may be found on the perianal skin and require recovery using a specialized scotch tape application technique.

Parasites dwelling within the tissue and blood of the host are more difficult to identify. Direct examination of the blood is useful for the detection of malarial parasites, Leishmania, trypanosomes, and filarial progeny (microfilariae). The concentration of organisms in the bloodstream may fluctuate, however, and require the collection of multiple specimens over several days. Both wet mount and stained preparations of thin and thick blood smears (see Chapter 51) are used. Timing of blood collection is important in diagnosing filarial infections because they may display marked periodicity. Lung flukes and occasionally other helminths discharge their offspring in the sputum and may be found there with appropriate concentration techniques. In others, larvae can be recovered with skin (onchocerciasis) or muscle (trichinosis) biopsy.

In some infections, parasite recovery is uncommon. Immunodiagnostic and nucleic acid hybridization techniques provide diagnostic alternatives for these situations. Although tests for circulating antibodies have long been available for many parasitic diseases, they have often lacked sensitivity and specificity. The replacement of crude, antigenically complex parasitic extracts with purified homologous antigens, together with the adaptation of highly reactive test systems, has significantly increased the sensitivity and specificity of such tests. Currently, reliable serologic procedures are available for amebiasis, cysticercosis, echinococciasis, paragonimiasis, schistosomiasis, strongyloidiasis, toxocariasis, toxoplasmosis, and trichinosis. More will undoubtedly follow in the near future.

Techniques for the detection of parasitic antigens in blood, body fluids, tissues, and excreta also have been developed. Commercial immunofluorescent and immunosorbent kits for T vaginalis (genitourinary fluids), E histolytica, Giardia, and Cryptosporidium (feces) are now commonly found in clinical laboratories. Less generally available are systems for the detection of malaria antigens in blood and T gondii in tissue.

Even with many recent advances, the acknowledged limitations of both microscopic and serologic techniques in diagnosing parasitic infections have stimulated a widespread interest in resorting to gene amplification techniques to affect a more sensitive and specific diagnosis of these infections. The advent of the polymerase chain reaction (PCR) in its various formats has had a tremendous impact on detecting many parasite infections. Highly specific probes are available for the detection of malaria, Chagas disease, the African trypanosomes, leishmaniasis, toxoplasmosis, cryptosporidiosis, schistosomiasis, cysticercosis, and the etiologic agents of lymphatic filariasis. In some instances, PCR can be multiplexed and conducted in real time (RT-PCR). This permits the simultaneous detection of several parasites from one sample. The probes for many of these parasites have demonstrated sensitivities that match or exceed those of traditional techniques. The major limitations of PCR probes as diagnostic tools largely relate to the technical aspects of the hybridization procedure and, with time, will undoubtedly be overcome.