APPENDIX 1

Trypanosoma cruzi

The full taxonomic name of the Chagas’ disease parasite is Trypanosoma (Schizotrypanum ) cruzi (Chagas 1909). In its biological classification, Trypanosoma cruzi (T. cruzi ) belongs to subkingdom Protozoa, phylum Sarcomastigophora, subphylum Mastigophora, class Zoomoastigophorea, order Kinetoplastida, family Trypanosomatidae, genus Trypanosoma, and species cruzi. Each of these categories reveals something interesting about this parasite.

Trypanosomes fit into the order Kinetoplastida because they are flagellar organisms with a kinetoplast, an organelle unique to this order which contains the mitochondrial DNA and gives rise to the mitochondrion. Sausage‑ or disc‑shaped, a kinetoplast has a single mitochondrion that contains enzymes for respiration and energy production. The DNA fibers run in an anterior‑posterior direction and are organized into a network of linked circles, with up to 20,000 mini‑circles and 20 to 50 maxi‑circles in the kinetoplast network (Battaglia et al. 1983). Like a minuscule computer, these circles program the replication rate and survival activity of the mitochondrion during the cell cycle. Trypanosomes have one nucleus. Although there is indirect evidence of sexuality, it has not been observed directly (Tait 1983), and the organisms reproduce asexually by binary fission.

Trypanosoma cruzi belong to the family Trypanosomatidae, or trypanosomes, which are classified together because during one stage of their lives they live in the blood and/or tissues of vertebrate hosts, and during other stages they live in the intestines of bloodsucking invertebrate vectors (Schmidt and Roberts 1989). They also belong to the subgenus Schizotrypanum, which is a category adopted for trypanosomes that multiply in vertebrates via intracellular stages. Technically speaking, all species are hemoflagellate and heteroxenous and (except T. equiperdum) are transmitted to animal hosts.

Trypanosoma cruzi belong to the stercorian section (A. stercoria ), a subspecies, because its infective stages develop in the vector’s digestive tract and contaminate the mammalian hosts through the vector’s feces. Important factors in the transmission of T. cruzi to humans are lack of personal hygiene, invasion of human habitations by vectors, and a close temporal relationship between the taking of the blood meal and defecation by the vector. Chagas’ disease also has been transmitted by insect feces falling from the ceiling and contaminating foods. Triatoma infestans is a particularly efficient vector because it defecates close to the bite wound, providing T. cruzi easy access to enter the wound.

In comparison, T. rangeli, which is not associated with human pathogenicity, belongs to the salivary section (B. salivaria) because it infects hosts through saliva as well as feces. Because the two parasites can be confused in diagnosis, one way to distinguish T. rangeli from T. cruzi is to examine by xenodiagnosis if the parasite is found in the insect’s saliva. If so, it is T. rangeli, which does not cause Chagas’ disease. The highly pathogenic (to humans) salivary trypanosomes belong to the T. bruceigroup, which are transmitted through the saliva of tsetse flies and cause human sleeping sickness.

Trypanosoma cruzi pass through different forms at various stages in their life cycle, of which three are important in the human context: trypomastigotes, amastigotes, and epimastigotes (see Figure 5). T. cruzi become metacyclic trypomastigotes in the hindgut of the vinchuca bug, and they are the infectious forms for animals and humans. Metacyclic trypomastigotes are 20 microns long, 3 microns wide, and can change positions very rapidly. (A micron equals .001 millimeters.) They are flat on the sides, with a free flagellum (tail) attached to an undulating membrane on the body (Manson‑Bahr and Bell 1987). They have a central nucleus and a posterior kinetoplast. Trypomastigotes occur in peripheral blood of hosts and multiply by splitting lengthwise, with the kinetoplast and nucleus dividing before the cytoplasm and a new flagellum developing from the new kinoplast. Amastigotes are intracellular round and oval forms, 1.5 to 5 microns long, without an external flagellum. They occur within hosts.

All trypanosome species are either elongate with a single flagellum or rounded with a very short, nonprotruding flagellum. The flagellum arises from a kinetosome (core or basal body), is attached to the parasite’s membrane, and pulls the organism by undulating along its side and, at certain life stages, extending forward from its prow. This makes the organisms agile swimmers and intruders into cells. The flagellum may also be used to attach the organism to the insect’s gut wall or salivary glands.

Epimastigotes are easily distinguishable from trypomastigotes, because, in epimastigotes, the flagellum begins near the center of the parasite between the nucleus and kinetoplast, extends along the anterior undulating membrane, and protrudes from the prow (Schmidt and Roberts 1989:57). In trypomastigotes, the kinetoplast and kinetosome are found in the posterior tip, from where the flagellum begins, flows along the membrane of the parasite, and extends from the prow. The epimastigotes in the midgut of the insect are small, 10 to 20 microns, multiply profusely by binary fission, provide a reservoir of parasites, and thus maintain the infection in the bug (Molyneux and Ashford 1983:168).

Longer epimastigotes (35 to 40 microns) travel from the midgut to the rectum of vinchucas, where they adhere to the epithelium of the rectal glands with their flagella. Epimastigotes develop into metacyclic trypomastigotes, which swim freely in the rectal lumen. Metacyclics are 17 to 22 microns long. They no longer divide and are very active. This life cycle takes anywhere from six to fifteen days, depending on the stage of the bug which ingests the parasite and the temperature at which the bug lives (Molyneux and Ashford 1983:168). This reproductive and infective cycle of T. cruzi continues throughout the life of the bug and apparently does no harm to it.

In observation, T. cruzi have no stages outside the gut of insects and the blood and cells of vertebrates. Drugs aside, once host and vector are infected, they generally remain so throughout their lives. This is because T. cruzi can reproduce asexually by binary fission in both the vector and the definitive host.

After metacyclic trypomastigotes enter a mammal’s cells, they reproduce by binary fissions into amastigotes. Amastigotes cluster into cyststiny, entangled snakelike bundles of evolving forms that burst cells, exploding amastigotes into the bloodstream, once again to repeat the process in another cell with such speed that industrial mass production looks slow in comparison. Meanwhile, they also produce stumpy trypomastigotes that circulate in the blood in order that they can be absorbed by blood‑ingesting vinchucas. This reproductive cycle is constant, so that infected humans have trypomastigotes permanently circulating within their blood and amastigotes reproducing in their cells.

Amastigotes frequently develop into short and stumpy trypomastigotes, which are found in chronic phases of the disease. Stumpy trypomastigotes are thought to be the most infective stage to triatomine bugs (Molyneux and Ashford 1983:167). As with African trypanosomes, the morphological form of the trypomastigotes that the insect ingests with blood influences the level of infection: “stout” forms seem to be more infective than are “slender” forms (WHO 1991:22). During the acute phase, triatomines are less likely to ingest trypomastigotes in the blood of the host because the host’s immune system is attacking these forms and driving them into the sanctuary offered by host cells. In the chronic phase, the slower, short, stumpy trypomastigotes can leisurely wait for ingestion by “kissing bugs,” because immunosuppression has reduced the risks of attack in the circulation system of the host. Although the numbers of trypomastigotes seldom reach the levels seen in the blood during the acute phase of Chagas’ disease or during African trypanosomiasis, the trypomastigotes of T. cruzi are able to increase their numbers in the peripheral circulation system of the chronic patient.

The advantage of circulating in the blood is that T. cruzi can then readily transmit its progeny to insects, who provide another environment for development and transportation to other hosts. This strategy assures that even though T. cruzi ultimately destroy the host in which they presently reside, their offspring will carry on in another host. Their migration between cells within the same host ensures a ready supply of new nurseries for their offspring, while transmission via insects promotes their movement from one host to another, whether sylvatic or domestic animals or humans. The disadvantages of circulating in the blood are that T. cruzi are under attack by the humoral immune system, and one strategy the organism has developed is to spend as little time in the blood as possible.

Trypanosome cruzi employ an important adaptive strategy seen with many parasites in that they try to maintain their own population levels within the carrying capacity of the host organism. It is not to their advantage to destroy their hosts too quickly, or, as George Stewart says, “They don’t want to burn down their home before they have acquired a replacement.”[77]With sound practicality, then, Chagas’ disease has a relatively low incidence of parasitemia in its acute phase.

After the vinchuca bug has ingested trypomastigotes, another reproductive process takes place within the insect’s gut. Insects do not get sick or suffer from T. cruzi– except for the loss of nutrients (WHO 1991:22). In contrast, however, a closely related parasite, Trypanosoma rangeli, which is harmless to humans, has a pathogenic effect on insects. After trypomastigotes have traveled to the anterior intestine, they take on epimastigote forms, then travel to the midgut and change into amastigotes and proliferate. Amastigotes next give birth to metacyclic trypomastigotes in the rear of the intestines and from there are transported through the feces the next time the insect takes a blood meal. Delay time between ingestion of a blood meal and defecation is a large factor in infection rates of triatomine vectors; Triatoma infestans, vinchucas, have a rapid time period.

Within the insect, T. cruzi go through their reproductive cycle in eight to ten days and produce as many as 100 protozoa. The number produced is affected by the blood‑meal size, the number of parasites ingested, the stage and age of the insect vector, the ability of the parasite to establish rectal gland infections in the vector, and the kinetics of parasite transformation in the insect’s digestive tract (WHO 1991:22).

The susceptibility of triatomine vectors to infection with T. cruzi varies greatly among different vector species and with their interaction with strains of T. cruzi (WHO 1991:22). The presence of three main isoenzymic strains (zymodemes) within T. cruzi was discovered in Brazil (Miles et al. 1977; Ready and Miles 1980) and more have been found in Bolivia (Tibayrenc et al. 1984) and Chile (Gonzalez et al. 1995). The geographic frequency of occurrence of the principal isoenzymic strains has been described in these countries and is important to pathogenicity (Brénière et al. 1989, Miles, Provoa, Prata, et al. 1981) and genetic variability of T. cruzi (see Appendix 2).