chagas disease - a review - teixeira e cols - 2006

8/2/2019 Chagas Disease - A Review - Teixeira e Cols - 2006

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REVIEW

Chagas diseaseA R L Teixeira, N Nitz, M C Guimaro, C Gomes, C A Santos-Buch. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Postgrad Med J2006;82:788798. doi: 10.1136/pgmj.2006.047357

Chagas disease is the clinical condition triggered byinfection with the protozoan Trypanosoma cruzi. Theinfection is transmitted by triatomine insects while bloodfeeding on a human host. Field studies predict that onethird of an estimated 18 million T cruzi-infected humans inLatin America will die of Chagas disease. Acute infectionsare usually asymptomatic, but the ensuing chronic T cruziinfections have been associated with high ratios ofmorbidity and mortality: Chagas heart disease leads tounexpected death in 37.5% of patients, 58% develop heartfailure and die and megacolon or megaoesophagus hasbeen associated with death in 4.5%. The pathogenesis ofChagas disease appears to be related to a parasite-induced mutation of the vertebrate genome. Currently,treatment is unsatisfactory.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article for

authors affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr A R L Teixeira, ChagasDisease MultidisciplinaryResearch Laboratory,Faculty of Medicine,University of Bras lia, POBox 04536 70919-970,Federal District, Brazil;[email protected]

Received 6 March 2006Accepted 13 July 2006. . . . . . . . . . . . . . . . . . . . . . .

American trypanosomiasis is an enzooticinfection caused by the kinetoplastid fla-gellate Trypanosoma cruzi. The spread of the

enzootics coincides with the distribution of thetriatomine vectors from parallel 42 N in thesouthern US to 42 S in Patagonia, Argentina.1

Fossil remnants have suggested that humansarrived on the American continent about

50 000 years ago. It may be supposed that thetriatomines involved in the life-cycle of T cruzi,eventually transmitted the infection to Amerindians approximately 9000 years ago,probably when triatomines adapted to humandwellings in Chile and Peru, adjacent to the Atacama desert.2 Paleoparasitological studiesshowed that 97 (40.6%) of 238 mummies yieldedmolecular data consistent with T cruzi infection.It is reasonable to suppose that Americantrypanosomiasis was a serious burden to Amerindians in pre-Columbian days. It is nowknown that T cruzi infection was readily trans-mitted to European and African settlers livingclose to triatomine wild-life ecotopes in the

American continents.Chagas disease is the clinical condition asso-

ciated with T cruzi infection, described in thepioneering work of Dr Carlos Chagas, a Brazilianphysician who worked at the Oswaldo CruzInstitute, Rio de Janeiro.3 He described thedividing epimastigote and the metacyclic trypo-mastigote forms ofT cruzi in the fore-gut and thehind-gut, respectively, of a Panstrongylus megistuscaptured in a human hut in the hinterland ofMinas Gerais State, Brazil, in 1909. Next, hereported that he had found trypomastigotes inthe blood of a mammal, a domestic cat.

Serendipitously, he investigated a 1-year-oldchild with fever of unknown origin and showedT cruzi trypomastigotes in a peripheral bloodsmear by direct microscopic examination.4

T C R U Z I COLONISATION OF THE HUMANBODYInsect vectors of T c ru z i infectionAt least 40 triatomine species can harbour T cruzi,thus they are all potential transmitters ofinfection.5 The Triatoma sp, which adapted tothe rural peri-domicile areas and domiciles of dryecosystems of Central and South America,6 arethe main transmitters of the infection (fig 1A, B).These species have been implicated in 1012

million cases of Chagas disease in humans. Nextin epidemiological importance are the Rhodniussp, dwelling in humid tropical climates, and theubiquitous Panstrongylus sp.1 5 After having ablood meal, the triatomine becomes engorgedand the infective metacyclic trypomastigotes arepassed in the faeces. The parasitic form entersthe body when the individual scratches the skinwound inflicted by the insects mouth-part, or itmay enter the body through permissive mucosaor conjunctival membranes. On entry into thebody, the trypomastigotes (fig 2) that invadetissue histiocytes survive the acidic parasitophor-ous vacuoles and leave the hostile environmentto freely enter the host-cell cytoplasm. The portof entry of the infection in the skin or in the eye

conjunctiva is clinically seen as an indurate,chronic inflammatory process typical of adelayed-type hypersensitivity reaction. The fla-gellates round up into amastigotes that undergomany cycles of multiplication by binary fission.The cell fills with amastigotes (fig 3), which willdifferentiate into mobile trypomastigotes (fig 4)that burst out reaching the blood stream ready toinfect other cells of the body. Early papers haveshown a direct relationship between host cellmembrane receptor density and parasite load intissues.7 8 In fact, every cell in the human bodywith the exception of neurones could be affectedby T cruzi colonisation in vivo. T cruzi infectionpersists in the human body for life.

Blood transfusionT cruzi infection can be readily transmitted byblood transfusion.9 10 The migration of peopleinfected by T cruzi poses a threat to thosecountries where this insect-transmitted diseasedoes not occur. Chagas disease has become apotential problem associated with migrationfrom endemic areas to the US, Canada, EasternEurope, Australia and Japan.11 About 8 (3%) of250 patients from Central America were found to

Abbreviations: kDNA, kinetoplast DNA

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be seropositive in Brooklyn and Queens in New York City (CASantos-Buch, unpublished observation), and a similar pro-

portion was found among Nicaraguan immigrants inWashington, DC.12 Appropriate selection of blood donors,the use of sensitive screening tests and the application of amandatory quality assurance system have maintained thesafety of blood banks in most Latin American countries.13 Theaddition of cresyl violet to donor blood while in storage inBuenos Aires blood banks was effective in preventing thetransmission of T cruzi. The prevention of transfusion-associated protozoan infections depends mainly on selectionof donors.14 Additionally, the use of more sensitive andaccurate advanced molecular diagnostic tests has greatlyreduced the risk of acquisition of blood-borne Chagasdisease.15

Congenital transmission of T cruzi has been reported inLatin American countries where Chagas disease is prevalentin those of reproductive age. Congenital transmission rangesfrom 2.5%16 in northeastern Brazil to 9.5% in Bolivia.17

Additionally, T cruzi can be transmitted by organ transplant,by laboratory accidents and by accidental self-inoculation byhospital personel.

DiagnosisDirect demonstration of the parasite in a living mammalianhost can be achieved only in the acute phase, by microscopicobservation of a fresh blood smear having the motileparasites amid red cells. Chronic infections require diagnosticprocedures that rely on parasite amplification before they canbe detected by microscopy. Xenodiagnosis consists of allow-ing a parasite-free triatomine to have a blood meal from theclinically suspected candidate and examining the bugsexcrements 30 and 60 days later for the presence of theparasite.18 A parallel diagnostic attempt can be made by

inoculating citrate-treated blood in axenic culture mediumfollowed by a microscopic search for the parasite at set times.The sensitivity of both methods varies with the ability of alow number of parasites to multiply in fresh medium andrequires long periods of time. The immediate detection ofchronic Chagas disease relies on the indirect demonstrationof specific anti-T cruzi antibodies in patient serum or onnucleic acid-based tests.18 High-fidelity tests19 20 to detectspecific antibodies include indirect haemagglutination, indir-ect immunofluorescence and the enzyme-linked immuno-sorbant assay. The sensitivity of individual tests ranges from96.5% to 100% and their specificity from 87% to 98.9%.However, cross-reactive antibodies to T cruzi-specific antigenscan be present in the serum of some patients with other

A

B

Figure 1 Chagas disease and lifecycle of Trypanosoma cruzi.(A) Geographical extent of human Chagas disease in Central and South

America. Dark areas indicate regions showing high ratios of insect-vector-transmitted T cruziinfections. Adapted from Silveira.6 (B) Sylvaticand peri-domicile lifecycles of T cruzi in early mammalian hosts and inhumans. Haematophagous bugs contaminated with the parasite can beingested by or feed upon opossum, armadillo and rat (left). Alternatively,the triatomine can feed on man to initiate the peri-domestic cycle ofChagas disease (right). Note the ports of entry of the parasites into thehuman body as evidenced by Romanas sign (top) and chagoma(bottom).

fp

bb

nDNA

kDNA

Figure 2 Electron microscopy of the Trypanosoma cruziamastigote.Note the nucleus (nDNA), the kinetoplast (kDNA), the basal body of theflagellum (bb) and the flagellar pocket (fp). Magnification 64800.

Transmission

N Protozoan Trypanosoma cruzi is the causal agent.

N The disease is transmitted by blood-feeding triatominebugs.

N Blood transfusion is the second most important mode oftransmission.

N Placental transmission of Chagas disease can occurfrom the mother to child. Transmission by organtransplant and by accident is not unusual.

Chagas disease 789


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kinetoplastid infections, with some intracellular parasite orbacterial diseases (eg, malaria, toxoplasmosis, paracoccidioi-domycosis, tuberculosis, leprosy, syphilis and pemphygus)and with some autoimmune conditions (eg, rheumatoidarthritis, systemic lupus erythematosus, etc), albeit at muchlow titres.21 22 Differential diagnosis can be achieved byimmunoblot assays.23 Diagnoses can be confirmed by theuse of nucleic acid tests such as polymerase chain reactionamplification with specific primer sets followed by hybridisa-tion with internal probes.2426 In view of the major caveatsleading to false-negatives and false-positives, it has beenagreed that at least two immunological tests are required toconfirm a diagnosis of Chagas disease. 20 27

Epidemiology and controlThe unquestionable benefits of major insecticide-basedcontrol programmes28 showing abrupt drops of T cruziinfection in young populations need to be emphasised. It isexpected that reported success will be reflected in low deathrates three decades after dislodgment of domestic Triatomainfestans. The progress achieved thus far provides a window of

opportunity before the triatomine develops resistance to the widespread and indiscriminate use of pyrethroid insecti-cides.29 More measures for control aimed at a sustainablereduction in prevalence of Chagas disease in the tropical rain-forest ecosystems of Central and South America have beenenvisaged3033. Above all, control cannot be satisfactorilyachieved by means other than social development andimprovement of human living conditions. Particularly,interventions to prevent infection by T cruzi and improvechildrens health mean poor housing has to be improved.34

However, given the large pool of primary hosts for thisenzootic disease, complete control of T cruzi arthropod vectortransmission to the human population seems daunting.Demographic change in Latin America has been marked over

the past four decades. For example, in the 1960s, 75% of thepopulation lived in rural areas, whereas at present 81% live inmetropolitan areas. As a consequence of the rural exodus,millions of people with Chagas disease live in the largestcities.35 The rural exodus appears to be related to themigration of people with Chagas disease to the cities for jobsand education, and thus the disease has spread to moresocioeconomic classes. The spread of Chagas disease in urbanareas needs to be reviewed separately on the basis of activevector-transmitted T cruzi infection in populations inhabiting

the outskirts of major cities in many Latin Americancountries.3640 Furthermore, localised transmission of acuteChagas disease has been associated with the oral route ofcontamination.4145 Overall, Chagas disease in urban areas hasbeen associated directly with about 1 death in 10 amongpeople aged between 25 and 64 years.46

Effective prophylaxis against T cruzi appears to be aformidable task as there are elusive biological challenges.An effective immunoprophylaxis against intracellular infec-tion is unattainable because specific, concomitant, steady-state immunity poses a somewhat effective barrier againstparasite circulation in the blood, but does not eliminateparasites that colonise non-phagocytic muscle cells, allowingthe protozoa to persist throughout its hosts life.1 Thus, vaccination against Chagas disease is not feasible with thecurrently available biotechnologies.9 29 On the other hand,chemotherapy for T cruzi infection may be ideal because it would curtail parasitism in reservoir hosts, diminishingperidomestic insect vector dissemination. Anti-trypanosomenitroderivatives that suppress blood parasitism show severetoxicity, requiring careful prescription.27 4753

T cruzi infections occur in a circumscribed region of the world that represents a relatively small market. Accordingly,pharmaceutical companies have been reluctant to investresources for the development of vaccines and drugs tocontrol and treat Chagas disease.

CLINICAL MANIFESTATIONS Acute Chagas disease Acute cases of Chagas disease are characterised by thepresence of the parasite in the patients blood, thus allowingthe demonstration of T cruzi trypomastigotes by directmicroscopic examination of a fresh blood smear. Often, theacute phase of the infection is not perceived by the patient;95% of the cases are asymptomatic. In the remainingsymptomatic cases, the clinical manifestations of acuteChagas disease are fever, malaise, muscle and joint pains,somnolence, cramps and diarrhoea, oedema, respiratorydisturbances, cyanosis and comma. Considering that ,5%

Diagnosis

N Acute infection: parasite detection in blood smears andimmunglobulin (Ig)M antibody tests.

N Chronic infection: xenodiagnosis, haemoculture andIgG immunological assays.

N Two consistently positive immunological tests arerequired.

N Nucleic acid tests are required to confirm diagnosis insome cases.

k n

f

Figure 4 Trypanosoma cruzi trypomastigote blood form showing itsnucleus (n), kinetoplast (k) and the flagellum (f). Giemsa stain, 61000.

Figure 3 Dividing forms of Trypanosoma cruzi in cells. Multiple heartcells are filled with amastigote forms of the parasite (arrows).

790 Teixeira, Nitz, Guimaro, et al


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of patients with acute symptoms die,54 it can be estimatedthat mortality in this phase of the infection is between 1:2500and 1:5000. Death in the acute phase of the disease is causedby myocarditis or meningoencephalitis, with attendantcomplications such as bronchopneumonia.55 The acute phase

ofT cruzi infection usually goes into remission spontaneouslyand the infection enters the chronic stage within 34 monthsof its onset.

Chronic Chagas disease Acute cases with or without symptoms of T cruzi infectionlead to the chronic stages, which can present in any agegroup.56 Interestingly, two thirds of 18 million peopleharbouring chronic T cruzi infection do not show anydetectable clinical manifestation of Chagas disease. Peopleconsidered to be in the indeterminate chronic phase of theinfection do not die of Chagas disease. About one third of thechronically infected human population develop clinicalmanifestations of the disease.56 The symptomatic diseaseaffects the heart in 94.5% of cases; these patients are

considered to have chronic Chagas heart disease. Heartinsufficiency is related to the cause of death in 58% ofpatients, whereas arrhythmias have been associated withunexpected deaths in 36.5%. The remaining 4.5% of patients with chronic Chagas infection show mega syndromes, adisease state that involves the oesophagus (megaoesophagus)and the colon (megacolon).57 Chronic Chagas heart diseaseaffects both sexes equally, frequently at between 30 and45 years of age. In the group of patients showing progressivechanges on electrocardiogram, unexpected death can occur in37.5% of cases. Importantly, 58% of the patients with chronicChagas heart disease show ominous signs of heart insuffi-ciency and frequently die 7 months to 2 years after the onsetof symptoms.56 People with Chagas disease with cardiac orgastrointestinal manifestations of the disease may also show

widespread lesions involving the sympathetic and parasym-pathetic peripheral nervous systems. These chagasic nervoussystem lesions specifically explain the physiopathologicalcondition of the heart and the mega syndromes observed atthe bedside.58 59

Chagas disease and AIDSThe natural course of T cruzi infection can be modified bycoinfections. Chagas disease and HIV mutually affect eachother.60 HIV infection may lead to reactivation of T cruziinfection.61 The disease in some T cruzi-HIV-infected patientsmay have a long silent clinical course, whereas in others itmay present with meningoencephalitis or myocarditis.

Therefore, these coinfections should be investigated inendemic and non-endemic regions of the world.62

PATHOGENESISTwo theories have been set forth to explain the pathogenesisof the lesions in Chagas disease. The parasite persistencetheory postulates that Chagas lesions could be a directconsequence of the mechanical rupture of the parasitisedhost cell and subsequent inflammation. The autoimmunetheory ascribes Chagas lesions to the rejection of parasite-free

target cells by immune lymphocytes. Here we present datashowing that these theories are not mutually exclusive.

Parasite persistenceMost observers agree that parasite persistence in the humanhost may lead to the rupture of colonised cells in the acutephase of the infection. The microscopic finding of parasite-free target cell lyses by immune effector lymphocytes has castdoubt on whether mechanical rupture is the main feature ofthe pathogenesis of acute disease. The difficulty in establish-ing a direct relationship between the intracellular presence ofparasitic T cruzi forms and concomitant tissue destruction canbe clearly shown in endemic areas of Chagas disease, whereapproximately one third of the infected human populationsuccumb to Chagas disease. Moreover, 80% of patients with

Chagas disease who died of the disease did not showamastigote nests of the parasite in histopathological sectionsof the heart. Furthermore, a lack of physical proximitybetween the parasite nest and the inflammatory lesions wasclearly shown among chronic Chagas hearts with intracel-lular amastigotes. There are, however, several questions thatremain unanswered: (1) Why are acute infections usuallyasymptomatic and remit spontaneously? (2) If severepathology is present in every patient with Chagas disease, isit also present in the cryptic T cruzi infection? (3) Why dopatients with cryptic T cruzi infection have high rates ofmorbidity and mortality?

Answers to these questions have been sought in long-itudinal field studiesfor example, among 190 adultspositive for antibody tests for T cruzi infection, 56 patientshad parasitaemias detected by xenodiagnoses, but the

remaining 134 patients with Chagas disease did not. In thisseries, progressive Chagas heart lesions were shown in 30% ofthe patients with detected parasitaemias, but in 28.8% of thepatients there was no detectable parasitaemia.63 Thesefindings have suggested that the severity of heart lesionsand the evolution of the disease may not be associated withthe prevalence of parasitaemias in patients with chronicChagas disease.

AutoimmunityExperimental data have shown that autoimmunity is animportant component of the pathogenesis of Chagas dis-ease.64 The observed rejection of target heart fibres by the

Epidemiology and prophylaxis

N Chagas is the most lethal endemic infectious disease inthe western hemisphere.

N Pyrethroid insecticides eliminate triatomines in perido-miciles and domiciles.

N Bed nets and shields prevent the bugs invasion ofhuman domiciles.

NImprovement of housing conditions.

N Social and economic development.

N Education, information and communication.

N Excluding candidate blood donors through epidemio-logical history.

N Effective chemotherapy would diminish transmission inhouseholds.

Clinical course

N Acute infections usually subside spontaneously.

N Chronic infections are asymptomatic in two thirds ofthe human population.

N Chronic Chagas disease affects mostly the heart andthe digestive tract.

N Arrhythmias and congestive heart failure are ominous

signs of the disease.N Megaoesophagus and megacolon cause dysphagia

and constipation, respectively.

Chagas disease 791


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immune system mononuclear effector cells (fig 5) appears tofavour autoimmunity. The accelerated rejection of embryonicrabbit cells by immune lymphocytes derived from chronicallyT cruzi-infected rabbits has been considered evidence ofautoimmunity in Chagas disease.64 65 Immune factors andpossible mechanisms triggering the lesions of Chagas diseasehave stirred discussions on the role ofT cruzi antigens in thepathogenesis of the disease, such as crossreactivity andmolecular mimicry.66 A lot more information on this subjectcan be found in previous review papers.67 68 We believe that

the roles played by parasite persistence in the body and byautoimmunity are essential to the pathogenesis of Chagasdisease.

The observation of progressive Chagas heart disease in Tcruzi-infected rabbits that had been treated with anti-trypanosome nitroderivatives generated an unavoidablequestion: what could be sustaining those destructive lesionsin the heart cells of treated rabbits? We sought an answer tothis question by examining the hypothesis that horizontalDNA transfer from the parasite to the mammal host (amutation) may explain the autoimmune lesions of Chagasdisease.

HORIZONTAL DNA TRANSFERHorizontal transfer of T cruzi kinetoplast DNA (kDNA)

minicircles to the genome of mammal hosts has beendetected.6872 Furthermore, a truncated kDNA sequence wasfound in the genome of a T cruzi-infected baboon (Papyohamadryas), which flanked the host DNA (GenBank accessionnumber DQ241812). Southern blots of genomic DNA fromthe heart, skeletal muscle and intestine of a chronic Chagasrabbit hybridised with a kDNA probe. Interestingly, the2.2 kb kDNA band formed with the rabbit genome DNAshowed a configuration different from the typical 1.4 kbband formed with the parasite DNA. These specific bands didnot hybridise with other parasite nuclear DNA internalprobes.25 73 The sequencing showed that both ends of thekDNA were flanked by rabbit DNA (GenBank accessionnumbers AF400668, AF399841 and AF415293) and that thekDNA integrated in direct CACCAACC repeats within therabbit genome. The host DNA, flanking the kDNA, showed

homology with the clone LBNL-125D4, which is a LINE-1retrotransposon, loaded with short SINE repeats interspersedin the vertebrate genome.74 An open reading frame thatbegan in the rabbit DNA extended into the integrated kDNA.DataBank analysis revealed a transcript potentially codingthe chimera antigen r45 (GenBank accession number AAR24603.1). These observations have suggested that per-sisting T cruzi infections can be a cumulative source ofparasite-induced kDNA mutations. We suggest that thenewly formed open reading frames translating chimeraproteins trigger the pathology of Chagas disease.

kDNA INHERITANCEWhen germ cells of an 18-month-old boy were foundcolonised by T cruzi amastigotes, we predicted that the

parasite could invade stem cells (ARL Teixeira, unpublishedobservation). The permissiveness of stem cell lines to T cruziinfection was an indication that genital crest cells couldacquire kDNA mutations during the differentiation stagefrom the 4th to the 8th day of gestation. In fact, kDNAintegration in the offspring of chronically T cruzi-infectedrabbits was shown. Genomic DNA extracted from variousbody tissues showed that the experimental animals were onlykDNA positive by nucleic acid tests. The cloning andsequencing of kDNA-positive samples showed minicirclesequences integrated in the genome of the rabbits(GenBank accession numbers AY488498AY488503). Todetermine whether a live infection is required for integration,

kDNA minicircle sequences were inoculated intravenously inT cruzi-infected rabbits. Minicircle sequences were polymer-ase chain reaction amplified only during the following3 weeks, thus showing that living infection is required forsustained vertical transfers of kDNA mutations. Hallmarkchagasic histopathological condition (minimal rejectionunits) was found in the heart, skeletal muscles andparasympathetic nervous system of the offspring. In thecontrol group, no lesions were found in the tissues of kDNA-negative rabbit offspring.

Birds are refractory to T cruzi infection,1

and thus representa clean model to study the vertical transfer of kDNAmutations. Therefore, experiments were conducted in Gallus

gallus because T cruzi infections could be established early inembryonic life. kDNA mutations were shown to be present in24% of the chickens which hatched from T cruzi-infectedeggs. The mutated chickens yielded sperm and unfertile ova, whose DNA contained kDNA amplification products.Crossing of kDNA-positive hens with roosters resulted inkDNA-mutated chickens in all instances. Sequencing showedintegration of kDNA in a retrotransposon (CR-1) at chromo-some 4 (GenBank accession number AY237306).Interestingly, some growing kDNA-positive chickens devel-oped muscle weakness whereas others showed signs of heartfailure. When these birds died, they showed the typical

minimal rejection units of Chagas disease found in rabbitsand in humans. The results therefore strongly suggest thatthe pathology observed in FO, F1 and F2 kDNA-mutatedbirds is parasite-independent. Typically, the lesions in theheart (fig 5), skeletal muscle and the parasympatheticganglia (fig 6) of kDNA-mutated birds were comprised ofminimal rejection unit lesions (fig 7) with features similar tothose seen in the human host. We believe that kDNAintegration in the vertebrate-host genome has the potentialto trigger an autoimmune response that results in host-tissuelesions, and explains the pathogenesis and variable clinicalmanifestations of Chagas disease.1

HISTOPATHOLOGICAL STUDY Acute Chagas disease

T cruzi can parasitise any nucleated cell in the human body.Therefore, connective tissue, striated and smooth muscles,germinal cells, and phagocytic mononuclear cells can beintensely parasitised. Also, epithelial cells of the liver,kidneys, thyroid, pancreas and skin are permissive to T cruzi.Schwann cells of the peripheral nerve fibres, ganglia and glial

Figure 5 Myocarditis in a kinetoplast DNA-mutated chicken. A typicalfeature of the Chagas lesion is the immune system mononuclear celladherence and lyses of parasite-free target heart myofibres.Haematoxylin and eosin (H&E) stain,6200.



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cells (usually astrocytes) of the brain are parasitised, butneurones are spared. Often, amastigote parasitic forms thatcolonise muscle cells become inaccessible to the immunesystem effector cells and, therefore, can hibernate in thoseniches for an unknown period of time. Grossly, the heart inhumans with acute disease increases in size and becomesdilated, flabby and congested. The lymph nodes near theemergence of the aorta and the pulmonary artery become

engorged. In the epicardial surface, the coronary and thelymphatic vessels are dilated. These gross findings predict thepresence of intense inflammatory infiltrates in the heart.Microscopically, muscle fibres and occasionally histiocytesshow pseudocysts (cysts without a wall) formed by intracel-lular T cruzi amastigotes. However, a conspicuous finding inthe acute phase of the infection is that of inflammatorymononuclear cell infiltrates and parasite-free host cell lyses(fig 8). These inflammatory cells, mainly macrophages andactivated lymphocytes, invade muscle structures and theparasympathetic and sympathetic ganglia in the body. Thelysis of parasite-free neurones is associated with adherence ofthe immune system mononuclear cells to the target cell outerplasma membrane. Electron microscopic studies of tissuelesions have shown inflammatory infiltrates in association

with glial cell elements. Heart cell destruction by immunelymphocytes and depopulation of the neurones are hallmarksof the pathology of acute-phase Chagas disease. Although thepathology of the acute phase shows the presence of scatteredparasites in the heart and nervous system, its maincomponents are the inflammatory infiltrates and target-celllyses. Sometimes the immune system mononuclear cellspalisade the perforating meningeal blood vessels in Virchow-Robin spaces. In the brain, T cruzi amastigotes are morefrequently found in astrocytes. These lesions are character-istic of meningoencephalitis.

Indeterminate phaseDuring the indeterminate phase of chronic T cruzi infections,patients do not show clinic manifestations related to heart ordigestive tract lesions.75 People who were in the indetermi-

nate phase of the infection and died of fatal accidents had noheart lesions or megaoesophagus or megacolon. Nonetheless,the indeterminate phase of the infection was established bythe direct demonstration of the parasite or by phenotype andgenotype markers of the cryptic infection. Microscopically,biopsies of 20 patients with these cryptic infections showeddiscrete, very low intensity inflammatory heart lesions.76

Chronic Chagas diseaseThe size of the heart of a patient with congestive heart failureis markedly increased and often reaches twice the normal size(fig 9). A typical gross finding of chronic Chagas heartdisease is apex effacement of the left ventricle or aneurysm

formation. Whenever either of these occurs, a thrombusappears in different stages of organisation. The other site ofthrombus formation is the huge right atrium of the Chagasheart. Thrombi at these locations can travel to the brain andthe lungs, and are frequent causes of death.

The main microscopic findings always present in a patient with chronic Chagas heart disease are inflammatory infil-trates and target heart cell lyses (fig 10). These inflammatoryinfiltrates are formed by macrophages, and small and largelymphocytes that migrate from the dilated lymphatic vessels

to the epicardial surfaces. These immune system mono-nuclear cells invade the heart and produce lyses of parasite-free target heart cells. The finding of T cruzi amastigote nestsin heart cells can be detected microscopically in 1020% ofchronic cases. Importantly, in these cases, the parasitesintracellular nests are more often found in areas of the

Figure 6 Ganglionitis and neuronal cell lyses (arrows) in a kinetoplastDNA-mutated chicken. Haematoxylin and eosin (H&E) stain,6200.

Figure 7 The minimal rejection unit in the heart of a kinetoplastDNA-mutated chicken. Parasite-free target cells are lysed by activatedimmune lymphocytes. Haematoxylin and eosin (H&E) stain,6400.

Pathogenesis

N Parasite persistence cannot be excluded.

N Autoimmunity appears to explain parasite-free targetcell destruction.

N Mechanism triggering autoimmunity needs further

clarification.N The horizontal transfer of T cruzi kDNA has been

detected.

N Parasite-refractory chickens show kDNA mutations andevident pathology.

N Chagasic condition is present in F1 and F2 kDNA-mutated chicken.

N New genes coding chimera proteins may triggerautoimmunity.

N Investigation of the cause of autoimmunity will be adaunting task.

Chagas disease 793


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myocardium free of inflammatory infiltrates. Consistently,nucleic acid tests show the remnants of parasite DNA in theaffected tissues of all chronically infected patients.27

Therefore, the hallmark of the microscopic pathology inChagas heart disease is the destruction of the target heartcells by immune effector lymphocytes. This microscopicfeature defines a minimal rejection unit (fig 11). Theconfluence of numerous rejection units signifies chronicChagas myocarditis. The destroyed heart fibres are graduallyreplaced by fibrous scars as the inflammation subsides.

Inflammatory cells infiltrate the heart conducting system inthe same manner as the contractile myocardium. Theintensity of the auto-destructive process varies from one siteto another in the heart; whereas some lesions are fresh,others evolve into fibrous scars as the inflammatory processfades away. With ageing, fibrous scars accumulate in themyocardium and can be found in every heart chamber,particularly in the left ventricle during late stage insuffi-ciency.

Histochemical analysis of the sympathetic and parasympa-

thetic nerve endings of patients with chronic Chagas diseasehas shown a low quantity of catecholamines and deficientacetylcholinesterase activity. Both findings are consistent with progressive autonomic denervation.77 Furthermore, theautonomous intracardiac nervous system in every Chagas

FC

L

Mi

I

L

Figure 8 Electron microscopy of the heart of a patient who died of

acute Chagas disease. Notice lymphocytes (L) invading the cytoplasm ofa cardiac fibre (FC), adhering to the microfilaments (Mi) andconcomitant lyses (courtesy of Dr Washington Luiz Tafuri, Faculdade deMedicina da Universidade Federal de Minas Gerais). Magnification67000.

A B

Figure 9 Gross pathology of chronic Chagas heart disease. (A) Posterior surface of a large heart showing whitish soldiers patch and dilatedcoronary vessels. (B) Internal view showing dilation of the right and left chambers of the heart.

Figure 10 Myocarditis in the heart of a patient with Chagas disease.Infiltration and adherence of mononuclear cells to target myofibres andlyses. Haematoxylin and eosin (H&E) stain,6200.



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heart shows typical lesions with varying degrees of intensity.

The inflammatory infiltrates are evident in the parasympa-thetic ganglia and in the sympathetic nerve endings in themyocardium. Ganglion cells and peri-ganglion areas areassociated with mononuclear cell infiltrates and proliferationof glial cells. Inflammatory cells palisade the neurones, whichshow degeneration and lysis. Neuronal lysis with degenera-tion and depopulation of neurones are the typical findings ofChagas heart disease.

Megaoesophagus and megacolon consist of gross dilationof the hollow viscera and thickening of their walls. Suchlesions are associated with loss of coordination of the motilityof these digestive tube segments.7883 These changes stemfrom inflammatory infiltrates of the internal and externallayers of smooth muscle cells of the digestive tract wall.However, inflammatory lesions particularly affect the para-sympathetic neurones within the walls (fig 12). Neuronal celldepopulation occurs randomly in all segments of thedigestive tract, but this is clinically more often evident inthe oesophagus and the colon. Usually, a mega syndrome

manifests when neurone loss reaches 55% of the units in theintramural parasympathetic ganglia. In some patients,neuronal depopulation can average 76.5% as compared withcontrol neurone counts.80 The use of nucleic acid tests hasshown the persistence of remnants of T cruzi DNA in tissuesamples taken from mega lesions.83 Although the parasitepersists in tissues showing mega syndromes, the literaturedoes not document colonisation of neurones by T cruzi. Asneurotoxin release by the parasite has not been shown, it ispresumed that neuronolysis and depopulation are not

associated with parasitism of these target cells. In contrast,neuronolysis has been associated with adherence of immunesystem mononuclear cells, as described for a minimalrejection unit. These observations suggest the rejection unitis the common denominator of Chagas disease pathology.

TREATMENTNifurtimox (4-(5-nitro-furylidenoamine)-tetrahydro-4-4-1,4-thiazine-1-1-dioxode) and o-benznidazole (N-benzyl-2-nitro-imidazolacetamide) are anti-trypanosome drugs used to treatthe T cruzi infections.84 85 The cytotoxic and genotoxic effectsassociated with the drugs are related to their chemicalstructures, resulting in the release of electrophilic radicals onenzymatic nitro-reduction. The free radicals have beenidentified as the nitro-anion, H2, K O2 and hydrogenperoxide.8690 Drug toxicity affects the parasite and also anyother innocent bystander cells in the human body.85 The nitro-group reduction alters the number of electrons at the externalorbital, thus augmenting the mutagenic potential because freeradicals bind to DNA double strands, forming adducts. 8590

The use of nitroderivatives in the treatment of acuteChagas disease has had limited success.9196 Physicians haveused these drugs sparingly because they do not eradicate Tcruzi infections in 83.5% of acute cases. Further, an indirectindication of parasite persistence is provided by positive after-treatment immunological tests. These observations explainwhy caution is required before using these drugs, in additionto reports showing that the use of anti-trypanosomenitroderivatives does not halt progression of the disease inpatients treated for Chagas disease.27 For example, electro-cardiographic changes in patients treated for Chagas disease

did not differ statistically from those recorded in a placebo-treated cohort of patients with acute Chagas disease.27

Furthermore, chagasic changes on electrocardiogram havebeen recorded in patients 24 years after treatment with anitroderivative compound.9496 Moreover, in patients acutely

Figure 11 The minimal rejection unit in the skeletal muscle of apatient with Chagas disease. Note the immune system mononuclear cellpalisade of the target cell sarcolemma and concomitant lyses of themyofibres in the absence of parasite colonies in situ.

Figure 12 Ganglionitis and neurone drop-out of the parasympatheticganglion of a kDNA-positive patient with Chagas disease andmegacolon.

Pathological study

N Acute-phase pathology shows T cruzi forms in tissuesand inflammatory infiltrates.

N Chronic pathology shows striking inflammation in theabsence of the parasite in situ.

N Lesions affect various organs and tissues in the human

body.N Muscle tissues and parasympathetic ganglia are often

destroyed.

N Lesions are characterised by mononuclear cell infil-trates and target cell lysis.

N Parasite-free neurone lyses are produced by inflam-matory infiltrates.

N The typical lesion in a patient with Chagas disease isthe destruction of parasite-free target cells.

N The minimal rejection unit is the typical pathologicallesion in Chagas disease.

Chagas disease 795


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infected with T cruzi, heart biopsies showed myocarditis,97

even though the patients had been treated with a nitroder-ivative drug. These findings strongly suggest that nitroder-ivative drug treatment does not prevent the onset of severeheart lesions.

In the chronic indeterminate phase, the only acceptablecriterion for cure resulting from specific drug therapy for Tcruzi infections is the lack of detectable specific antibodiesand the results of nucleic acid tests to determine evidence ofthe parasites phenotype and genotype. Results of these

studies showed that 94.7% of nitroderivative drug-treatedpatients with chronic Chagas disease tested positive forspecific antibodies to T cruzi antigens.93 In one series that hadreported 76% success in the treatment of patients withchronic Chagas disease with a nitroderivative compound, amere 8% drop was observed in the prevalence of positivenucleic acid tests 618 years after chemotherapy.Importantly, it was shown that mortality was similar intreated and untreated groups of patients with chronic Chagasdisease 10 years after chemotherapy.27

CONCLUSIONSDrug treatment for T cruzi infections is considered unsatis-factory because it does not halt the progressive lesions of theheart and digestive tract of patients with chronic Chagasdisease. It appears that effective treatment of Chagas disease

would require a drug that does not produce undesirableeffects and eradicates the infection or improves the prognosisof a sick patient. Specific chemotherapy with the drugs thatare now available may not be cost effective. Nevertheless,treatment with a nitroderivative compound is clearlyindicated in acutely infected patients, during the course ofimmune suppression and during organ transplantation of apatient with Chagas disease.

The controversy over the efficacy of treatment underlines theimportance of sustained therapeutic research to control Chagasdisease.2 The results of studies reported here suggest that thepathogenesis of the disease is related to kDNA mutations of thevertebrate hosts genome. There is a vast field of research on which to base the development of drugs to prevent T cruzi-induced kDNA mutations.3 The persistence of lifelong infec-tions providing a source of kDNA that may result in cumulativemutations over time requries newly developed, targeted drugs.This is an area of scientific research that has the potential togenerate health benefits for millions of people already infectedwith T cruzi, one third of whom will develop clinicalmanifestations of Chagas disease.

SELF ASSESSMENT QUESTIONS (TRUE (T) FALSE (F);ANSWERS AT THE END OF THE REFERENCES)

1. Acute Chagas disease

(A) Is usually seen in children below 15 years of age

(B) Is often not perceived by the individual or by the doctor

(C) Is recognised by the finding ofT cruzi in blood smears

(D) May be asymptomatic and subsides spontaneously

(E) In ,5% of the cases is associated with heart failure ormeningoencephalitis

2. Chronic Chagas disease

(A) Is usually seen 23 decades after the onset of T cruziinfection

(B) Is eradicated by anti-trypanosome drugs

(C) Is cured by treatment as indicated by seroconversion ofimmunological tests and negative nucleic acid assays

(D) Effects of treatment are beneficial because they preventprogressive Chagas heart disease

(E) Produces megaoesophagus and megacolon in the earlystages of the infection only

3. Pathogenesis

(A) Results from a parasite-released toxin that killsneurones

(B) Is often associated with a neurone infection and celldeath

(C) Appears to associate minimal rejection units withautoimmunity

(D) Is always related to the parasites mechanic destructionof target cells

(E) Suggests that immune system cells are not involved

4. Pathological study of chronic Chagas disease

(A) Is related to the parasites pseudocyst which is easilyseen in histopathological sections

(B) Leads to small lesions without clinic repercussion

(C) Does not cause significant rates of morbidity

(D) Is usually fatal within 2 years after the onset ofsymptoms of heart failure

(E) Can occur in the absence of T cruzi infections and ofspecific phenotype and genotype markers of the

parasite.

5. Prophylaxis

(A) Is partly obtained by spraying pyrethroids in thedomicile and peridomicile

(B) Requires control of mosquitoes

(C) Is controlled by vaccination in endemic areas

(D) Is not affected by economic development

(E) Cannot be attained by improving housing conditions

ACKNOWLEDGEMENTSWe thank Meire Maria de Lima for critical reading of the manuscript.NN is in receipt of a Post-Doctoral Fellowship from the Ministry of

Education-CAPES, Brazil. MCG and CG are holders of scholarshipsfrom the Brazilian National Council-CNPq.

Authors affiliations. . . . . . . . . . . . . . . . . . . . .

A R L Teixeira, Multidisciplinary Research Laboratory, Faculty ofMedicine, University of Brasilia, Federal District, BrazilN Nitz, M C Guimaro, C Gomes, Chagas Disease MultidisciplinaryResearch Laboratory, University of Bras lia, Federal District, BrazilC A Santos-Buch*, Cornell University Medical College, New York, New

York, USA

*Retired.

Treatment

N Nitroderivative drugs curtail parasitaemias and clinical

signs of acute infection.N Treatment does not eradicate T cruzi infection.

N Progressive chronic Chagas heart disease persists intreated patients.

N Amelioration of symptoms can be achieved withmodern clinical and surgical techniques.



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Funding: This work has been partially supported by Financiadora deEstudos e ProjetosFINEP, Brazil, and by NIH grant I-R03-AI067334-01.

Competing interests: None declared.

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chagas disease - a review - teixeira e cols - 2006

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