Clinical Analysis and Parasite Genetic Diversity in Human Immunodeficiency Virus/Chagas' Disease Coinfections in Brazil
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Am J. Trop. Med. Hyg., 61(2). 1999. pp. 198-206 Copyright O 1999 by ?he American Society of Tmpical Medicine and Hygiene CLINICAL ANALYSIS AND PARASITE GENETIC DIVERSITY IN HUMAN IMMUNODEFICIENCY VIRUS/CHAGAS' DISEASE COINFECTIONS IN BRAZIL LORE" PEREZ-RAMIREZ, CHRISTIAN BARNABÉ, ANA MARLI C. SARTORI, MARCELO S. FERFEIRA, JOSÉE. TOLEUNO, ELVETH V. "ES, MARClUS K. BURGARELLI, AGUINALDO C. SILVA, MARIA A. SHMANAI- YASUDA, JOSUE N. LIMA, ALDA M. DA-CRUZ, OSWALDO C. OLJYEIRA, CARMEN GUILHERME, BRIGITTE BASTRENTA, AM^ MICHEL TIBAYRENC Centre d'Etudes sur le Polymorphismes des Microorganismes, Unite Mixte de Reclierclie, Centre National de la Recherche ScientifqudInstitut Français de Reclierclie Scientijque pour le Développement en Coopération, Montpellier, France; Departmento , de Doenças Infecciosas e Parasitarias, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil: Departmento de Clínica Médica, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais, Brazil; Departmento de Parasitologia. Instituto Adolfo Lutz. São Paulo, Brazil; Departmento de Molestias Infecciosas, Universidade de Campinas, Campinas, Brazil; Hospital Casa do HeniofilicoLnboratorio de Imunidade Celular e Humoral em Protozooses, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil Abstract. To evaluate the possible role of parasitemia on Chagas' disease reactivation in Chagas' diseasehuman immunodeficiency virus (W)coinfection cases and the impact of HCV coinfection on Trypanosoma crirzi genetic diversity, 71 patients with Chagas' disease (34 W+and 37 HIV-) were surveyed. Moreover, 92 T. cruzi stocks from 47 chronic chagasic patients (29 HIV+ and 18 HIV-) were isolated and analyzed by multilocus enzyme elec- trophoresis and a random amplified polymoriphic DNA procedure. High parasitemia appeared to play a major role in cases of Chagas' disease reactivation. In HIV+ patients, the genetic diversity and population structure (clonality) of T. cmzi was similar to that previously observed in HIV- patients, which indicates that immunodepression does not modify drastically genotype repartition of the parasite. There was no apparent association between given T. cruzi genotypes and specific clinical forms of Chagas' diseasemm associations. Chagas' disease, the American trypanosomiasis, is wide- In the present study, we have explored the clinical diver- spread from the southern United States (where the indige- sity of Chagas' disease/HIV associations in Brazil and have nous cases are rare) to northern Argentina. It is characterized tested the following hypotheses: 1) that reactivation .of Cha- by two successive phases, an acute one, with high parasit- gas' disease in HN+ patients is due to high parasitemia emia, and a chronic one, with low parasitemia. Although independent of specific T. cruzi genotypes; 2) that these ge- efficient measures of transmission control have been imple- notypes play a specific role in reactivation of Chagas' dis- mented in some countries (by eliminating the insect vector), ease in HIV+ patients; and 3) that apparent T. cruzi clonal Chagas' disease still is a priority health problem in Latin population structure is due to immune defenses of the host America. Chagas' diseaselhuman immunodeficiency virus rather than to true clonal evolution and therefore disappears @IV) associations appear to be a growing threat in large in HIV+ immunocompromised patients. It is worth noting Latin American cities, and can be extremely pathogenic, that these patients probably acquired Chagas' disease prior with severe cases involving the central nervous system to the HIV infection. (CNS) with ei!her tumoral lesions or meningoencephalitis.'- 5 The causative agent of Chagas' disease is Trypanosoma MATERIALS AND METHODS cruzi, a flagellate protozoan. This parasite exhibits consid- Patients. Patients were recruited in four Brazilian hospi- erable genetic diversity,6v7 and it has been proposed that spe- cific clinical forms of Chagas' disease were due to this di- tals: Services of Infectious Diseases of the University Hos- versity.* Genetic diversity of T. cruzi appears to be governed pitals of Sa0 Paulo, Campinas, and Uberlândia, and The by a predominant clonal evolution.6 Clonality in T. cruzi and Hospital for Hemophilic Patients in Rio de Janeiro. They were included in the study if they showed positive results in in other pathogens has important medical implication^.^ two First, if clonal evolution is predominant, the multilocus ge- at least of three serologic assays for Chagas' disease notypes of the pathogen propagate themselves unchanged as (indirect hemagglutination, indirect immunofluorescence, genetic photocopies, which makes them relevant as powerful and ELISA). Seventy-two chronic chagasic patients were se- markers for epidemiologic surveys. However, if genetic re- lected according to this criterion (Table l).Within this group combination is predominant, the multilocus genotypes of the of 72 patients, an HIV+ and an HIV- group was defined pathogen are unstable and cannot be used as epidemiologic according to positivity or negativity for HIV serologic re- markers. Second, in case of clonal evolution, the natural actions (ELISA and Western blot). We were able to isolate clones correspond to distinct evolutionary units that tend to and characterize the parasite stocks from 47 (29 HN+ and accumulate divergent mutations, including those genes that 18 HIV-) of 72 patienta. The criterion for reactivation of control properties such as virulence or resistance to drugs. Chagas' disease was the presence of infective trypomasti- A challenging hypothesis to clonality is that the immune gote forms of the parasite detected by microscopic exami- defenses of the host eliminate most of the genotypes of the nation of peripheral blood. The Medical Ethics Committee pathogen, which generates pseudoclones and apparent link- of each participating center approved the study protocol. In- age disequilibrium (nonrandom association between geno- formed consent was obtained from all study patients. types occurring. --...- at different loci) even if the_pathogenis not Xenodiagnosis. This technique uses T. cruzi-free triatom- clonal. 1I ine vectors (reared in- the laboratory). Thirty third-stage' TABLE1 Characteristics of the 72 patients: age, geographic origin, clinical forms, parasitemia, and code of Trypanosoma cruzi corresponding stocks* HIV-positive patients HIV-negative patients Patient Age Birth No. of Patient Age Birth No. of code (years) localityf Clinical form Parasitemia* stocks Codee code (years) localityt Clinical form Parasitemiat stocks Code5 HC1 37 MG Indeterminate 61.9% 3 TlO-T6-T7 u1 79 MG Cardiac 9.170 1 T1 HC2 29 BA Cardiodigestive 27.7% 2 N7-T3 u2 58 MG Cardiac 26.3% 1 T2 HC3 47 SP Cardiodigestive 24% 1 T2 u3 40 MG Indeterminate 4.5% 1 T1 HC4 27 BA Cardiac 53.8% 4 T3-T4-T9-T11 U8 53 CE Digestive O NS HC5 33 PE Cardiodigestive 6.7% 1 TI u10 59 SP Cardiac O NS HC6 31 BA Indeterminate O 2 T3-T4 u1 1 65 MG Cardiodigestive 43.4% 2 T5-TlOr HC7 59 SP Cardiac 38% . 3 Tl-Tgf-TlO . U13 31 MG Cardiac 40% 2 T1-T6 HClO 57 BA Indeterminate O NS U15 70 MG Cardiac O NS HC12 42 CE Indeterminate 8.2% 1 T1 U16 37 GO Cardiodigestive O NS HC13 42 MG Cardiodigestive 26% 2 Tl-T2 U17 71 SP Cardiodigestive 3.8% 1 T1 HC14 37 SP Myocarditis 65.4% 4 T 1-T2-bN17-bN 19 U18 72 MG Cardiodigestive 65.5% 2 T12-TI6 HC18 31 MG Indeterminate 14.28% 1 TI u19 76 MG Cardiodigestive O NS HC19 55 AL Indeterminate 20% 2 Tl-T2 u20 63 MG Cardiac O NS HC20 32 AL Indeterminate O NS. u21 ' 51 MG Cardiodigestive O NS HC21 42 GO Neurologic 66.6% 3 Tl-T7-T9 u22 64 MG Cardiac 4.8% 1 T1 ' HC22 27 GS Neurologic 100% 2 T5-T8 U23 47 GO Cardiac O NS HC23 28 BA Indeterminate NP 3 T6-TIl-Tl3 U24 67 MG Cardiac O NS HC24 43 BA Cardiac NP 2 T1-T4 U25 57 MG Cardiac O NS HC25 25 BA Cardiodigestive NP 2 Tl-T2 U26 72 MG Cardiac O NS HG27 42 EA Indeterminate NP 1 T1 U27 52 MG Cardiac 0. NS CP1 38 MG Indeterminate O NS U28 78 MG Cardiac 23.8% 2 T2-T5 CP2 58 GO Indeterminate 19.2% 1 T3 U29 69 MG Cardiac O NS CP3 48 SP Indeterminate O NS U30 49 MG Cardiac O NS CP5 46 PA Cardiac O 1 bT2 U3 1 67 BA Cardiac 41.2% NS CP8 40 SP Indeterminate NP 2 T5-TlO U32 50 MG Cardiac O NS CP9 43 SP Indeterminate NP 2 Tl-T4 u33 40 MG Cardiac O NS u4 42 MG Indeterminate O 4 bT2-cT3-cT6-cT8 u34 67 MG Cardiac 4% NS u5 47 MG Cardiac 60% 4 N 10-T4-bT8-bT9 u35 65 MG Cardiodigestive O NS U6 36 MG Neurologic 100% 7 TIO-TI 1-T16-T25- U36 58 MG Cardiac O NS bT22-bT3-bT5 u7 43 MG Indeterminate 7.7% 2 T1-T2 HC17 40 MG Indeterminate O NS u12 48 MG Neurologic 89.3% 4 T5-T19f-Tmf-T23d HCC25 38 BA Indeterminate NP 1 T1 FU1 28 AL Indeterminate 47.570 3 13-14-T2 17HCC 30 BA NP 1 T2 RJ2 44 ES Indeterminate 40% 1 I1 HCC40 28 MG Digestive NP 1 T1 HCC41 29 MG . Ca;diac NP 1 T3 HCC42 45 MÇ NP 2 T3-T4 HCC43 42 BA Indeterminate NP 1 T1 HCC44 31 MG -;Cardiac NP 1 T1 HCC26 NP 1 T1 HCC3 1 39 SP Indeterminate NP 1 N2 * HIV = human immunodeficiency virus. t MG = Minas Gerais: BA = Bahia: SP = §ão Paulo: CE = Ceara: PE = Pemanbuco: GO = Goias: AL = Alaaoas: GS = Rio Grande do Sul: PA = Parana: ES = Esoiritu Santo. *Number of positive nymphsltotal nymphs. NP = parasitemia not determined. 5 NS = no stock isolated. ,-. ... .. .. ... .. .. .._ (I ~ .. .. - . I 200 PEREZ-RAMIREZ AND OTHERS nymphs of Triatoma infestam, a major vector in South since ME corresponds to two different loci? For a given T. America, are fed on the putative infected patient, and the cruzi stock and for a given locus, each isoenzyme band was feces of this vector are checked for the presence of T.