Conferencia "Machado - Guerreiro" Congenital Chagas’ disease in Chile: Incidence, Diagnosis and treatment evaluation by PCR Presidente: Dra. Luisa Giménez Presentación

Conferencista: Dra. Myriam Lorca Conferencia

Es un honor para mí, presidir esta Conferencia de Chagas Congénito a cargo de un experto de la jerarquía de la Dra. Myriam Lorca, que la tituló "Congenital Chagas’ disease in Chile: Incidence, Diagnosis and treatment evaluation by PCR". La misma lleva el nombre de Machado-Guerreiro en Homenaje a los creadores de la reacci ón de fijación de complemento, que se usó durante tanto tiempo en el diagnóstico de la Enfermedad de Chagas crónico. Una de las formas reconocidas en la actualidad de trasmisión de la enfermedad de Chagas, es la connatal que ya en los primeros años fue registrada por Carlos Chagas. Es conocida la importancia de establecer el diagnóstico precoz de ésta, ya que permite instaurar el tratamiento inmediato, obteniendo altos índices de curación de los pacientes. También conocemos que las estimaciones existentes de esta nosología, que son pocas, varían entre un 0,5 al 6 % para los hijos de madres chagásicas de areas endémicas y no endémicas. Es importante resaltar y reiterar que para establecer el diagnóstico de Chagas Congénito es necesario conocer su existencia, que aún hoy día, no es tenida en cuenta en muchos servicios de obstetricia y la investigación de la madre y el niño. De allí la importancia del aporte que la Dra. Lorca nos brindará. Dra. Luisa Gimenez Encargada del Sector Chagas Servicio de Cardiología del Hospital Alvarez Buenos Aires, Argentina.

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Congenital Chagas’ disease in Chile: Incidence, Diagnosis and Treatment Evaluation by PCR Dr. Myriam Lorca Associated Professor of Parasitology Biomedical Sciences Institute Faculty of Medicine, University of Chile

Congenital Chagas’ disease is produced after passage of Trypanosoma. cruzi from the infected mother to the fetus via the placenta. (1) Since the disease through the vector has been practically eliminated in Chile and others countries in Latin America, we have been considered important to examine certain aspects of this mechanism of infection which should be taking in account in futures programs of intervention in the transmission of the parasite to the human beans EPIDEMIOLOGY OF THE DISEASE The prevalence of Chagas’ disease in pregnant women ranges from 8% (Uruguay) to 50% (Bolivia), the last studies conducted in Argentina have been demonstrated a 9% of the maternal infection and in Paraguay 7.7 to 10.5%. ( 1,2,3 )In Chile the infection level reported ranges from 1 to 10% in women at reproductive age; however prevalence of 20% were diagnosed in several regions of the country ( 4-7 ). The incidence of the congenital infection ranges according the different studies conducted in Latin America between1,9 and 10% (2,3,4,5 ) CLINICAL ASPECTS The infection by T. cruzi follows different patterns when compared to toxoplasmosis. The infection of the fetus may occur at any time of the gestation (acute or chronic), it may infect the fetus in a second opportunity, or twins at the same time. The infection may produce pathology in the growing fetus but usually not abortion. However in HIV infected woman with Chagas’ disease infection the abortion has been demonstrated ( 8). At birth, the child may show a broad spectrum of clinical manifestations, ranging from asymtomatic to acute syndrome that could be fatal. Approximately 80% of infected mother may not transmit the parasite to the fetus during gestation, and will give birth a healthy child. Ten to 20% of the cases, T. cruzi will cross the placenta and infect the fetus (9). The consequences of the infection on the newborn child will depend upon the time of infection, resulting in the birth of asymtomatic or symptomatic infected and inclusively a death child. In the case of infected child the outcome of the disease will determine the future of the child: cure, chronic Chagas’ disease or death are some of the possibilities. Several studies conducted in mother-child have been realized in several regions of Chile, including areas with high and low prevalence of the disease. In the areas with high frequency the number of positive cases ranger from 2.4 to 26.5% in pregnant women (4-7). In those areas with low frequency, the prevalence is usually between 1 to 2%. However the transmission in both areas reaches from 13 to 22 % (10). Approximately 80% of the children born to infected mothers will be asymptomatic and normal. In the case of symptomatic infected child, they will show low body weight at birth, ictericia, anemia, hepato-splenomegaly, meningoencephalitis, myocarditis,

and ocular lesions. In extreme cases death may occur. The early diagnosis of the infection has the advantage to allow opportune treatment, which will prevent further lesions. When the treatment start before six months of age, the child will recover from the symptoms and the parasites will be eliminated from the organism. This outcome has been confirmed inpatients observed for four years after the completion of the treatment. In cases treated after two years of age (post-infection), the child developed cardiopathy, megacolon and neurologic symptoms (11 -12). The factors that influence the transmission of the infection form mother to fetus have not been clearly identified. Several questions have been formulated, and the answer may require extensive investigation: Are there several T. cruzi strains infecting the population or it is due to characteristics of the pregnant women, during particular period of gestation that may favor the extreme variation in the infection rates of the fetus? Are the differences of prevalence in the different regions due to the presence of several T. cruzi strains? The variability of the clinical manifestations of the disease is due several T. cruzi strains? Studies conducted in Chile during 1980 showed that transplacental infection in the general population was less than 3% in the regions with high exposure to the parasite and the vector. However in the regions with low exposure level, the rate of infection was higher (10%) in the same group of people (Table 1). These differences were considered the result of the use of different methodologies to evaluate the disease frequency or an acquired infection by the vector in the high endemic area. Recent studies (13) have demonstrated that the risk of infection has been reduced to a minimum due to effective elimination of the vector in those areas with high risk.

Table 1 Trypanosoma cruzi infection in pregnant women in Chile, evolution of the prevalence of the maternal infection and the incidence of congenital transmission in endemic areas of Chile Year

Author

1990

Muñoz et al

1997 2000

Bahamonde et al Garcia et al

Chagasic Endemic area High Low High High

Pregnant woman N° % + 547 26.5 4,237 1.2 1,667 2.4 938 7.8

% Congenital transmisión 2.1 9.8 12.5 16.4

Low

5,495 1.4

28.2

MOLECULAR ASPECTS Several reports indicate the existence of T cruzi strains in Latin America. The identification has been done using zymodemes, schizodemes and recently PCR-RFLP, PCR and hybridization with specific radionucleotid probe against clonets of T. cruzi Initial work that was conducted revealed the existence of distinct selvatic and wild T. cruzi strains. Currently it has been determined that certain strains show preference for nervous tissues over other organs. Genetic studies of T. cruzi suggest that there are isolates that show variation in the level of virulence to the host (14-18). Three main clonets have been differentiated up to this moment. Clonet 19/20 produce mainly encephalitis, infect striated muscle and only a 20% of the cardiac tissue, show also a high prevalence in the vector and low frequency in humans beans. On the other side clonet 39 infect specially striated muscle and mainly ( 83%) cardiac tissue and liver and spleen cells. This clonet also show a high frequency in human beans and a medium prevalence in the vector.

Congenital infection by T. cruzi has been extensively studied in Chile to determine the genetic background of the parasite as well as the possible influence on the consequences of a chronic infection or the evolution after the treatment. The results of the genetic background of the strains, which infected congenitally child, are showed in table 3. According the results the main clonet present in congenital cases in Chile is Clonet 39 in high and low endemic area. The clonet 19/20 is less frequent and is present only in low endemic area ( 3 cases). This result could explain some of the differences in the frequency the transmission in both areas.There are not relationship between the clonets and the clinical form of presentation of the congenital cases. Moreover all the sudied cases ( 21) have been asymtomatic. Table 2 Genetic Background of Typanosoma cruzi clones in congenital cases of Chagas disease Cases N° 12 9

Procedence High Endemia Low Endemia

Congenital transmission 16.4 28,2

Clonet 39

Clonet 19/20

12 6

0 3

DIAGNOSIS Two methods are used to diagnose T. cruzi infection: Direct and indirect or serologic techniques. (9) - Direct methods are used in newborn to confirm connate infection, including microstout and xenodiagnosis. Both methods are based on concentration of parasites in the blood. They show low yield particularly in asymtomatic cases with low parasitemia. The use of PCR has facilitated the diagnosis and increased the sensitivity by 97%, independent of the type of infection (3,19-23) ( Table 2) Table 3 Trypanosoma cruzi: PCR results in children born from infected mothers N° + Mothers 73 78

Endemic Area High Low

PCR NB N° + % 73 12 16,4 78 22 28,2 151 22,5

Xeno +% 1 1,4 3 3,9 4 2,6

IgG IgG Clinical Serology at birth Serology 6month Manifestation N° % N° % Asym. Sym 73 100 5 6,3 12 0 78 100 N/D 22 0

- Indirect or serologic methods include ELISA and immunoflourescense methods. They allow the detection of IgG, IgM, and IgA antibodies to the parasite (10, 11). The presence of IgG to T. cruzi in the blood of a newborn does not indicate neonatal infection unless the titers are 2 titer higher those measured in the mother and they persist for 6 months after birth. Recently developed methods using recombinant or synthetic antigens allowed a better characterization of neonatal Chagas’ disease.(24-25) Congenic infection is confirmed by the finding the protozoa using direct methods. In addition, serologic methods will show significant levels of IgG and IgM. IgG titers should be at least 2 titers over the maternal levels for at least six months after birth. Before the initiation of treatment, it is recommended to conduct the following general tests: Hemogram, liver function, glycemia, electrocardiogram, echocardiogram and brain echogram. In addition, it is suggested to examine retina, determine protein content in spinal fluid, together with bacteria culture, and T. cruzi IgG determination using ELISA or immunofloruescense techniques. Nutritional status of the patient should also be evaluated before the initiation of the treatment. TREATMENT Current the treatment during the acute stage involves the use of niturtimox for up to 60 days using incrementing dosages. The patients should be monitored using clinical examination every 15 days to determine adverse effects and conduct blood and liver function diagnostic tests. In addition, the patient should be evaluated for antibodies against T. cruzi using ELISA or immunoflourescense tests, PCR and xenodiagnosis after 1, 6, 12, 24 and 36 months after the treatment period. I most of the cases, it is possible to obtain complete cure of the disease and negative results in parasitological diagnosis. The most frequent adverse reactions observed include hypersensitivity, gastrointestinal symptoms, peripheral nervous symptoms and convulsions that respond to Phenobarbital medication. In children born from infected mothers that show evidences of T. cruzi infection, it is considered to be a transplacental infection

and should receive treatment.(4) SURVEILLANCE AND INTERVENTION PROGRAM The program will target transplacental transmission diagnosis in mother and newborn child, followed by treatment if needed, close control of positive cases for a year after medication, epidemiological study of the family and report to the National Health Service. Medical treatment should not be initiated in pregnant or lactating women. It is recommended for women living in an area with high Chagas’ disease risk to undergo medical evaluation, treatment if needed, sanitary education and antibody study of the immediate family. CONCLUSIONS 1. The success in the control of vector transmitted Chagas’ disease and screening programs in blood banks in the reduction of the frequency of human cases, has uncovered the Public Health importance of transplacental transmission of T. cruzi . 2. The early diagnosis of the infection and the adequate treatment result in The serological, clinical and parasitological cure of the infection in the Newborn. 3. PCR have been demonstrated to be an excellent method of the diagnosis of congenital Chagas disease with high sensitivity and specificity. 4. Two main clonets of Trypanosoma cruzi are present in the infected child and associated with the transmision and the geographical procedence 5. Up to this moment there are not evidence of relationship between the clone and the clinical manifestation of the infection 6. The cost of surveillance and intervention program is lower than the medical treatment required for chronic Chagas’ disease cases. 7. Early treatment of T. cruzi infected children will produce complete cure and interruption of the cycle. 8. In countries where other surveillance and intervention programs for mother and child are in place, the inclusion of Chagas’ disease monitoring will maximize the impact of the activities that target this segment of the population. REFERENCES 1. At ías, A.M. Enfermedad de Chagas. Cap.28 en Parasitolog ía Médica. Atías, A. 1ª 337-45. 2. Blanco SB, Segura EL. Gurtler RE. Control of congenital transmission of Trypanosoma cruzi in Argentina. Medicina (B. Aires), 1999, 59 Suppl. 2; 138-42. 3. Russomando G, de Tomassone MM, de Guillen I, Acosta N, Vera N, Almiron M, Candia N, Calcena MF, Figueredo A. Treatment of congenital Chagas’s disease diagnosed and followed up by the polymerase chain reaction. Am J Trop Med Hyg, 1998 Sept., 59; 3, 487-91. 4. Muñoz, P., Thiermann, E., Atías, A., & Acevedo, C. Enfermedad de Chagas congénita sintomática en recién nacidos y lactantes. Rev. Chil. Pediat. 1992: 65(4); 196-202. 5. Lorca, M., Beroiza, A., Mu ñoz, P. & cols. Estudio materno infantil de enfermedadde Chagas en zonas endémicas III: Salamanca, Valle del Choapa Chile. Parasitol aldía. 1987: 11; 97-100. 6. Bahamonde, M.I., Muñoz, P., Urarte, E., Veloso, C., & Lorca, M. Enfermedad de Chagas Cong énita. Situaci ón actual en área de baja endemia chagásica, Hospital San Juan de Dios. 1993. IV Jorn. Anual. Parasitol., Cartagena, Chile. 1994: 36. 7. Schenone, H. & col. Enfermedad de Chagas congenita en Chile. Estudio longitudinal de la reproductividad de mujeres chagásicas y no chagásicas y de algunos par ámetros parasitológicos y clínicos de ellas y de sus correspondientes hijos. Bol. Chile. Parasitol. 1985: 40; 24 29. 8. Nisida IV, Amato Neto V, Braz LM, Duarte MI, Umezawa Es. A survey of congenital Chagas ’ disease, carried out at three health institutions in Sao Paulo City, Brazil. Rev Inst Med Trop Sao Paulo, 1999 Sep, 41: 5, 305-11. 9. Lorca M, Veloso C. & Thiermann E. Congenital Chagas ’ disease and its serological diagnosis through conventional serology and methods of molecular Biology of Parasitism. Ehrlich R, & Nieto A. Eds. Ediciones Trilce Montevideo, Uruguay. 1994: 155-166. 10. "Diagnóstico de la infección transplacentaria por Trypanosoma cruzi, por medio de la técnica de Reacción en Cadena de la Polimerasa. Análisis génico-epidemiológico de las cepas infectantes". Programa de Magister Ciencias Biol ógicas, menci ón Parasitología, Escuela de Postgrado, Facultad de Medicina, Universidad de Chile.2000 11. Lorca, M., González, A., Reyes, V., Veloso, C., Vergara, U. & Frasch C. El diagn óstico de la enfermedad de Chagas crónica mediante antígenos recombinantes de Trypanosoma cruzi. Rev. Med. Chile. 1993: 121; 363 -368. 12. Muñoz, P. & Acevedo, C. Cap. 10 en Enfermedad de Chagas y el sistema nervioso.Pub. Cien. Nº 547, 1994. OPS. Buenos Aires, Argentina. 13. Eliminación del vector de la enfermedad de Chagas. Éxito de la Salud Pública Chilena. Lorca M, García A, Bahamonde M I , Fritz A, Tassara R Rev. Med. Chile. 2000( submitted) 14. Ayala FJ. Trypanosomacruzi and Leishmania have clonal population structure of epidemiological significance. Biol. Res. 1993: 26; 47-63. 15. Revollo, S., Oury, B., Laurent, JP., Barnabe, C., Quesney, V., Carriere, V., Noel, S., Tibayrenc, M. Trypanosoma cruzi: impact of clonal

evolution of the parasite on its biological and medical properties. Exp. Parasitol. 1998: 89(1); 30 -39. 16. de Diego, JA:, Palau, MT., Gamallo, C., Penin, P. Relationships between histopathological findings and phylogenetic divergence in Trypanosoma cruzi. Trop. Med. Int. Health. 1998: 3(3); 222-223. 17. Tibayrenc, M. & Breni ère, SF. Trypanosoma cruzi: major clones rather than principal zymodemes. Mem. Inst. Oswaldo Cruz. 1988: 83; 249-255. 18. Souto, RP., Fernandes, O., Macedo, C., Campbell, DA. & Zingales, B. DNA markers define two major phylogenetic lineages of Tripanosoma cruzi. Mol. Biochem. Parasitol. 1996: 60; 71-80. 19. Centurion-Lara, A., Barrett, L. & Van Boris, W.C. Quantitation of parasitemia by competitive polymerase Chaín reaction amplification of parasite kDNA minicircles during chonic infection with Trypanosoma cruzi. J. Infect. Dis. 1994: 179(5); 1334 -1339. 20. Wincker, P., Telleiria, J., Bosseno, M.F., Cardoso, M.A., Marques, P., Yaksic, N.,Aznar, C., Liegeard, P., Hotebeyrie, M., Noireau, F., Morel, C.M. & Brenier, S.F. PCR-based diagnosis for Chagas`disease in Bolivian children living in an active transmition area: comparison with conventional serological and parasitological diagnosis. Parasitol. 1997: 114(4); 367-373. 21. Wincker, P., Bosseno, MF., Britto, C., Yacksic, N., Cardoso, MA., Morel, CM.,Brenière, SF. High correlation between Chagas' disease serology and PCR -based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area. FEMS Microbiol. Lett. 1994: 124(3);419-423. 22. Kiechhoff, LV., Votava, JR., Ochs, DE., Moser, DR. Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi. J. Clin. Microbiol. 1996: 34(5); 1171 -1175. 23. Junqueira, AC., Chiari, E., Wincker, P. Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern brazil. Trans. R. Soc. Trop. Med. Hyg. 1996: 90(2); 129-132. 24. Reyes, B., Lorca, M., Muñoz, P., Frasch, ACC. Fetal IgG specificities against Trypanosoma cruzi antigens in newborns. Proc. Natl. Acad. Sci 1990: 87; 2846-2850. 25. Lorca, M., Veloso, C. et al. Synthetic peptides of Trypanosoma cruzi antigens in the diagnosis of congenital Chagas disease. Res. & Rev in Parasitol. 1998: 58; 43-47.

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