JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3824–3826 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00048-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.
Congenital Trypanosoma cruzi Infection in Neonates and
Infants from Two Regions of Chile Where
Chagas’ Disease Is Endemic
Chagas’ disease, or American trypanosomiasis, whose eti- cities of Ovalle (region IV) and La Serena (region V) and in ological agent is the protozoan parasite Trypanosoma cruzi, the hospitals of Los Andes, San Camilo, and Gustavo Fricke, is an important public health concern in Latin America. At located in region V. The blood samples were collected system- least 8 million people acquired the infection via the arthro- atically as part of a chagasic infection screening program pod vector or another mechanism of transmission, such as launched by the Health Ministry of Chile (MINSAL) in these blood transfusion or vertical transmission from mother to geopolitical regions of the country. Samples were taken under child (9). In Chile (11), the parasitose is endemic and en- the guidance of the ethics committee for scienti？c research of zootic in the northern and central biogeographical regions the ISP-MINSAL. The ages of the children ranged from 1 day (18?30 S to 34?36 S). to 2 years 8 months. Eighty-two (45.8%) children were less Three species of Reduviidae insect are transmitters of Cha- than 3 days of age, 15.1% (27/179) were between 4 and 15 days gas’ disease to mammals, including humans, in Chile: Triatoma old, 30.2% (54/179) were more than 15 days and less 1 year old, infestans, Triatoma spinolai, and Mepraia gajardoi (3, 13). and 8.9% (16/179) were 1 year to 2 years 8 months old; 98 were Modes of transmission of Chagas’ disease other than infections female (54.7%), and 81 were male (45.3%). Samples were through insect vectors have been documented to occur in taken from peripheral blood and stored until shipment to the Chile, including accidental laboratory infections, infections laboratory where they were processed. With each sample, we caused by blood transfusion, and transplacental infections. In- performed an indirect immuno？uorescence assay (IF) and an fections by the oral route in humans have not been described enzyme-linked immunosorbent assay (ELISA) for detecting to occur in Chile (12). serum-speci？c antibodies to T. cruzi (2, 14). PCR was per- Epidemiological studies conducted in previous decades, formed as described by Wincker et al. (16), with the following mainly with blood banks in areas where Chagas’ disease is modi？cations. DNA was extracted with a commercial kit (Favor- endemic, with hospital maternity wards, and with speci？c age Prep blood genomic DNA extraction minikit) in accordance groups, especially children, as well as an intensive campaign of with the instructions of the supplier. With this extracted DNA, T. infestans disinfestations promoted by the Chilean health PCR was performed using 2.5 l of each sample. We used authority, have led to accurate knowledge of the epidemiolog- primer 121 (5 TAA TGT AAA ACG GGG GAG ATG CAT ical aspects of Chagas’ disease and the control of transmission GA 3 ) (10 mol/liter) and primer 122 (5 GGT TCG ATT by those insect vectors and in blood banks in areas of the GGG GTT GGT GTA ATA TA 3 ) (10 mol/liter) to amplify country where Chagas’ disease is endemic and enzootic (4, 8, a region of the kinetoplast DNA of T. cruzi. As a control for 11). In 1999, this led to the declaration of the interruption of the integrity of the extracted DNA and inhibition of PCR, we transmission of Chagas’ disease via the T. infestans vector in ampli？ed a DNA segment of the human -globin gene by use Chile (1). However, the emergence of new cases of T. cruzi of the following primers: b_glo1 (5 CCT CCT TTG AAG TTC infections in Chile both in blood banks and via transplacental TCC AA 3 ) and b_glo 2 (5 CCT CTT CAC TCA TGG CTT infection maintains the importance of systematic studies on the AG 3 ). In each run, a negative (no-DNA) sample and positive epidemiological and epizootic aspects of this parasitic disease controls (T. cruzi DNA, Tulahuen strain) were included. Am- in Chile (4). Congenital Chagas’ disease is considered the pli？cation products were visualized by agarose gel electro- principal mode of T. cruzi infection in geographical areas phoresis in 2% ethidium bromide, using a running time of 55 where transmissions by insect vectors and blood transfusion min at 107 mV. Samples were considered positive when a are controlled (15). 330-bp product was ampli？ed by primers 121/122. Primers for PCR used in laboratory diagnosis of Chagas’ disease is con- b_glo1/b_glo2 ampli？ed a 239-bp product (10). In region IV of Chile, most of the samples studied were from sidered a sensitive and speci？c test and additionally a useful probe for evaluating the ef？cacy of treatment of infected pa- the Ovalle hospital. In region V, most samples were derived tients (14, 15). Since there have been few studies of Chagas’ from the G. Fricke and Los Andes hospitals. All serum sam- disease in recent years in Chile, in the present research we ples were IF and ELISA positive ( 1:20 serum dilution). A describe the use of kinetoplast KNA (kDNA) PCR to detect T. positive PCR result, detecting kDNA of T. cruzi in blood sam- cruzi infections in neonates and infants born from mothers with ples of the 179 neonates and infants, was found in 15 cases Chagas’ disease in two regions of Chile where Chagas’ disease (8.4%). Of the 15 PCR-positive samples, 9 were from region V. is endemic. The purpose of this research was to determine the The frequencies of T. cruzi infections observed in the two frequency of congenital infections in children delivered from biogeographical regions were not signi？cant by a chi-square chagasic mothers by using PCR for kDNA as a laboratory test (Table 1). We also observed no signi？cant difference in the diagnostic tool. percentages of transplacental T. cruzi infection by sex: females, 2 During the years 2007 and 2008 at the Reference Laboratory 9.2% (9/98), and males, 7.4% (6/81) (0.18; P 0.67). of Parasitology of the Institute of Public Health of Chile (ISP), Figure 1 shows the results for the 15 positive PCR results, with blood samples from 179 children less than 2 years of age from a 330-bp band of kDNA of T. cruzi. The -globin control regions IV and V of Chile were received. In those regions, ampli？cation showed a band of 239 bp.
samples were collected in maternity wards of hospitals in the The 8.4% rate of positive PCR results for kDNA of T. cruzi
VOL. 48, 2010 LETTERS TO THE EDITOR 3825
TABLE 1. PCR for Trypanosoma cruzi kDNA for 179 chagasic cruzi ranged from 1 to 12% (6, 15). Our results are in children 2 years old from biogeographical regions IV and V concordance with this reported frequency of transmission. of Chile (2007 and 2008) Our research provides valuable evidence that PCR for T.
cruzi kDNA is an appropriate tool for diagnosis of transpla- No. of chagasic No. of positive Region Hospital children PCR results (%) cental infections and con？rms the frequencies reported for other countries where Chagas’ disease is endemic or not en- 91 IV Ovalle demic (6, 15). In a comparative study done with chagasic blood IV La Serena 2 adonors in Chile, it was observed that some patients with pos- 6 (6.5) Total (IV) 93 G. Fricke itive xenodiagnoses presented negative PCR results for kDNA V 34 V Los Andes 36 (4). Thus, negative PCR results for children delivered from V San Camilo 16 chagasic mothers will not discon？rm the presence of T. cruzi a9 (10.5) Total (V) 86 infection (6); consequently, an underestimation of the trans-
mission frequency is possible when PCR is used exclusively. Total 179 15 (8.4) Further studies on the use of PCR as a diagnostic test for a Chi-square result, 0.94; P value, 0.34 (no statistical difference). transplacental Chagas’ infection are required to determine with certainty the performance of this test, especially in com- bination with other direct or indirect parasitological diagnostic observed in children most likely indicates the occurrence of T. tests. cruzi transplacental infection in these two geopolitical regions of Chile. The majority of the positive cases were detected in We thank Susana Verdugo (Hospital de Ovalle), Raul Higueras children who were only a few days old and whose mothers had (Hospital de Los Andes), and Litzy Villalon (Hospital G. Fricke) for given birth in the maternity wards of hospitals in these ？eld participation in the study. regions and not in their homes, where they might have been exposed to the insect vector by being bitten (12). Moreover,
transmission of Chagas’ disease by T. infestans vectors inside REFERENCES dwellings is considered virtually nonexistent in Chile, reaf- 1. Anonymous. 1999. Inform of the Certi？cation Committee. National Program ？rming that children with positive PCR results had an in for the Control of the Chagas Disease. Ministry of Health of Chile, Santiago, utero infection transmitted by their mothers (1). PCR was Chile. run only with venous blood samples of the children, avoiding 2. Breniere, S., R. Carrasco, H. Miguez, J. L. Lemesre, and Y. Carlier. 1985. the use of umbilical cord blood samples, where contamina- Comparison of immunological test for the serodiagnosis of Chagas disease in Bolivian patients. Trop. Geogr. Med. 37:231–238. tion with T. cruzi DNA from the mother could be possible. 3. Carvajal, A., J. Orellana, W. Wigant, C. Borquez, and I. Lobato. 2007. Few studies of transplacental transmission caused by T. cruzi Prevalence of triatomines infected with Trypanosoma cruzi in the coast of have been done in Chile. In 1989, Schenone et al. (11) found Arica city. Parasitol. Latinoam. 62:118–121. that 7.1% (24/336) of the newborn of mothers with Chagas’ 4. Galaz, P., S. Garcia, R. Mercado, E. Orrego, B. Pagliero, M. Contreras, P. disease showed a positive xenodiagnosis test result, a ？gure Salinas, and C. Arancibia. 2007. Parasitological and epidemiological aspects that rose to 14.5% (53/364) when they studied the persis- of Trypanosoma cruzi seropositive blood donors. Rev. Med. Chile 135:1291– 1295. (In Spanish.) tence of speci？c anti-T. cruzi antibodies up to 18 months 5. Garcia, A., M. I. Bahamonde, S. Verdugo, J. Correa, C. Pastene, A. Solari, after birth, as determined by an indirect hemagglutination and M. Lorca. 2001. Trypanosoma cruzi transplacental infection. Situation in reaction in the blood samples of the children (10). In an- Chile. Rev. Med. Chile 129:330–332. other study, Mercado et al. used xenodiagnosis to detect the 6. Jackson, Y., C. Myers, A. Diana, H. P. Marti, H. Wolff, F. Chappuis, L. parasite in 297 newborn delivered in the maternity ward of Loutan, and A. Gervaix. 2009. Congenital transmission of Chagas disease the hospital of Salamanca in region IV of Chile; they ob- in Latin American immigrants in Switzerland. Emerg. Infect. Dis. 15:601– 603. served that 29 (9.8%) had serum-speci？c antibodies against 7. Mercado, R., C. Rodriguez, S. Astete, M. Contreras, and H. Schenone. 1996. T. cruzi but described no transplacental T. cruzi infections The modi？ed Strout method used as a systematical diagnostic test of the (7). In the only other published study on congenital Chagas’ congenital Chagas disease in Chile. Field trial at a high endemic zone. Am. J. disease in Chile using PCR, Garcia et al. reported 21.2% Trop. Med. Hyg. 55(Suppl.):291. (32/151) cases as positive. This frequency is signi？cantly 8. Neghme, A., H. Schenone, F. Villarroel, and A. Rojas. 1991. Experimental higher than and not consistent with that determined by us antitriatomic programm in Santiago, Chile. Bol. Chil. Parasitol. 47:47–57. 9. Rassi, A., Jr., A. Rassi, and J. A. Marin-Neto. 2010. Chagas disease. Lancet (5). In other South American countries (Argentina, Bolivia, 375(9723):1388–1402. Brazil, and Paraguay) and in a country where Chagas’ dis- 10. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, and H. A. ease is not endemic, rates of transplacental infection by T. Erlich. 1992. Enzymatic ampli？cation of beta-globin genome sequences and restriction site analysis for diagnosis of sickle cell anaemia. Biotechnology 24:476–480. 11. Schenone, H., M. Contreras, J. M. Borgon?o, A. Rojas, P. Tello, and P. Salinas. 1989. Some epidemiological, clinical and parasitological character- istics of the congenital Chagas disease in Chile. Rev. Pediatr. (Santiago) 32:65–72. 12. Schenone, H., M. Contreras, J. M. Borgon?o, R. Maturana, P. Salinas, and L. Sandoval. 1991. Overview of the epidemiology of the Chagas disease in Chile. Bol. Chil. Parasitol. 46:19–30. 13. Schenone, H., F. Villarroel, and A. Rojas. 1995. Presence of Triatoma spi- nolai in human dwellings. Bol. Chil. Parasitol. 50:76–79. FIG. 1. Positive ampli？cation of T. cruzi kDNA extracted from 14. Solari A., S. Ortiz, A. Soto, C. Arancibia, R. Campillay, and M. Contreras. blood samples of 15 chagasic children. Columns: 1, molecular weight 2001. Treatment of Trypanosoma cruzi-infected children with nifurtimox: a 3 standard; 2, T. cruzi kDNA ampli？cation positive control (330 bp); 3, year follow-up by PCR. J. Antimicrob. Chemother. 48:515–519. no-T. cruzi DNA control; 4, -globin control ampli？ed band (239 bp); 15. Virreyna, M., F. Torrico, C. Truyens, C. Alonso-Vega, M. Solano, and Y. Carlier. 2003. Comparison of polymerase chain reaction methods for reliable 5 to 19; positive cases of chagasic children.
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and easy detection of congenital Trypanosoma cruzi infection. Am. J. Trop. Morel. 1994. Use of a simpli？ed polymerase chain reaction procedure to Med. Hyg. 68:574–582. detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am. J. Trop. Med. Hyg. 51:771–777. 16. Wincker, P., C. Britto, J. B. Pereira, M. A. Cardoso, W. Oelemann, and C. M.
Maria I. Jercic
Parasitology Reference Laboratory Instituto de Salud Publica de Chile Avenida Marathon 1000 7780050 Santiago, Chile
Educational Parasitology Unit Faculty of Medicine Universidad de Chile Las Palmeras 299 Int. Quinta Normal Santiago, Chile
Rodrigo Villarroel Parasitology Reference Laboratory Instituto de Salud Publica de Chile Avenida Marathon 1000 7780050 Santiago, Chile
Fax: 56-2-6814499 E-mail: firstname.lastname@example.org
Published ahead of print on 4 August 2010.