- Email: [email protected]
Presence of Trypanosoma cruzi in pregnant women and typing of lineages in congenital cases
Acta Tropica 124 (2012) 243–246
Contents lists available at SciVerse ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Presence of Trypanosoma cruzi in pregnant women and typing of lineages in congenital cases Sylvia Ortiz a , Inés Zulantay a , Aldo Solari a , Margarita Bisio b , Alejandro Schijman b , Yves Carlier c , Werner Apt a,∗ a
Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, P.O. Box 427, Santiago 3, Chile Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, CONICET, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina c Laboratory of Parasitology, Faculty of Medicine, Free University of Brussels, 27 ULB CP 616, 808, Route de Lennik-B1070, Brussels, Belgium b
a r t i c l e
i n f o
Article history: Received 19 March 2012 Received in revised form 30 July 2012 Accepted 3 August 2012 Available online 10 August 2012 Keywords: Trypanosoma cruzi genotypes Chronic chagasic mothers Newborn PCR Hybridization
a b s t r a c t The objective of this study was to determine the presence of Trypanosoma cruzi in blood samples of mothers with chronic Chagas disease and their newborn by conventional PCR targeted to minicircle kinetoplastidic DNA (kDNA), and to determine the lineages in mother/newborn pairs of the congenital cases by hybridization assays with probes belonging to the TcII, TcI and TcV Discrete Typing Units (DTU). In 63 (57.2%) of the mothers the presence of circulating T. cruzi was demonstrated by PCR immediately before delivery and in three newborn (3%) congenital transmission was confirmed by serial PCR and conventional serology between 1 and 16 months of life, at which point treatment was started. The hybridization signals showed that two of the newborn had the same DTU as their mother (TcI, TcII and TcV), whilst in the third congenital case only TcV was detected in the cord blood, suggesting that in this infant TcI and TcII did not cross the placenta or the parasite was not present at a detectable level. Levels T. cruzi DNA was determined by TaqMan Probe based Real Time PCR assay targeted to nuclear satellite sequences in these three pairs of samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In Chile, maternal–foetal Trypanosoma cruzi transmission acquired extra relevance after the interruption of transmission by Triatoma infestans (the main domestic vector) and blood transfusion control in the late 1990s. In previous studies in an area endemic for Chagas disease in Chile it was reported that 3.7% of pregnant mothers are infected with T. cruzi and vertical transmission occurs in 2.5% of these pregnancies (Apt et al., 2010). Vertical T. cruzi transmission appears to depend on both parasite (strain, levels of parasitemia) and host characteristics (placental factor and/or immune response to infection) (Hermann et al., 2004; Bern et al., 2009). T. cruzi belongs to the order Kinetoplastida, characterized by mitochondrial DNA (kDNA) composed of maxicircles and minicircles; the latter are an ideal PCR target to amplify molecules for detection with high sensitivity, due to the high copy number per cell (Schijman et al., 2003; Virreira et al., 2006, 2007). The second application of PCR-DNA based detection of T. cruzi minicircles from biological samples is the use of the amplified products as molecular markers for T. cruzi genotyping. Minicircle amplification yields
∗ Corresponding author. Tel.: +56 2 9786122; fax: +56 2 9786122. E-mail address: [email protected] (W. Apt). 0001-706X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2012.08.001
a product from the variable region, a highly polymorphic sequence present in different minicircle classes, useful for T. cruzi typing by means of hybridization tests with a panel of well characterized variable regions as kDNA probes (Veas et al., 1991; Breniére et al., 1998). The aim of this study was to investigate the presence of T. cruzi in mother/newborn binomials by PCR targeted to minicircle kinetoplast DNAs (kDNA), and to determine the T. cruzi lineages present in the binomials of congenital cases by hybridization assays.
2. Materials and methods 2.1. Population study Between 2006 and 2010 we studied by PCR in peripheral blood the presence of circulating T. cruzi in 110 mothers with chronic Chagas disease with average age of 32 years (range 17–45). 27% of the mothers were primiparous, 48% had one or two previous pregnancies and 25% three or more. All mothers were from the Province of Choapa, IV Region, which is located between 29◦ 02 and 32◦ 16 S in the area of transverse valleys of Chile. The presence of T. cruzi by PCR in cord blood of 100 of their newborn was also investigated. The Informed Consent for this study was approved by the Ethics Committee of the Faculty of Medicine of the University
244
S. Ortiz et al. / Acta Tropica 124 (2012) 243–246
of Chile. Maternal infection was confirmed by ELISA and IFI IgG as previously described (Zulantay et al., 2011), assessed during the first quarter of pregnancy. 2.2. Minicircle PCR assay The samples of peripheral blood of the mothers and from umbilical cords of the newborn were preserved in guanidine–EDTA as described by Wincker et al. (1994) and boiled for 15 min at 98 ◦ C before extraction and purification of DNA using the Favorgen kit according to the manufacturer’s instructions (Biotech, Corp., Selangor, Malaysia), and maintained at −20 ◦ C until use. 5 L of supernatant was used for DNA template of PCR. The reactions were performed in triplicate with oligonucleotides 121 and 122, which anneal to the four conserved regions present in minicircles of T. cruzi (Wincker et al., 1994), including a positive and negative control in each test. We conducted a random sampling of specimens with negative PCR to perform a control test inhibition with  globin. The 330-base pair PCR product was separated by electrophoresis in 2% agarose gels and visualized by staining with ethidium bromide. In all the newborn cases with positive PCR at birth, serial PCR assays and conventional serology by ELISA and IFI were performed at 1–16 months of life. Additionally, the positive kDNA-based PCR results in blood samples of binomials of mother/congenitally infected newborn were quantified by a TaqMan Probe-based Real Time PCR assay targeted to nuclear satellite sequences (Schijman et al., 2011; Bravo et al., 2012). 2.3. Southern blot and hybridization assays T. cruzi DTU genotyping in the three mother/newborn binomials in which congenital transmission was confirmed was performed by Southern blot of kDNA amplicons, as described previously (Veas et al., 1994). Briefly, 10 L of each PCR product was subjected to electrophoresis, transferred onto Hybond N+ nylon membranes (Amersham, Little Chalfont, United Kingdom) and cross-linked with ultraviolet light to fix the DNA. The membranes were pre-hybridized for at least 2 h at 55 ◦ C and hybridized with different probes of T. cruzi minicircle 32 Plabeled DNA (1 × 106 cpm/membrane). Nylon membranes were then submitted to successive washing at different conditions of stringency (Veas et al., 1991). For genotyping, different T. cruzi stocks were used to generate the DNA probes to determine the parasite lineage or mixture infecting each patient. Construction of specific probes sp104c11 (TcI, clonet 19), NRc13 (TcV, clonet 39) and CBBc13 (TcII, clonet 32) was performed by amplification of the variable region of T. cruzi minicircles; primers for probe generation were CV1 (5 -GATTGGGGTTGGAGTACTAT-3 ) and CV2 (5 -TTGAACGGCCCTCCGAAAAC-3 ), which produced a 270-bp fragment (Veas et al., 1994). The DNA probes were labeled using the random primer method with [␣32 P] dCTP and the hybridization profiles were analysed. 3. Results 63 mothers (57.2%) studied immediately before delivery presented the specific 330 bp band of kDNA of T. cruzi. Congenital transmission was confirmed in 3 of 100 newborn (3%) studied by serial PCR and conventional serology in peripheral blood determination between 1 and 16 months of life. In the newborn with negative PCR, conventional serology between 1 and 24 months of life was performed to discount completely congenital infection. All these cases were negative at the end of the follow-up period. The genotyping of T. cruzi DTUs in the three mother/newborn binomials with congenital infection showed that two of the newborn had the same T. cruzi DTU as their mother (TcI, TcII and TcV), whilst in the
Fig. 1. (A) Minicircle PCR amplified analysis with primers 121–122. (a) Electrophoresis pattern obtained for amplicons of T. cruzi kDNA stained with ethidium bromide from three binomial samples of mothers (M) and their congenital newborn (NB), (b) pattern of hybridization with sp104 (TcI) probe, (c) pattern of hybridization with NR (TcV) probe and (d) pattern of hybridization with CBB (TcII) probe. (B) Hybridization probe specificity controls. M: molecular weight marker.
third congenital case only TcV could be detected in the cord blood (Fig. 1). Finally, Fig. 2 shows the levels of T. cruzi DNA in three binomial mothers immediately before delivery and their newborn with congenital infection by TaqMan Real Time PCR assays. The parasite range fluctuated between <1–18.37 and 1.35–12.56 parasites/ml in the newborn and mothers, respectively. 4. Discussion Some authors have significantly correlated the likelihood of congenital infection with parasite density in the mother’s blood (Brutus et al., 2010), while other authors have contradicted this hypothesis (Hall et al., 2010). In this study, 63 mothers with chronic Chagas disease had positive parasitemia immediately before delivery; nevertheless only three transmitted the infection to their newborn. None of the cases with positive PCR at birth was treated immediately, since we have demonstrated that there could be false positive results in cord blood of newborn of mothers with Chagas disease. This criterion was established due to lack of adhesion to perform direct parasitological examination in cord blood of the newborn. In the three congenital cases the IFI and ELISA IgG was positive after one year of age and in at least two controls the specific band of kDNA of T. cruzi was detected by PCR. By real time PCR it was possible to quantify the levels of T. cruzi DNA circulating in the three mother/congenital cases immediately before delivery and at birth, which fluctuated between <1 and 18.37 parasites/ml. Unlike other studies, the parasite load of T. cruzi in mother/newborn binomial demonstrated parasitemia of <10 parasites/ml and >1000 parasites/ml of the mothers and 76.3% of the newborn, respectively. In this study, some neonates also showed lower parasitemia (Virreira et al., 2007). Finally, congenital cases studied between three months and seven years of life by qPCR showed parasite loads between 0.46 and 512 parasites/ml (Duffy et al., 2009). These differences may be related to the timing of transplacental transmission of parasites during gestation, a weaker neonatal parasitism, or the transmission might have occurred closer to delivery (Virreira et al., 2007). The three congenital newborn were asymptomatic at birth, independent of the mother’s parasitemia and the T. cruzi genotype found. It was not possible to study the evolution of T. cruzi populations in the three newborn, since they were treated with nifurtimox and became parasitologically negative in the post-therapy follow up. The present study reports mixed infection with T. cruzi lineages in the mothers and transmission of these mixtures in two out of three congenital cases, suggesting that in this infant TcI and TcII did not cross the placenta or the parasite was not present at a detectable level. Other DTUs such as TcIII and TcIV have not been found in previous
S. Ortiz et al. / Acta Tropica 124 (2012) 243–246
245
Fig. 2. Levels of Trypanosoma cruzi DNA in three mothers before delivery and their newborn with congenital Chagas disease, determined by real time PCR assay with nuclear satellite primers (Tc1–Tc2).
studies of Chile, even though large sample sizes were used and TcVI were found at a very low rate (Miles et al., 1984; Apt et al., 1987; Barnabé et al., 2001). Even though the size of sample analysed here is limited, in the three mother/newborn binomials with circulating T. cruzi we were able to detect the most prevalent DTUs circulating in the region where the study was done, which were previously shown to be TcI, TcII, TcV and TcVI (Solari et al., 2001; Torres et al., 2004). We cannot rule out the possibility of the presence of other DTU as it was not test probe for DTU III, IV and VI, since DTU VI is one of the circulating in the studied area. It has been estimated that congenital Chagas disease will persist as a public health problem in the endemic countries at least for 30 years or more, until the infected women of childbearing age are reduced to very few (Dias, 2009). Nevertheless, up to now we do not know the importance of the level of parasitemia and the DTU of maternal T. cruzi in the prognoses of congenital transmission; this is still a unsolved challenge for clinicians and investigators. Acknowledgments This work was supported by Grant 1080445 and 1100768 from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT-Chile), DI-SAL 05/17-2 of the University of Chile and Project 06/09 Valona Region, Belgium. The authors give special thanks to the health teams of the Choapa Province, IV Región, Chile. Finally, we are grateful to Miguel Saavedra, Eduardo Araya and Gabriela Martínez, for invaluable technical support. References Apt, W., Aguilera, X., Arribada, A., Gómez, L., Miles, M.A., Widmer, G., 1987. Epidemiology of Chagas disease in Northern Chile: isoenzyme profiles of Trypanosoma cruzi from domestic and sylvatic transmission cycles and their association with cardiopathy. American Journal of Tropical Medicine and Hygiene 37, 302–307. Apt, W., Zulantay, I., Solari, A., Ortiz, S., Oddó, D., Corral, G., Truyens, C., Carlier, Y., 2010. Vertical transmission of Trypanosoma cruzi in the Province of Choapa, IV Region, Chile. Preliminary Report (2005–2008). Biological Research 43, 269–274. Barnabé, C., Neubauer, K., Solari, A., Tibayrenc, M., 2001. Trypanosoma cruzi: presence of the two major phylogenetic lineages and of several lesser discrete typing units (DTUs) in Chile and Paraguay. Acta Tropica 78, 127–137.
Bern, C., Verastegui, M., Gilman, R.H., Lafuente, C., Galdos-Cardenas, G., Calderon, M., Pacori, J., Del Carmen Abastoflor, M., Aparicio, H., Brady, M.F., Ferrufino, L., Angulo, N., Marcus, S., Sterling, C., Maguire, J.H., 2009. Congenital Trypanosoma cruzi transmission in Santa Cruz, Bolivia. Clinical Infectious Diseases 49, 1667–1674. ˜ C., Nazal, N., Saavedra, M., Martínez, G., Araya, E., Apt, W., Zulantay, Bravo, N., Munoz, I., 2012. Real-Time PCR in fecal samples of Triatoma infestans obtained by XD: proposal for an exogenous internal control. Parasites & Vectors 5, e59. Breniére, S.F., Bosseno, M.F., Telleria, J., Bastrenta, B., Yacsik, N., Noireau, F., Alcazar, J.L., Barnabé, C., Wincker, P., Tibayrenc, M., 1998. Different behavior of two Trypanosoma cruzi major clones: transmission and circulation in young Bolivian patients. Experimental Parasitology 89, 285–295. Brutus, L., Castillo, H., Bernal, C., Salas, N.A., Schneider, D., Santalla, J.A., Chippaux, J.P., 2010. Detectable Trypanosoma cruzi parasitemia during pregnancy and delivery as a risk factor for congenital Chagas disease. American Journal of Tropical Medicine and Hygiene 83, 1044–1047. Dias, J.C.P., 2009. Elimination of Chagas disease transmission: perspectives. Memórias do Instituto Oswaldo Cruz. Rio de Janeiro 104, 41–45. Duffy, T., Bisio, M., Altcheh, J., Burgos, J.M., Diez, M., Schijman, A.G., 2009. Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients. PLoS Neglected Tropical Diseases 3, e419. Hall, C.A., Pierce, E.M., Wimsatt, A.N., Hobby-Dolbeer, T., Meers, J.B., 2010. Virulence and vertical transmission of two genotypically and geographically diverse isolates of Trypanosoma cruzi in mice. Journal of Parasitology 96, 371–376. Hermann, E., Truyens, C., Alonso-Vega, C., Rodriguez, P., Berthe, A., Torrico, F., Carlier, Y., 2004. Congenital transmission of Trypanosoma cruzi is associated with maternal enhanced parasitemia and decreased production of interferon-gamma in response to parasite antigens. Journal of Infectious Diseases 189, 1274–1281. Miles, M., Apt, W., Widmer, G., Povoa, M.M., Schofield, C.J., 1984. Isoenzyme heterogeneity and numerical taxonomy of Trypanosoma cruzi stocks from Chile. Transactions of the Royal Society of Tropical Medicine and Hygiene 78, 526– 535. Schijman, A., Altcheh, J., Burgos, J.M., Biancardi, M., Bisio, M., Levin, M., Freilij, H., 2003. Aetiological treatment of congenital Chagas disease diagnosed and monitored by the polymerase chain reaction. Journal of Antimicrobial Chemotherapy 52, 441–449. Schijman, A., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejía Jaramillo, A.M., et al., 2011. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Neglected Tropical Diseases 5, 931. Solari, A., Campillay, R., Ortiz, S., Wallace, A., 2001. Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients. Experimental Parasitology 97, 226–233. ˜ Torres, J.P., Ortiz, S., Munoz, S., Solari, A., 2004. Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses. Parasitology 128, 161–168. Veas, F., Breniere, S.F., Cuny, G., Brengues, C., Solari, A., Tibayrenc, M., 1991. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cellular and Molecular Biology 37, 73–84.
246
S. Ortiz et al. / Acta Tropica 124 (2012) 243–246
Veas, F., Cuny, G., Breniere, S.F., Tibayrenc, M., 1994. Subspecific kDNA probes for major clones of Trypanosoma cruzi. Acta Tropica 48, 79–82. Virreira, M., Alonso-Vega, C., Solano, M., Jijena, J., Brutus, L., Bustamante, Z., Truyens, C., Schneider, D., Torrico, F., Carlier, Y., Svoboda, M., 2006. Congenital Chagas disease in Bolivia is not associated with DNA polymorphism of Trypanosoma cruzi. American Journal of Tropical Medicine and Hygiene 75, 871–879. Virreira, M., Truyens, C., Alonso-Vega., C., Brutus, L., Jijena, J., Torrico, F., Carlier, Y., Svoboda, M., 2007. Comparison of Trypanosoma cruzi lineages and levels of parasitic DNA in infected mothers and theirs newborns. American Journal of Tropical Medicine and Hygiene 77, 102–106.
Wincker, P., Britto, C., Pereira, J.B., Cardoso, M.A., Oelemann, W., Morel, C.M., 1994. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. American Journal of Tropical Medicine and Hygiene 51, 771–777. Zulantay, I., Corral, G., Guzmán, M.C., Aldunate, F., Guerra, W., Cruz, I., Araya, A., ˜ C., Apt, W., 2011. The investigation of congeniTapia, V., Márquez, F., Munoz, tal infection by Trypanosoma cruzi in an endemic area of Chile: three protocols explored in a pilot project. Annals of Tropical Medicine and Hygiene 105, 123– 128.
Contents lists available at SciVerse ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Presence of Trypanosoma cruzi in pregnant women and typing of lineages in congenital cases Sylvia Ortiz a , Inés Zulantay a , Aldo Solari a , Margarita Bisio b , Alejandro Schijman b , Yves Carlier c , Werner Apt a,∗ a
Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, P.O. Box 427, Santiago 3, Chile Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, CONICET, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina c Laboratory of Parasitology, Faculty of Medicine, Free University of Brussels, 27 ULB CP 616, 808, Route de Lennik-B1070, Brussels, Belgium b
a r t i c l e
i n f o
Article history: Received 19 March 2012 Received in revised form 30 July 2012 Accepted 3 August 2012 Available online 10 August 2012 Keywords: Trypanosoma cruzi genotypes Chronic chagasic mothers Newborn PCR Hybridization
a b s t r a c t The objective of this study was to determine the presence of Trypanosoma cruzi in blood samples of mothers with chronic Chagas disease and their newborn by conventional PCR targeted to minicircle kinetoplastidic DNA (kDNA), and to determine the lineages in mother/newborn pairs of the congenital cases by hybridization assays with probes belonging to the TcII, TcI and TcV Discrete Typing Units (DTU). In 63 (57.2%) of the mothers the presence of circulating T. cruzi was demonstrated by PCR immediately before delivery and in three newborn (3%) congenital transmission was confirmed by serial PCR and conventional serology between 1 and 16 months of life, at which point treatment was started. The hybridization signals showed that two of the newborn had the same DTU as their mother (TcI, TcII and TcV), whilst in the third congenital case only TcV was detected in the cord blood, suggesting that in this infant TcI and TcII did not cross the placenta or the parasite was not present at a detectable level. Levels T. cruzi DNA was determined by TaqMan Probe based Real Time PCR assay targeted to nuclear satellite sequences in these three pairs of samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In Chile, maternal–foetal Trypanosoma cruzi transmission acquired extra relevance after the interruption of transmission by Triatoma infestans (the main domestic vector) and blood transfusion control in the late 1990s. In previous studies in an area endemic for Chagas disease in Chile it was reported that 3.7% of pregnant mothers are infected with T. cruzi and vertical transmission occurs in 2.5% of these pregnancies (Apt et al., 2010). Vertical T. cruzi transmission appears to depend on both parasite (strain, levels of parasitemia) and host characteristics (placental factor and/or immune response to infection) (Hermann et al., 2004; Bern et al., 2009). T. cruzi belongs to the order Kinetoplastida, characterized by mitochondrial DNA (kDNA) composed of maxicircles and minicircles; the latter are an ideal PCR target to amplify molecules for detection with high sensitivity, due to the high copy number per cell (Schijman et al., 2003; Virreira et al., 2006, 2007). The second application of PCR-DNA based detection of T. cruzi minicircles from biological samples is the use of the amplified products as molecular markers for T. cruzi genotyping. Minicircle amplification yields
∗ Corresponding author. Tel.: +56 2 9786122; fax: +56 2 9786122. E-mail address: [email protected] (W. Apt). 0001-706X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2012.08.001
a product from the variable region, a highly polymorphic sequence present in different minicircle classes, useful for T. cruzi typing by means of hybridization tests with a panel of well characterized variable regions as kDNA probes (Veas et al., 1991; Breniére et al., 1998). The aim of this study was to investigate the presence of T. cruzi in mother/newborn binomials by PCR targeted to minicircle kinetoplast DNAs (kDNA), and to determine the T. cruzi lineages present in the binomials of congenital cases by hybridization assays.
2. Materials and methods 2.1. Population study Between 2006 and 2010 we studied by PCR in peripheral blood the presence of circulating T. cruzi in 110 mothers with chronic Chagas disease with average age of 32 years (range 17–45). 27% of the mothers were primiparous, 48% had one or two previous pregnancies and 25% three or more. All mothers were from the Province of Choapa, IV Region, which is located between 29◦ 02 and 32◦ 16 S in the area of transverse valleys of Chile. The presence of T. cruzi by PCR in cord blood of 100 of their newborn was also investigated. The Informed Consent for this study was approved by the Ethics Committee of the Faculty of Medicine of the University
244
S. Ortiz et al. / Acta Tropica 124 (2012) 243–246
of Chile. Maternal infection was confirmed by ELISA and IFI IgG as previously described (Zulantay et al., 2011), assessed during the first quarter of pregnancy. 2.2. Minicircle PCR assay The samples of peripheral blood of the mothers and from umbilical cords of the newborn were preserved in guanidine–EDTA as described by Wincker et al. (1994) and boiled for 15 min at 98 ◦ C before extraction and purification of DNA using the Favorgen kit according to the manufacturer’s instructions (Biotech, Corp., Selangor, Malaysia), and maintained at −20 ◦ C until use. 5 L of supernatant was used for DNA template of PCR. The reactions were performed in triplicate with oligonucleotides 121 and 122, which anneal to the four conserved regions present in minicircles of T. cruzi (Wincker et al., 1994), including a positive and negative control in each test. We conducted a random sampling of specimens with negative PCR to perform a control test inhibition with  globin. The 330-base pair PCR product was separated by electrophoresis in 2% agarose gels and visualized by staining with ethidium bromide. In all the newborn cases with positive PCR at birth, serial PCR assays and conventional serology by ELISA and IFI were performed at 1–16 months of life. Additionally, the positive kDNA-based PCR results in blood samples of binomials of mother/congenitally infected newborn were quantified by a TaqMan Probe-based Real Time PCR assay targeted to nuclear satellite sequences (Schijman et al., 2011; Bravo et al., 2012). 2.3. Southern blot and hybridization assays T. cruzi DTU genotyping in the three mother/newborn binomials in which congenital transmission was confirmed was performed by Southern blot of kDNA amplicons, as described previously (Veas et al., 1994). Briefly, 10 L of each PCR product was subjected to electrophoresis, transferred onto Hybond N+ nylon membranes (Amersham, Little Chalfont, United Kingdom) and cross-linked with ultraviolet light to fix the DNA. The membranes were pre-hybridized for at least 2 h at 55 ◦ C and hybridized with different probes of T. cruzi minicircle 32 Plabeled DNA (1 × 106 cpm/membrane). Nylon membranes were then submitted to successive washing at different conditions of stringency (Veas et al., 1991). For genotyping, different T. cruzi stocks were used to generate the DNA probes to determine the parasite lineage or mixture infecting each patient. Construction of specific probes sp104c11 (TcI, clonet 19), NRc13 (TcV, clonet 39) and CBBc13 (TcII, clonet 32) was performed by amplification of the variable region of T. cruzi minicircles; primers for probe generation were CV1 (5 -GATTGGGGTTGGAGTACTAT-3 ) and CV2 (5 -TTGAACGGCCCTCCGAAAAC-3 ), which produced a 270-bp fragment (Veas et al., 1994). The DNA probes were labeled using the random primer method with [␣32 P] dCTP and the hybridization profiles were analysed. 3. Results 63 mothers (57.2%) studied immediately before delivery presented the specific 330 bp band of kDNA of T. cruzi. Congenital transmission was confirmed in 3 of 100 newborn (3%) studied by serial PCR and conventional serology in peripheral blood determination between 1 and 16 months of life. In the newborn with negative PCR, conventional serology between 1 and 24 months of life was performed to discount completely congenital infection. All these cases were negative at the end of the follow-up period. The genotyping of T. cruzi DTUs in the three mother/newborn binomials with congenital infection showed that two of the newborn had the same T. cruzi DTU as their mother (TcI, TcII and TcV), whilst in the
Fig. 1. (A) Minicircle PCR amplified analysis with primers 121–122. (a) Electrophoresis pattern obtained for amplicons of T. cruzi kDNA stained with ethidium bromide from three binomial samples of mothers (M) and their congenital newborn (NB), (b) pattern of hybridization with sp104 (TcI) probe, (c) pattern of hybridization with NR (TcV) probe and (d) pattern of hybridization with CBB (TcII) probe. (B) Hybridization probe specificity controls. M: molecular weight marker.
third congenital case only TcV could be detected in the cord blood (Fig. 1). Finally, Fig. 2 shows the levels of T. cruzi DNA in three binomial mothers immediately before delivery and their newborn with congenital infection by TaqMan Real Time PCR assays. The parasite range fluctuated between <1–18.37 and 1.35–12.56 parasites/ml in the newborn and mothers, respectively. 4. Discussion Some authors have significantly correlated the likelihood of congenital infection with parasite density in the mother’s blood (Brutus et al., 2010), while other authors have contradicted this hypothesis (Hall et al., 2010). In this study, 63 mothers with chronic Chagas disease had positive parasitemia immediately before delivery; nevertheless only three transmitted the infection to their newborn. None of the cases with positive PCR at birth was treated immediately, since we have demonstrated that there could be false positive results in cord blood of newborn of mothers with Chagas disease. This criterion was established due to lack of adhesion to perform direct parasitological examination in cord blood of the newborn. In the three congenital cases the IFI and ELISA IgG was positive after one year of age and in at least two controls the specific band of kDNA of T. cruzi was detected by PCR. By real time PCR it was possible to quantify the levels of T. cruzi DNA circulating in the three mother/congenital cases immediately before delivery and at birth, which fluctuated between <1 and 18.37 parasites/ml. Unlike other studies, the parasite load of T. cruzi in mother/newborn binomial demonstrated parasitemia of <10 parasites/ml and >1000 parasites/ml of the mothers and 76.3% of the newborn, respectively. In this study, some neonates also showed lower parasitemia (Virreira et al., 2007). Finally, congenital cases studied between three months and seven years of life by qPCR showed parasite loads between 0.46 and 512 parasites/ml (Duffy et al., 2009). These differences may be related to the timing of transplacental transmission of parasites during gestation, a weaker neonatal parasitism, or the transmission might have occurred closer to delivery (Virreira et al., 2007). The three congenital newborn were asymptomatic at birth, independent of the mother’s parasitemia and the T. cruzi genotype found. It was not possible to study the evolution of T. cruzi populations in the three newborn, since they were treated with nifurtimox and became parasitologically negative in the post-therapy follow up. The present study reports mixed infection with T. cruzi lineages in the mothers and transmission of these mixtures in two out of three congenital cases, suggesting that in this infant TcI and TcII did not cross the placenta or the parasite was not present at a detectable level. Other DTUs such as TcIII and TcIV have not been found in previous
S. Ortiz et al. / Acta Tropica 124 (2012) 243–246
245
Fig. 2. Levels of Trypanosoma cruzi DNA in three mothers before delivery and their newborn with congenital Chagas disease, determined by real time PCR assay with nuclear satellite primers (Tc1–Tc2).
studies of Chile, even though large sample sizes were used and TcVI were found at a very low rate (Miles et al., 1984; Apt et al., 1987; Barnabé et al., 2001). Even though the size of sample analysed here is limited, in the three mother/newborn binomials with circulating T. cruzi we were able to detect the most prevalent DTUs circulating in the region where the study was done, which were previously shown to be TcI, TcII, TcV and TcVI (Solari et al., 2001; Torres et al., 2004). We cannot rule out the possibility of the presence of other DTU as it was not test probe for DTU III, IV and VI, since DTU VI is one of the circulating in the studied area. It has been estimated that congenital Chagas disease will persist as a public health problem in the endemic countries at least for 30 years or more, until the infected women of childbearing age are reduced to very few (Dias, 2009). Nevertheless, up to now we do not know the importance of the level of parasitemia and the DTU of maternal T. cruzi in the prognoses of congenital transmission; this is still a unsolved challenge for clinicians and investigators. Acknowledgments This work was supported by Grant 1080445 and 1100768 from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT-Chile), DI-SAL 05/17-2 of the University of Chile and Project 06/09 Valona Region, Belgium. The authors give special thanks to the health teams of the Choapa Province, IV Región, Chile. Finally, we are grateful to Miguel Saavedra, Eduardo Araya and Gabriela Martínez, for invaluable technical support. References Apt, W., Aguilera, X., Arribada, A., Gómez, L., Miles, M.A., Widmer, G., 1987. Epidemiology of Chagas disease in Northern Chile: isoenzyme profiles of Trypanosoma cruzi from domestic and sylvatic transmission cycles and their association with cardiopathy. American Journal of Tropical Medicine and Hygiene 37, 302–307. Apt, W., Zulantay, I., Solari, A., Ortiz, S., Oddó, D., Corral, G., Truyens, C., Carlier, Y., 2010. Vertical transmission of Trypanosoma cruzi in the Province of Choapa, IV Region, Chile. Preliminary Report (2005–2008). Biological Research 43, 269–274. Barnabé, C., Neubauer, K., Solari, A., Tibayrenc, M., 2001. Trypanosoma cruzi: presence of the two major phylogenetic lineages and of several lesser discrete typing units (DTUs) in Chile and Paraguay. Acta Tropica 78, 127–137.
Bern, C., Verastegui, M., Gilman, R.H., Lafuente, C., Galdos-Cardenas, G., Calderon, M., Pacori, J., Del Carmen Abastoflor, M., Aparicio, H., Brady, M.F., Ferrufino, L., Angulo, N., Marcus, S., Sterling, C., Maguire, J.H., 2009. Congenital Trypanosoma cruzi transmission in Santa Cruz, Bolivia. Clinical Infectious Diseases 49, 1667–1674. ˜ C., Nazal, N., Saavedra, M., Martínez, G., Araya, E., Apt, W., Zulantay, Bravo, N., Munoz, I., 2012. Real-Time PCR in fecal samples of Triatoma infestans obtained by XD: proposal for an exogenous internal control. Parasites & Vectors 5, e59. Breniére, S.F., Bosseno, M.F., Telleria, J., Bastrenta, B., Yacsik, N., Noireau, F., Alcazar, J.L., Barnabé, C., Wincker, P., Tibayrenc, M., 1998. Different behavior of two Trypanosoma cruzi major clones: transmission and circulation in young Bolivian patients. Experimental Parasitology 89, 285–295. Brutus, L., Castillo, H., Bernal, C., Salas, N.A., Schneider, D., Santalla, J.A., Chippaux, J.P., 2010. Detectable Trypanosoma cruzi parasitemia during pregnancy and delivery as a risk factor for congenital Chagas disease. American Journal of Tropical Medicine and Hygiene 83, 1044–1047. Dias, J.C.P., 2009. Elimination of Chagas disease transmission: perspectives. Memórias do Instituto Oswaldo Cruz. Rio de Janeiro 104, 41–45. Duffy, T., Bisio, M., Altcheh, J., Burgos, J.M., Diez, M., Schijman, A.G., 2009. Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients. PLoS Neglected Tropical Diseases 3, e419. Hall, C.A., Pierce, E.M., Wimsatt, A.N., Hobby-Dolbeer, T., Meers, J.B., 2010. Virulence and vertical transmission of two genotypically and geographically diverse isolates of Trypanosoma cruzi in mice. Journal of Parasitology 96, 371–376. Hermann, E., Truyens, C., Alonso-Vega, C., Rodriguez, P., Berthe, A., Torrico, F., Carlier, Y., 2004. Congenital transmission of Trypanosoma cruzi is associated with maternal enhanced parasitemia and decreased production of interferon-gamma in response to parasite antigens. Journal of Infectious Diseases 189, 1274–1281. Miles, M., Apt, W., Widmer, G., Povoa, M.M., Schofield, C.J., 1984. Isoenzyme heterogeneity and numerical taxonomy of Trypanosoma cruzi stocks from Chile. Transactions of the Royal Society of Tropical Medicine and Hygiene 78, 526– 535. Schijman, A., Altcheh, J., Burgos, J.M., Biancardi, M., Bisio, M., Levin, M., Freilij, H., 2003. Aetiological treatment of congenital Chagas disease diagnosed and monitored by the polymerase chain reaction. Journal of Antimicrobial Chemotherapy 52, 441–449. Schijman, A., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejía Jaramillo, A.M., et al., 2011. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Neglected Tropical Diseases 5, 931. Solari, A., Campillay, R., Ortiz, S., Wallace, A., 2001. Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients. Experimental Parasitology 97, 226–233. ˜ Torres, J.P., Ortiz, S., Munoz, S., Solari, A., 2004. Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses. Parasitology 128, 161–168. Veas, F., Breniere, S.F., Cuny, G., Brengues, C., Solari, A., Tibayrenc, M., 1991. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cellular and Molecular Biology 37, 73–84.
246
S. Ortiz et al. / Acta Tropica 124 (2012) 243–246
Veas, F., Cuny, G., Breniere, S.F., Tibayrenc, M., 1994. Subspecific kDNA probes for major clones of Trypanosoma cruzi. Acta Tropica 48, 79–82. Virreira, M., Alonso-Vega, C., Solano, M., Jijena, J., Brutus, L., Bustamante, Z., Truyens, C., Schneider, D., Torrico, F., Carlier, Y., Svoboda, M., 2006. Congenital Chagas disease in Bolivia is not associated with DNA polymorphism of Trypanosoma cruzi. American Journal of Tropical Medicine and Hygiene 75, 871–879. Virreira, M., Truyens, C., Alonso-Vega., C., Brutus, L., Jijena, J., Torrico, F., Carlier, Y., Svoboda, M., 2007. Comparison of Trypanosoma cruzi lineages and levels of parasitic DNA in infected mothers and theirs newborns. American Journal of Tropical Medicine and Hygiene 77, 102–106.
Wincker, P., Britto, C., Pereira, J.B., Cardoso, M.A., Oelemann, W., Morel, C.M., 1994. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. American Journal of Tropical Medicine and Hygiene 51, 771–777. Zulantay, I., Corral, G., Guzmán, M.C., Aldunate, F., Guerra, W., Cruz, I., Araya, A., ˜ C., Apt, W., 2011. The investigation of congeniTapia, V., Márquez, F., Munoz, tal infection by Trypanosoma cruzi in an endemic area of Chile: three protocols explored in a pilot project. Annals of Tropical Medicine and Hygiene 105, 123– 128.