BioMed CentralCases Journal ssOpen AcceCase Report Fatal congenital Chagas' disease in a non-endemic area: a case report María Flores-Chávez1, Yamile Faez2, José M Olalla2, Israel Cruz1, Teresa Gárate1, Mercedes Rodríguez1, Pilar Blanc2 and Carmen Cañavate*1 Address: 1Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Pozuelo-Majadahonda Km 2, 28220 Majadahonda, Madrid, Spain and 2Hospital Carlos Haya, Av. Carlos Haya s/n, 29010 Málaga, Spain Email: María Flores-Chávez - mflores@isciii.es; Yamile Faez - yamilefh@hotmail.com; José M Olalla - jomaolalla@yahoo.es; Israel Cruz - cruzi@isciii.es; Teresa Gárate - tgarate@isciii.es; Mercedes Rodríguez - mero@isciii.es; Pilar Blanc - pblanci@telefonica.net; Carmen Cañavate* - ccanave@isciii.es * Corresponding author Abstract The early diagnosis of congenital Chagas' disease is very important if infected newborns, whether symptomatic or not, are to receive adequate treatment. This paper describes the complications arising in the diagnosis of a newborn with fatal congenital Chagas' disease in Spain, a non-endemic area where visceral leishmaniasis is present. Introduction Chagas' disease is endemic from the south of the United States to southern Argentina and Chile, where it is mainly transmitted by reduvid insects. The protozoan that causes the disease – which has a wide range of clinical manifesta- tions – is T. cruzi, a parasite that shows great genetic vari- ation. Large numbers of protozoa are detected during the acute phase of the disease, while numbers are much lower during the chronic phase. However, it is during this latter phase when the humoral immune response is patent. Vertical or congenital transmission of T. cruzi is also pos- sible. This route is associated with an infection rate of 0– 10%, depending on the geographical area in question. Recent studies indicate the majority of children born to infected mothers to be asymptomatic, although some 2– 10% present with severe respiratory distress, hepat- osplenomegaly, myocarditis and meningoencephalitis [1]. Without specific treatment, the mortality rate among such children is high. Since the transmission of T. cruzi during pregnancy cannot be prevented, early diagnosis in newborns is essential so that appropriate etiological treat- ment can be administered; such treatment can be 100% effective. Since maternal IgG antibodies and excretion- secretion antigens that stimulate IgM or IgA production can cross the placenta [2], serological tests cannot discrim- inate between infected and non-infected newborns. How- ever, the presence of anti-T. cruzi antibodies in maternal serum identifies mothers whose newborns are at risk of having been infected [2]. Accordingly, to confirm the par- asite infection, mobile T. cruzi trypomastigotes should be sought microscopically in the cord blood or peripheral blood of newborns after a concentration procedure (the microhaematocrit test) [3]. Case presentation A 37 year-old Argentine woman with a background of bipolar disease attended an appointment for a fetal ultra- sound scan during the third trimester of pregnancy at a teaching hospital. The results revealed her unborn child to Published: 7 November 2008 Cases Journal 2008, 1:302 doi:10.1186/1757-1626-1-302 Received: 7 August 2008 Accepted: 7 November 2008 This article is available from: http://www.casesjournal.com/content/1/1/302 © 2008 Flores-Chávez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 5 (page number not for citation purposes) Cases Journal 2008, 1:302 http://www.casesjournal.com/content/1/1/302be suffering a 3–4 weeks delay in growth, unilateral ven- triculomegaly, and signs of stress. Birth was induced at 34 weeks (delivery was by Caesarean section); the newborn weighed 1130 g and was in a convulsive state. Physical examination of the child revealed scant spontaneous activity, moderate responses to stimuli, poor peripheral perfusion, severe microcephaly, small palpebral slits, foci of choroiditis, vitreous opacity, and hepatosplenomegaly. Cranial ultrasonography revealed a left subependimal hemorrhage and dilation of the occipital horns of the lat- eral ventricles. A cranial computed tomography (CT) scan confirmed the dilation of the lateral ventricles and revealed colpocephaly and megacisterna magna. Electro- encephalography indicated a low to moderate activity voltage and discrete signs of dysrhythmia in the frontal zone. Ultrasonography detected hypertrophy of the left ventricle of the heart; systolic function was conserved. In abdominal ultrasound scans the liver was hyperecho- genic; moderate hepatosplenomegaly was also seen. Blood tests revealed leukopenia, neutropenia and persist- ent thrombocytopenia, hypoproteinemia, hyponatremia and hypocalcemia, elevated non-conjugated bilirubin lev- els, elevated concentrations of glutamyl oxaloacetic transaminase, glutamyl pyruvic transaminase, gamma- glutamyl transpeptidase and alkaline phosphatase. To rule out a possible intrauterine infection or connatal sep- sis, blood and cerebrospinal fluid (CSF) were cultured with negative results. The latex agglutination test for Strep- tococcus group B bacteria and the shell-vial test for cytome- galovirus were performed on urine samples; all results were negative. Serological tests ruled out TORCHES, Epstein-Barr virus, parvovirus (B19), enterovirus and ade- novirus infections. During the newborn's stay in the intensive care unit, fever peaks were recorded. A lower uri- nary tract infection caused by Escherichia coli was identi- fied; treatment with ampicillin and cefotaxime was administered. Newborn blood, urine, fecal and CSF sam- ples were examined by the Centro Nacional de Microbiología (CNM), a Reference Center for Infectious Diseases for the Spanish Healthcare System, to test for viral infections and toxoplasmosis; all tests were negative. Tests of samples of maternal serum ruled out rubella, cytomegalovirus, syph- ilis, toxoplasmosis and HIV; only anti-herpes IgG anti- bodies were detected (IgM negative). The child died after 2 1/2 months of poor clinical progress during which no response to symptomatic treatment was seen. Autopsy revealed generalized nodular encephalitis. The entire cer- ebral cortex showed inflammatory nodules around areas of necrotic tissue (Fig. 1A). The cytoplasm of the mononu- clear phagocytes in the cortex and brainstem showed round structures compatible with Leishman-Donovan bodies (Fig. 1B and 1C). The same structures were not seen, however, in histological sections of the myocar- dium, liver or spleen. Taking into account these results, the post mortem diagnosis was leishmaniasis. To investi- gate the possible route of Leishmania infection, the mother and the rest of her family underwent serological tests for this pathogen. A commercial indirect immunofluorescent antibody test (IFAT) kit (Leishmania Spot IF, bioMérieux) detected IgG anti-Leishmania antibodies in the mother and one of the child's two siblings (Table 1). To confirm these latter results, sections of the child's brain, spleen, liver and heart embedded in paraffin wax were examined by the Parasitology Department of the CNM. The serums of the family members (stored at -20°C until use) were also analyzed. DNA was extracted from the embedded tissues and from the child's serum, and PCR was performed using oligonucleotides R221-R332 and R223-R333 (Ln-PCR) [4] to test for Leishmania spp., and Tcz1-Tcz2 [5] to test for Trypanosoma cruzi. The latter path- ogen was detected in all newborn's samples examined, but Leishmania was not detected. The T. cruzi genotype was determined by PCR of miniexon and 24Sα rRNA genes [6,7], Tc IId was identified. In addition, in-house IFAT using L. infantum as antigen showed an absence of anti- Leishmania antibodies in all family members tested, except for a doubtful positive result for one of the deceased child's siblings. Further testing found no anti- bodies to rk39 [8], a specific antigen of the Leishmania donovani complex, in any family member. IgG anti-T. cruzi antibodies were detected by IFAT and ELISA (using T. cruzi Table 1: Detection of anti-Trypanosoma cruzi and anti-Leishmania antibodies in the family members of the deceased child. T. cruzi tests Leishmania tests In-housea ELISA In-houseb IFAT rk39-ELISAa In-houseb IFAT Commercialb IFAT Mother 2.09 > 1/320 0.04 N 1/1280 Father 0.05 N 0.01 N N Brother 1 2.10 1/320 0.07 ± 1/80 1/320 Brother 2 0.04 N 0.01 N N Deceased child 1.89 > 1/320 0.03 N nd NOTE. - aResults are expressed as absorbances. The cut-off in the T. cruzi ELISA test was 0.5, and 0.2 in the rk39 ELISA test. bThe IFAT results show the serum dilutions in which fluorescent parasites were observed. The cut-off for T. cruzi was 1/40, and 1/80 for Leishmania. N = negative, ± = parasites observed with and without fluorescence, nd = not done.Page 2 of 5 (page number not for citation purposes) Cases Journal 2008, 1:302 http://www.casesjournal.com/content/1/1/302as antigen) in the mother and in the same sibling as above – a clear marker of T. cruzi infection. Anti-T. cruzi antibod- ies were also found in serum of the newborn (Table 1). Discusion In Spain, Chagas' disease is an emerging imported parasi- tosis. In recent years, the population of immigrants from areas where the disease is endemic has grown notably; indeed, these citizens represent some 34% of the country's entire immigrant population (1 735 025 are Latin Ameri- cans) [9]. This situation has influenced legislation regard- ing the selection of blood donors; thus since 2005 Spanish law requires all blood transfusion centers to per- form validated testing to rule out T. cruzi infection on individuals with epidemiological risk. However, no con- sensus has been reached with respect to the monitoring of pregnant women from endemic areas, and only some Spanish regions (Valencia, Catalonia and Murcia) per- form serological screening during pregnancy. To date, a number of cases of congenital transmission have been described, in which the administration of appropriate Detection of Trypanosoma cruzi parasites in samples of the deceased childFigure 1 Detection of Trypanosoma cruzi parasites in samples of the deceased child. A and B, sections of cerebral cortex stained with hematoxylin and eosin, and C, brain stem stained with Giemsa stain. The magnification is shown in the lower right corner; C is digitally magnified. Arrows indicate: in A, an inflammatory nodule, in B, parasites in the cytoplasm, and in C the nucleus (N) and kinetoplast (K) of one of the amastigotes. D, Results of Tcz1-Tcz2 PCR (lanes 1 to 5) and Ln-PCR (lanes 6 to 10) from serum. Lanes 1 and 6, serum; lanes 2, 5, 7 and 10, negative controls; lanes 3 and 8, T. cruzi positive control; lanes 4 and 9, Leishmania positive control; M, molecular weight markers (PCR marker 50 – 2000 bp). A) B) C) D) 1000x 40x 1000 150 50 10 750 500 300 bp 2 1 3 4 5 M 6 7 8 9 M 10 N K Page 3 of 5 (page number not for citation purposes) Cases Journal 2008, 1:302 http://www.casesjournal.com/content/1/1/302treatment was associated with favorable clinical progress [10,11]. The present paper describes a severe case of congenital Chagas' disease that was diagnosed post mortem. This high- lights the limitations of conventional microscopic and serological analyses when the epidemiological back- ground is not considered. Since information on the mother's origin was not made available while the child was alive, the possibility of Chagas' disease was not taken into account, therefore the newborn was monitored fol- lowing the normal Spanish protocols. The post mortem detection of T. cruzi DNA in a serum sam- ple from the newborn analyzed at the Parasitology Department of the CNM indicates that parasites had been circulating in the peripheral blood (Fig. 1D); a lack of experience in microscopical diagnosis of trypomastigotes probably explains why they were unnoticed in earlier blood tests. On the other hand, as the tissue amastigotes of T. cruzi are morphologically very similar to the Leishma- nia amastigotes, they can be confused in microscopic examinations. With respect to immunodiagnosis, although the conventional serology procedures for both protozoa show good sensitivity and specificity in terms of discriminating between infected and non-infected indi- viduals, cross reactivity is a problem. The present data show that the commercial IFAT kit used to test for anti- Leishmania antibodies suffers this disadvantage (Table 1). Therefore, the reliability of commercial serological kits for the diagnosis of Chagas' disease and leishmaniasis should be assessed in this new epidemiological context, espe- cially since parasitological diagnosis is of low sensitivity in the chronic phase of T. cruzi infection. It should be remembered that the recombinant antigen rk39 shows no cross reactivity with sera from patients with Chagas' dis- ease [8], therefore the absence of anti-rk39 antibodies in any of the newborn's family members confirms that what were thought to be anti-Leishmania antibodies detected by the commercial IFAT kit were a product of cross-reactivity. Since in Spain, the immigrant population is exposed to Leishmania infection, and given that cross reactivity can be a problem in serological analyses, PCR might offer the best way to discriminate between L. infantum and T. cruzi. Indeed, from year 2002 to 2007, 100 newborns' blood samples (whose mothers were seropositive to T. cruzi infection) were examined by T. cruzi-PCR at the Parasitol- ogy Department of CNM-ISCIII. It is important to high- light that three new cases were PCR positive in the last year (3/78). Neither the detection of parasites in the placenta nor maternal infection with any particular genotype are indi- cators of congenital infection. However, it has been reported that mothers who transmit the T. cruzi infection commonly have high parasitaemia and show an associ- ated reduction in γ-IFN production [12]. Although these factors were not examined in the present mother, it is probable that a state of immunodepression had been favored by her bipolar disease, which in turn would have favored a high parasite load and the infection of her fetus. The post mortem microscopical detection of the parasite in the child's brain tissue might explain the ultrasonography and cranial CT findings. Although no amastigotes were seen in the remaining tissues examined, the presence of T. cruzi DNA in all the necropsy samples suggests that the parasite was distributed all over the child's body, causing the pathological alterations recorded. At monitoring times during the first and second trimester, ultrasound scans revealed no fetal abnormalities, suggesting that intrauterine infection occurred during the third trimester. Conclusion In the absence of prognostic markers for the development of the infection, a procedure involving i) the serological screening of pregnant women from Latin America, ii) fetal monitoring by ultrasonography, and iii) parasitological diagnosis of the newborn, via direct microscopic observa- tion and molecular tests, may contribute towards the early detection of congenital T. cruzi infection in non-endemic areas, allowing newborns to receive timely treatment. Moreover, since reinfection is unlikely in Spain, providing post-partum anti-parasite treatment to infected mothers could help avoid further instances of vertical transmis- sion. Consent Written informed consent was obtained retrospectively from the patient's mother for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests. Acknowledgements We gratefully acknowledge financial support from the Fondo de Investi- gación Sanitaria (RETIC-RICET, RD06/0021/0009 and RD06/0021/0019). References 1. Torrico F, Alonzo-Vega C, Suarez E, Tellez T, Brutus L, Rodriguez P, et al.: Are maternal re-infections with Trypanosoma cruzi asso- ciated with higher morbidity and mortality of congenital Chagas disease? Trop Med Int Health 2006, 11:628-635. 2. Rodriguez P, Truyens C, Alonso-Vega C, Flores A, Cordova M, Suarez E, et al.: Serum levels for IgM and IgA antibodies to anti- Trypanosoma cruzi in samples of blood from newborns from mothers with positive serology for Chagas disease. Rev Soc Bras Med Trop 2005, 38:62-64. 3. Torrico MC, Solano M, Guzman JM, Parrado R, Suarez E, Alonzo-Vega C, et al.: Estimation of the parasitemia in Trypanosoma cruzi human infection: high parasitemias are associated withPage 4 of 5 (page number not for citation purposes) Cases Journal 2008, 1:302 http://www.casesjournal.com/content/1/1/302Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral severe and fatal congenital Chagas disease. Rev Soc Bras Med Trop 2005, 38(Suppl 2):58-61. 4. Cruz I, Cañavate C, Rubio JM, Morales MA, Chicharro C, Laguna F, et al.: A nested polymerase chain reaction (Ln-PCR) for diag- nosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg 2002, 96:S185-S189. 5. Moser DR, Kirchhoff LV, Donelson JE: Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reac- tion. J Clin Microbiol 1989, 27:1477-1482. 6. Fernandes O, Sturm NR, Derre R, Campbell DA: The mini-exon gene: a genetic marker for zymodeme III of Trypanosoma cruzi. Mol Biochem Parasitol 1998, 95:129-133. 7. Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B: DNA markers define two major phylogenetic lineages of Trypano- soma cruzi. Mol Biochem Parasitol 1996, 83:141-152. 8. Burns JM Jr, Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG: Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA 1993, 90:775-779. 9. INE. Avance del Padrón Municipal a 1 de enero de 2008 2008 [http://www.ine.es/prensa/np503.pdf]. 10. Muñoz J, Portus M, Corachan M, Fumado V, Gascon J: Congenital Trypanosoma cruzi infection in a non-endemic area. Trans R Soc Trop Med Hyg 2007, 101:1161-1162. 11. Riera C, Guarro A, Kassab HE, Jorba JM, Castro M, Angrill R, et al.: Congenital transmission of Trypanosoma cruzi in Europe (Spain): a case report. Am J Trop Med Hyg 2006, 75:1078-1081. 12. Hermann E, Truyens C, onso-Vega C, Rodriguez P, Berthe A, Torrico F, et al.: Congenital transmission of Trypanosoma cruzi is asso- ciated with maternal enhanced parasitemia and decreased production of interferon-gamma in response to parasite antigens. J Infect Dis 2004, 189:1274-1281.Page 5 of 5 (page number not for citation purposes)