Am. J. Trop. Med. Hyg., 78(2), 2008, pp. 294-297
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene
CASE REPORT
Molecular Identification of Trypanosoma cruzi I Tropism for Central Nervous System in Chagas Reactivation Due to AIDS
Juan M. Burgos,
Sandra Begher,
Helder M. Valadares Silva,
Margarita Bisio,
Tomas Duffy,
Mariano J. Levin,
Andrea M. Macedo, AND
Alejandro G. Schijman*
Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina; Unidad Clínica Médica e Infectología, Hospital Ignacio Pirovano, Gobierno de la Ciudad de Buenos Aires, Buenos Aires, Argentina; Departamento de Bioquímica e Imunologia, ICB/UFMG, Belo Horizonte, Brazil
ABSTRACT
Trypanosoma cruzi lineages, microsatellite allelic polymorphism, and mithocondrial gene haplotypes were directly typified from peripheral blood and cerebrospinal fluid specimens of a Bolivian patient with Chagas disease with accompanying AIDS and central nervous system severe involvement. Of note, the patient’s blood was infected by a mixture of T. cruzi I and T. cruzi IId/e polyclonal populations while the cerebrospinal fluid showed only a monoclonal T. cruzi I population. Our findings do not corroborate the original assumption of innocuity for T. cruzi I in the southern cone of the Americas and highlight lineage I tropism for central nervous system causing lethal Chagas reactivation.
INTRODUCTION
Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, affects about 18 million people in the Americas.1 The pandemic of acquired immunodeficiency syndrome (AIDS) opened the possibility of the appearance of Chagas disease reactivation in individuals seriously immunocompromised by the human immunodeficiency virus (HIV) and co-infected with T. cruzi.2–4 The two organic sites more frequently involved are the central nervous system (CNS) and the heart.2–4 In the former, it is observed as mono or multi-focal acute meningoencephalitis in about 75–80% of the cases. Fever, headache, vomiting, focal signs, seizures, and rarely, meningeal signs constitute the major clinical manifestations. Cerebrospinal fluid, in these cases, displays a mild lymphomononuclear pleocytosis, with raised proteins, and the parasite is usually present in this fluid. Also in this phase, the presence of T. cruzi is constant in the blood. Histologically, the brain shows severe meningoencephalitis, with intense inflammatory, predominantly perivascular infiltrate, with the presence of countless amastigotes of T. cruzi parasitizing glial cells and, rarely, neurons. The meninges also show parasitism within macrophages, which could explain the high frequency of the finding of tripomastigote forms in the cerebrospinal fluid.4
Natural T. cruzi populations have been classified into two major phylogenetic lineages: T. cruzi I and T. cruzi II.5 Five lower phylogenetic subdivisions have been identified within T. cruzi II lineage, designated T. cruzi IIa-e, whereas genotype subdivision within T. cruzi I is under discussion.6,7 Trypanosoma cruzi I has a large geographical distribution from North to South America, predominating in the Amazonian basin northward where domestic and sylvatic triatomine species are involved in transmission in several endemic regions located in Venezuela, Colombia, Central America, and Mexico.1 In these countries, T. cruzi I infects patients and different pathologic forms have been observed.8,9 However, information regarding the clinical relevance of this lineage in the southern cone of America is scarce.10–14
The role of T. cruzi parasitemia and genetic make-up on the onset of chagasic reactivation due to AIDS, as well as the impact of HIV infection in T. cruzi genetic diversity, are poorly explored fields of clinical parasitology. Comparative zymodeme studies of parasite stocks obtained from HIV-positive patients with T. cruzi co-infection did not reveal significant differences regarding the tissue tropism or repartition of the strains.15 However, as the parasite isolates were obtained after in vitro culturing of intestinal material from laboratory-reared triatomine bugs used for xenodiagnosis of clinical blood samples, the parasite genotypes recovered could be mostly representative of the most successful clones selected during the isolation procedures, and not represent the genotypes causing the clinical manifestations of disease.
CASE REPORT
In February 2005, a 41-year-old HIV-positive Bolivian man residing in Argentina was hospitalized at the neurosurgery service of Hospital Pirovano in Buenos Aires city, with fever, paresis of inferior limbs, ischuria, and presumptive diagnosis of transverse myelitis. The nuclear magnetic resonance of the vertebral column showed de-myelinization with medular edema. During hospitalization, the patient was first diagnosed with HIV infection with a CD4 count of 61 cells (8%). Serological tests for Hepatitis B and Ebstein-Barr viruses were negative. However, he had positive serological findings for Herpes Zoster Virus, cytomegalovirus, toxoplasmosis, and Trypanosoma cruzi infection (Indirect Hemoagglutination 1/64, ELISA R: 5.7, Strout positive). Ganciclovir, azitromicine, benznidazole, and HAART were indicated but the patient evolved to sepsis and died 2 days after diagnosis.
Peripheral blood and cerebrospinal fluid (CSF) samples were collected for differential PCR-based diagnosis of CSF infection. For detection of T. cruzi DNA, the CSF and blood samples were mixed with one volume of 2x Guanidinium Hidrochloride 6 M—EDTA 0.2 M, and the DNA was extracted as previously described for blood samples.11 For CMV, HSV, and T. gondii DNA detection, DNA was isolated from a CSF aliquot using the QIAamp DNA mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions for body fluid DNA extraction. CMV, HSV, and T. gondii-specific DNAs could not be amplified from the CSF specimen.16 In contrast, minicircle T. cruzi sequences were amplified from both blood and CSF samples, indicating CNS–Chagas reactivation.17 Accordingly, we aimed to identify the T. cruzi lineages and characterize their clonal complexity directly in the infected clinical samples using recently developed nucleic acid amplification strategies.11,17–19
Trypanosoma cruzi lineage.
The parasite phylogenetic lineage was investigated in blood and CSF using PCR strategies targeted to lineage-specific genomic markers, such as the intergenic spacer of the spliced-leader genes, the D7 domain of the 24s alpha ribosomal RNA genes, and the A10 fragment, as recently reported.11 Table 1
shows the findings from these studies. The analysis of the intergenic spacer of the spliced-leader genes involved 3 independent PCR assays that allowed classification of the infecting populations such as T. cruzi II b/d/e (425 bp) plus T. cruzi I (475 bp) in peripheral blood whereas a single T. cruzi I infection (475 bp) was detected in the CSF specimen. Trypanosoma cruzi IIa or IIc strains were not detected (150/157 bp, Table 1).11 Furthermore, the 24s alpha ribosomal (125 bp + 140 bp) and A10 nuclear markers (525 bp, Table 1) were indicative of bloodstream T. cruzi IId/e populations because the 580-bp A10 amplicon that is specific for T. cruzi IIb was not obtained (Table 1).11
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TABLE 1 Molecular characterization of natural Trypanosoma cruzi lineages and populations involved in Chagas disease reactivation in a patient with AIDS
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Polymorphism of the mitochondrial gene for cytochrome oxidase subunit II.
The haplotype of the gene encoding subunit II of cytochrome oxidase in the mitochondrial genome (CO II) was characterized by Alu I based RFLP-PCR, as recently described.18 This strategy confirmed the mixed T. cruzi I and T. cruzi II bloodstream populations because RFLP-PCR showed a profile of haplotypes A plus B (Figure 1A). In contrast, only haplotype A (T. cruzi I) was detected in the CSF specimen (Table 1
and Figure 1A), in agreement with the spliced-leader intergenic region PCR analysis. Furthermore, the profiling of the CO II subunit gene established that the patient’s blood was infected by a hybrid population corresponding to T. cruzi IId or T. cruzi IIe (CO II haplotype B) but not to T. cruzi IIb (CO II haplotype C) in accordance with the A10–PCR characterization (Table 1 and Figure 1A).
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FIGURE 1. A, Restriction fragment length polymorphism analysis of a gene fragment for the subunit II of mitochondrial cytochrome oxidase performed in clinical samples from a patient with cerebral Chagas disease and AIDS. PCR products were obtained as described in Freitas and others (2006), digested with Alu I, and separated by agarose gel electrophoresis. CSF and Blood: analysis of cerebrospinal fluid and peripheral blood patient‘s samples, respectively; Lanes HA, HB, and HC: analysis of reference stocks belonged to haplotype A, B, and C (K98, CL, and JG, respectively); Mw: molecular weight marker (numbers are expressed in bp). B, Analysis of allelic polymorphism of microsatellite loci in clinical samples from a patient with cerebral Chagas disease and AIDS. ALF (Pharmacia-GE) DNA sequencer electrofluorograms showing the amplified fragments obtained by TcTAC15 and TcTAT20 microsatellite loci analyses on blood and CSF samples. The numbers above the peaks refer to the size of the amplicons in bp. Peaks without sizes correspond to the molecular DNA ladder.
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Microsatellite loci polymorphism analysis.
The clonal complexity of the populations was profiled by means of Nested-PCR targeted to sequences flanking microsatellite repeats for the loci TcTAT20, TcTAC15, TcATT14, and TcAAAT6.19 To determine the allele sizes for each tested locus, 1 to 3 µL of the Nested-PCR fluorescent products were analyzed in a 6% denaturing polyacrylamide gel of an ALF sequencer (GE Healthcare, Milwaukee, Wisconsin) in comparison to fluorescent DNA fragments of 50–500 bp using Allelelocator software (GE Healthcare).
The microsatellite profiling showed only one peak for the loci TcTAT20, TcTAC15, and TcAAAT6 and 2 peaks for the locus TcATT14 (Table 1 and Figure 1B) suggesting the presence of a monoclonal population in the CSF sample, following the interpretation of Oliveira and others (1998).20 Recent analysis of locus TcTAC15 in 16 stocks belonging to T. cruzi I lineage as well as in 25 stocks belonging to T. cruzi II and T. cruzi III detected only the 96 bp allele in all tested T. cruzi I stocks but in none of those belonging to the other lineages.19 Thus, the TcTAC15 microsatellite profile in CSF points to a T. cruzi I infection, in agreement with the above-mentioned spliced-leader gene amplification (Table 1). In contrast, peripheral blood samples rendered 3 peaks corresponding to allelic sizes of 99, 132, and 135 bp, which are found in T. cruzi II but not in T. cruzi I lineages (Table 1 and Figure 1B).19 The 96-bp allele of T. cruzi I could not be detected in the blood sample.
DISCUSSION
In the present case report, it was possible to characterize and compare the parasitic genetic diversity directly in peripheral blood and CSF samples, allowing identification of a mixed population in bloodstream conformed by T. cruzi I and T. cruzi IId/e lineages whereas only T. cruzi I was found at the site of CNS reactivation, as determined by amplification of the dimorphic intergenic region of spliced-leader genes and the D7 domain of the 24s alpha ribosomal RNA genes (Table 1).11 The SL-PCR and 24s alpha ribosomal DNA PCR assays have a detection limit of 5 pg and 100 fg of total parasite DNA for both lineages per reaction tube, respectively,11 thus at least 5 T. cruzi I parasite cells were present in 1 µL of CSF sample (1 parasite cell approximately: 0.2 pg) whereas at least less than 1/10 parasite equivalents of T. cruzi II could be present in that sample, showing the predominance of T. cruzi I in CSF. We have also characterized the clonal complexity of the T. cruzi populations using microsatellite loci profiling, allowing detection of a polyclonal population in blood and a monoclonal strain in the CSF sample. The 96-bp TcTAC15 allele, typical of T. cruzi I stocks (Valadares Silva HM and others, unpublished data) could not be detected in peripheral blood, but alleles common for T. cruzi II populations (99, 132, and 135 bp) were amplified, suggesting a higher parasitic load for T. cruzi II than for T. cruzi I in bloodstream. However, in the CSF only the 96-bp TcTAC15 allele was amplified, in concordance with the lineage-specific amplification tests (Table 1 and Figure 1B). All together, these data point to a tropism of T. cruzi I for the CNS reactivation in this patient.
In a previous report, we described an Argentinean Chagas-AIDS patient with T. cruzi IId parasitemia whose cerebral parasite populations belonged to T. cruzi IIb.17 Thus, both reported cases have in common that bloodstream T. cruzi IId populations are not detectable at the sites of reactivation. A recent study of a cohort of congenital infants with Chagas disease showed the co-infection of T. cruzi I and T. cruzi IId in only 1 newborn out of 37 T. cruzi IId patients.11 Yet again, the particularity of this patient within the cohort was that he had perinatal AIDS.
All together, these observations suggest that (1) T. cruzi I might circulate at a higher frequency than it has been previously assumed on the basis of typing culture isolates from clinical specimens in Southern countries of America; (2) the low rate of detection of this lineage in patients with indeterminate or chronic Chagas disease might be due to its low bloodstream parasitic load as a consequence of a higher tropism for target organs and (3) the exacerbation of T. cruzi I parasitemia caused by immunosupression allows this lineage to become detectable.
Our findings associate for the first time a monoclonal T. cruzi I strain with CNS Chagas reactivation in a patient with a complex bloodstream infection by T. cruzi I and T. cruzi IId/e clones. This was possible because we have identified the natural populations from the CSF and blood samples by means of a battery of sensitive PCR-based strategies without the need of in vitro isolation, which might have underestimated the population complexity due to clonal selection during culture expansion.
Finally, these data do not support the original assumption of innocuity of T. cruzi I in southern endemic regions of America.
Acknowledgments: The authors thank R. Teijeiro, H. Lopez Alcoba, J. Levalle, and F. Deccarlini of Hospital Pirovano for management and follow-up of the patient and H. Freilij and J. Altcheh of the Laboratory of Chagas of Hospital Ricardo Gutierrez in Buenos Aires city, for serological confirmation of T. cruzi infection.
Financial support: This project received major support from WHO-TDR ID 20285, Bunge & Born Foundation, CONICET (PIP 5469), PICT 33955 from the National Agency of Science and Technology to AGS, and partial support from CNPq/FAPEMIG to AMM. AGS and MJL are members of CONICET Researcher’s Career and AMM of CNPq. JMB, MB, and TD are research fellows of CONICET and HMSV of CAPES.
Disclaimer: All contributing authors have participated in the study and concur with the submission. No conflicts of interests have been disclosed. The data presented in this manuscript have not been and will not be submitted for publication elsewhere.
* Address correspondence to Alejandro G. Schijman, Laboratorio de Biología Molecular de la Enfermedad de Chagas, INGEBI-CONICET, Vuelta de Obligado 2490, Buenos Aires 1428, Argentina. E-mail: schijman{at}dna.uba.ar 
This study fulfilled all criteria required by the Medical Code of Ethics and the Helsinki II statement and was approved by Ethical Committee. Written informed consent was obtained from the patient.
Authors’ addresses: Juan M. Burgos, Margarita Bisio, Tomas Duffy, Mariano J. Levin, and Alejandro G. Schijman, Laboratorio de Biología Molecular de la Enfermedad de Chagas, INGEBI-CONICET, Vuelta de Obligado 2490, Buenos Aires, 1428, Argentina, Telephone: 05411-47832871 ext. 50, Fax: 05411-47868578, E-mail: schijman{at}dna.uba.ar. Sandra Begher, Hospital Pirovamo, Monroe 3555, Capital Federal, Argentina. Helder M. Valadares Silva and Andrea M. Macedo, Universidade Federal de Mina Gerais, Av Antonio Carlos 6627, Belo Horizonte, HG, Brazil.
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