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Chagas disease is a tropical disease, endemic of the Americas and caused by the protozoan Trypanosoma cruzi. The classic transmission route of T. cruzi is based on the contact with feces from Triatominae insects containing trypomastigotes forms of the parasite. T. cruzi is also spread by oral and congenital forms, besides blood donation and organ transplant transmission. The parasite has a tropism to several organs and tissues, including heart, brain, digestive tract, muscles, placenta, and fetuses. The determination of parasite burden in organs is an important parameter for scientific assays related to parasite control and epidemiology. Among the procedures to detect and quantify T. cruzi, PCR demonstrates high sensitivity and specificity. In this study, we developed a simple and fast method to detect T. cruzi from fixed placentas and fetuses of Wistar rats. T. cruzi was detected only in tissues from infected animals, with different parasite burdens. Once the histology usually demonstrates some limitations such as low sensitivity and specificity to detect T. cruzi in fetuses, our methodology will be useful for fixed fetal tissues from organ banks. The detection of T. cruzi from gestational tissues will contribute to the elucidation of mechanisms related to congenital Chagas disease.
Trypanosoma cruzi is a flagellated protozoan (Trypanosomatidae family) with an important impact in populations of tropical and subtropical America. The parasite causes the Chagas disease in humans, usually leading to the degeneration of cardiac and/or digestive tissues in the chronic symptomatic phase of the infection. Chagas disease is partially controlled by drugs (Benznidazole and Nifurtimox) and several attempts have been developed for the control of T. cruzi. Most of the strategies for the evaluation of T. cruzi transmission, epidemiology and control are dependent on methods of detection in tissues. Samples from humans, animal models and Triatominae insects have been exhaustively analyzed for elucidation of mechanisms and control of Chagas disease.
The T. cruzi detection in tissues is usually performed using histology, immunohistochemistry, electron microscopy and PCR. Compared to the traditional detection techniques, PCR demonstrates higher sensitivity and specificity. Once standardized, PCR is easy to perform and allows the analysis of multiple samples with a superior reproducibility. PCR was applied for the detection of T. cruzi in diverse tissue sources, such as from Triatominae insects, domestic and wild animals, humans and assays related to immunology, epidemiology and chemotherapy. However, the T. cruzi detection in fixed samples was usually restricted to histologic procedures. The use of PCR for T. cruzi detection in fixed tissues was mainly reported for wild animals (raccoon and monkeys) and few attempts were developed for mice or rats, especially placentas and fetuses.
In this study, our aim was to detect T. cruzi in fixed placentas and fetuses from infected Wistar rats. The chronically infected animals were treated with zinc for 18 days and several immunologic parameters analyzed. Placentas and fetuses were collected and fixed with formaldehyde 3.72% for evaluation of parasite burden. The samples were treated with TE pH 9 buffer for breaking the cross-linkages between genomic DNA and proteins, which improved the integrity of purified material. The treated and purified genomic DNA was applied for detection of T. cruzi using the S35/S36 primers, amplifying fragments of 330 bp.
The mechanisms of congenital Chagas disease are not fully elucidated, demanding novel strategies for the control of the transmission of T. cruzi to fetuses. Moreover, there is interesting material from banks composed of fixed placenta and fetuses, primarily analyzed by histology. Therefore, the molecular analysis of fixed placentas and fetuses will expand the data applied for the comprehension of the congenital Chagas disease.
The aim of this study is to detect Trypanosoma cruzi from fixed placentas and fetuses using conventional PCR.
The first attempt to purify genomic DNA using a traditional method (Wizard SV Genomic DNA Purification System) from fixed placentas failed. When analyzed by agarose electrophoresis, a degraded DNA was observed (Fig. 1A, lane 2). The PCR reaction using this template was negative, generating only primer dimers (Figure not shown). Indeed, the Wizard SV Genomic DNA Purification System kit is not designed for fixed samples, which demanded a complementary procedure. The treatment with the TE9 buffer breaks the cross-linkages between genomic DNA and proteins, allowing the partial recovery of an integral material. After the treatment with TE9 buffer, a faint, but complete DNA band was observed (Fig. 1B, lane 2). The band was similar to the genomic DNA purified from the non-fixed placenta (Fig. 1B, lane 3). After the PCR reaction, a band of 330 bp was polymerized from samples using DNA from the fixed placenta (Fig. 1C, lane 2). The result was similar to the positive control composed by genomic DNA from epimastigotes (Fig. 1C, lane 3).
The PCR detection protocol was applied in fixed samples from animals infected with T. cruzi and treated with zinc. T. cruzi was detected in placentas and fetuses from infected and/or treated animals, with different levels of parasite burden (Fig. 1D and E, lanes 8–13). Although no T. cruzi parasites were detected in all fetuses tissues analyzed by histology, the detection with PCR was inconclusive in only two samples (Fig. 1E, lanes 8 and 12). However, the use of complementary assays, such as nested, real-time PCR or higher percentages of agarose gels will be useful for inconclusive samples. The effect of zinc treatment on the T. cruzi parasite loads of placentas and fetuses was inconclusive compared to the non-treated and infected counterparts. This result confirms the role of zinc as an immunomodulatory rather than an anti-T. cruzi molecule. Indeed, zinc improved the immune response against T. cruzi in pregnant infected animals, decreasing the amastigotes nests in hearts. However, the parasite burden in placentas and fetuses was not fully determined due to the histology limitations. For the complete elucidation of the effects of zinc on the parasite load of placentas and fetuses, quantitative assays (real-time PCR) will be required. Moreover, several methods have been described for DNA extraction from fixed samples, with different results. Besides the use of alkaline buffers for DNA extraction, there are several kits with similar applications. In further assays, we will perform an extensive comparison between our method and the kits, concerning parameters such as cost, DNA yield, and purity. These parameters will guide a simple, effective and low-cost method for DNA extraction from fixed samples.
Our results indicate a procedure to detect T. cruzi from fixed placentas and fetuses of Wistar rats. There is an interesting bank of fixed organs from laboratories which samples were evaluated using histology. Our study may offer a specific and sensitive methodology to evaluate and confirm the observations reported using classic methods. Once the histology demonstrates limitations to determine the parasite burden of fetuses, the PCR detection on fixed organs is a sensitive, reproducible and specific option, complementing the studies related to the congenital Chagas disease.
Our study allowed the detection of T. cruzi from fixed placentas and fetuses of Wistar rats. The methodology will be useful for fixed organs and tissues from banks.
The main limitation of the DNA extraction from fixed tissues is the low recovery of whole molecules. The quality of DNA is uncontrolled, which may generate false negative patterns. The use of complementary T. cruzi (e.g. TCZ) or rat primers (e.g. GAPDH) will be useful to confirm the DNA integrity.
This study describes a qualitative form to detect T. cruzi in fixed placentas and fetuses. The next step will be the adaptation of our methodology for quantitative PCR (real-time PCR). The use of primers for rat tissue detection (GAPDH, for example) should be also validated.
Placental and fetal tissues
Female Wistar rats (5 animals/group) were used and housed in the Facility House of the São Paulo University Campus of Ribeirão Preto, with water and food at libitum. The animal manipulation protocol was approved by the local Ethics Committee (protocol number 11.1.1210.53.2). The animals were infected with T. cruzi and after 30 days allowed to mate. The pregnancy was confirmed by the presence of the vaginal plug. The animals were treated for 18 days (from the day 1st to 18th of pregnancy) with zinc sulfate (20 mg/Kg/day). At the 18th day of pregnancy, the animals were euthanized and the placentas and fetuses collected. The animals were divided into 4 groups: non-treated and non-infected (PC), treated and non-infected (PCZ), non-treated and infected (PI) and treated and infected (PIZ). The placentas and fetuses were fixed with 3.72% formaldehyde for 18 h at room temperature. The formaldehyde was replaced for 80% ethanol for conservation.
The genomic DNA was extracted using the Wizard SV Genomic DNA Purification System kit (Promega), following the manufacturer's instructions. Firstly, the tissues (10 mg) were washed with PBS and used for DNA extraction without previous treatment. The placentas were cut into a wedge, while a part of the fetal thoracic region (heart) was collected. The same procedure was performed for the non-fixed controls (non-fixed placenta and 106 epimastigote forms). For the DNA-protein cross-linkages reversion, caused by the fixation process, a TE9 buffer (500 mM Tris, 20 mM EDTA, 10 mM NaCl, pH 9.0) was used. The tissues (10 mg) were washed with PBS and incubated with 1 ml of TE pH 9 buffer for 24 h at 37°C. After the incubation, the samples were washed with PBS and the DNA extracted as previously described. The DNA samples were quantified in a NanoDrop 2000 (ThermoFisher Scientific), normalized for 25 ng/ml in TE buffer and 50 ng was applied in PCR assays.
T. cruzi was detected by PCR using the primers S35/S36 (5’-AAATAATGTACGGGTGAGATGCATG – 3’ and 5’-GGGTTCGATTGGGGTTGGTG – 3’), which amplify a fragment of 330 bp. The PCR reaction (50 µl/reaction) was performed using the GoTaq Green Master Mix (Promega), following the manufacturer's instructions. The amplification was performed in a Eppendorf Mastercycler thermocycler, using a touchdown thermal profile consisting of 1 cycle of 94°C /2:00; 5 cycles of 94°C /0:30, 60°C /0:30, 72°C /1:00; 5 cycles of 94°C /0:30, 55°C /0:30, 72°C /1:00 and 25 cycles of 94°C /0:30, 50°C /0:30, 72°C /1:00 with a final extension at 72°C /10:00. The samples were visualized on 0.8% agarose gels stained with 0.5 µg/ml ethidium bromide.
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (Grant number: 2013/04205-6).
We would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
The experimental protocols involving animals were approved by the local Ethics Committee (Protocol No 11.1.1210.53.2).