Treatment of Trypanosoma cruzi-infected children with nifurtimox: a 3 year follow-up by PCR

A. Solaria,*, S. Ortíza, A. Sotoa, C. Arancibiaa, R. Campillaya, M. Contrerasb, P. Salinasb, A. Rojasb and H. Schenoneb

a Molecular and Cellular Biology Programme, and b Parasitology Programme, Biomedical Sciences Institute, Faculty of Medicine, University of Chile, Casilla 70086, Santiago 7, Chile


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients suffering from Chagas' disease, as determined by positive serological results, were tested for further evidence of Trypanosoma cruzi infection by xenodiagnosis and PCR. The patients included 67 children aged from 0 to 10 years and 75 adults. All children were positive by PCR on their pre-therapy sample, while only 69% of the seropositive adults and none of the 78 seronegative control adults were PCR positive. Xenodiagnosis was positive in 79% of the children, but only in 21% of the adults. A group of 66 children was treated with nifurtimox, and followed up every 3 months during the first year and every 6 months during the second and third year post-therapy, by PCR, xenodiagnosis and serology. We concluded that PCR was the most effective test to monitor children for 3 years post-chemotherapy, when all the cases converted from positive to negative. Conventional serology, however, remained positive after that period in most cases. In contrast, conversion to negative xenodiagnosis occurred very early after treatment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chagas' disease is caused by the protozoon Trypanosoma cruzi, which affects 16–18 million people in 21 endemic countries of America.1 Even though the parasitaemia can be subclinical in the chronic indeterminate phase, and difficult to detect by blood culture, xenodiagnosis or direct microscopic blood or tissue examination, the polymerase chain reaction (PCR) looked to be a more promising way to overcome the low sensitivity of conventional parasitological diagnostic methods.2,3

The crucial role of the parasite in the outcome of the disease has been demonstrated by successful treatment, particularly in the early stages, to prevent its progress.46 Therefore, it is necessary to use the most sensitive diagnostic methods as a criterion of cure after specific chemotherapy. Several conventional and non-conventional serological methods have been used and been proven to be successful in monitoring the efficacy of different therapeutic alternatives.79 However, some of these methods are less specific in some endemic areas where T. cruzi infection may be confused with Trypanosoma rangeli infection, or they are not being used in routine diagnosis. Moreover, in many cases such as newborns infected by their mothers and immunosuppressed patients, the conventional serological tests are of low value.10 For these reasons, a search for new and alternative methods of parasite detection is needed.

Several reports have described PCR directed at genomic DNA11 and ribosomal DNA12 to detect T. cruzi. The PCR method used here amplifies minicircle variable regions by means of oligonucleotides specific for the conserved regions present in all the minicircles.13

The present study is focused on the evaluation of treatment efficacy by conventional serology, xenodiagnosis and PCR, in a group of children who received chemotherapy with nifurtimox and were followed up for 36 months.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients

Chagasic patients were defined in this study as those individuals who were positive in two independent and conventional serological methods: ELISA-IgG and indirect haemagglutination test (IHAT) performed at the blood bank of the University of Chile Clinical Hospital in the Metropolitan region of Chile, or in our institution, as described previously.14 Essentialy both methods used a soluble extract of T. cruzi (Tulahuen strain) prepared by us. A cut-off titre of 16 (IHAT), and a cut-off of 0.35 (average of optical density plus three standard deviations) was used for ELISA-IgG using anti-human IgG conjugates to horseradish peroxidase and o-phenyl-diamine. Blood samples were obtained from 67 children aged from 0 to 10 years (mean 6 years) from endemic areas of Chile (III region, 27° S, and V and Metropolitan regions, 33° S) who met the case definition given above. Chagasic children were detected in the surveillance phase of the Southern Cone initiative to eradicate Triatoma infestans.15 The 75 adult cases were either mothers of the children who transmitted the infection to newborns or seropositive blood donors at the Clinical Hospital. All the chagasic patients were asymptomatic. The 78 negative control patients were based on negative results by both serology tests. Half of these were healthy blood donors, and the others were T. cruzi non-infected, with different chronic heart, oesophageal and/or colon conditions. No related infectious diseases were included (Leishmaniasis or T. rangeli infection) since they are absent in Chile. Sixty-six children received specific treatment with nifurtimox for 60 days at a dose of 7–10 mg/kg.16 Blood samples were obtained for repeated serology at approximately 3, 6, 9, 12, 18, 24, 30 and 36 months post-therapy; xenodiagnosis and PCR were also performed at these intervals. The study was approved by the Faculty of Medicine Ethics Committee and informed consent was obtained from each patient or responsible adult when children were involved.

Xenodiagnosis

This test was carried out twice using two cylindrical wooden boxes (seven third-instar nymphs of T. infestans each) essentially as described by Schenone.17 Microscopic examination of insect faeces was performed 30, 60 or 90 days after feeding when the previous examination was negative. All the microscopic fields in a 2.2 cm2 area were examined twice.

DNA extractions

A volume of 1 mL of blood was mixed with the same volume of a 6 M guanidine hydrochloride, 200 mM EDTA solution, boiled for 15 min and stored at 4°C. Samples of 500 µL were extracted with DNA phenol–chloroform and precipitated with ethanol as described.13 Samples were resuspended in 50 µL H2O, chromatographed in a P10 filtration microcolumn and preserved at –20°C.

PCR amplifications

PCR amplifications were carried out as described by Wincker et al.13 Briefly, 5 µL of each control sample was added to a final volume of 50 µL containing 0.25 mM each of dATP, dCTP, dGTP and dTTP, 200 ng each of T. cruzi-specific primers (121 and 122), 2.5 U of Taq DNA polymerase, 5 µL of 10x Taq DNA polymerase buffer and 6.7 mM MgCl2. Mineral oil (80 µL) was added to avoid reaction mixture evaporation. The samples were amplified for two cycles at 98°C for 1 min and 64°C for 2 min, 33 cycles at 94°C for 1 min and 64°C for 1 min, and a final incubation at 72°C for 10 min in a thermocycler. A similar protocol was used as PCR internal control for amplification of the human ß-globin gene sequences as described by Saiki et al.18 Routinely, on a set of five blood lysates from patients to be diagnosed, one negative blood sample from a non-infected person was included. Every 22 samples a single amplification of a DNA preparation originating from a confirmed chagasic patient was included as positive control, and a tube without DNA was included as a negative control. A 330 bp product represented a positive assay. However, larger PCR products of 680 and 1000 bp, which are multiples of the minicircle variable regions, usually appeared when DNA was in excess. The amplification was sufficiently specific. However, sometimes other DNA products were generated or the DNA band was poor, necessitating a confirmatory hybridization test. PCR assays were performed in duplicate. When contradictory results appeared, further DNA extraction and PCR amplifications were performed.

Electrophoretic analysis of PCR products

For most assays, 10 µL of the amplified sample was electrophoresed in 2% agarose gels and stained with ethidium bromide. The gels were analysed on a UV transilluminator. Amplified DNA was then transferred to a nylon membrane, denatured and crosslinked by UV irradiation. The membranes were prehybridized in 6x SSC and 1 mM EDTA, pH 7.4, 5x Denhardt's solution, 0.5% SDS, 20 µg/mL denatured salmon sperm DNA at 65°C, and hybridized overnight in the same solution with the labelled probe. Washing was carried out in 0.1x SSC. Development of the hybridization was carried out using alkaline phosphatase conjugate, following the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany), or by exposure of the membranes to X-ray film for 2 or 24 h.

DNA hybridizations

All hybridizations were carried out using total kinetoplast DNA (kDNA) from T. cruzi labelled with the non-radioactive digoxigenin methods (Boehringer Mannheim), or with [{alpha}-32P]dCTP and the Ready Prime kit (Pharmacia, Amersham, UK).

Statistics

All results were analysed using the {chi}2 statistical test, and P values <0.05 were considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A panel of 78 non-chagasic cases with or without other pathologies were all negative by PCR assay. Comparisons of xenodiagnosis and PCR results for the initial pretherapy samples are shown in the TableGo. One hundred per cent of children and 69% of adults were PCR positive; of these, 79.1% of children and 21.3% of adults were also positive by xenodiagnosis. Several discrepancies were observed in the adult results: five seropositive adults were PCR negative but xenodiagnosis positive. Although PCR is sensitive enough to detect T. cruzi DNA minicircles, the method relies on the use of a minimal amount of blood sample. Our results with several cases of positive xenodiagnosis and negative PCR in the adult patient group can be explained by the smaller amount of blood sample used here in the DNA extraction method compared with other studies.2,13 However, when 5 mL of blood was collected and processed for PCR in these five adults, three cases were positive by the test and the other two were positive also by hybridization tests. Therefore, the PCR sensitivity in adults could have been 75% rather than 69%.


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Table. The sensitivity of xenodiagnosis and PCR in chagasic children and adults
 
A group of 66 children from three geographical areas received specific anti-T. cruzi treatment with nifurtimox and were followed-up by serology, xenodiagnosis and PCR. Percentages of positive tests are shown in the FigureGo (a). The average number of samples studied was 56, since some samples were lost during the field work, particularly on the last period of follow-up. The results show that serology still remained positive and serum titres unchanged 36 months after treatment, except in the two youngest cases (1 and 14 months old) out of seven cases ranging from 1 to 20 months old. It is possible to evaluate the effectiveness of the treatment by xenodiagnosis and by PCR. While positive xenodiagnosis dropped rapidly 3–6 months after treatment from 79.1% to 1.6%, PCR conversion showed a unique behaviour depending on the geographical area studied. Longer follow-up detects an intermediate conversion to negative PCR in the III and Metropolitan regions, and a late conversion in the V region, as shown in the FigureGo (b). While in the former areas PCR converted from positive to negative not later than 16 months post-treatment, in the V region several cases converted after that period and some still remained positive after 2 years. The difference in rapidity of conversion to PCR negative in these geographical areas was statistically significative after the first year follow-up.



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Figure. Percentage of positive test results obtained over time after treatment with nifurtimox. (a) Results presented according to method of testing: PCR {blacksquare}; xenodiagnosis {blacksquare}; and serology {blacksquare}. (b) Results obtained by PCR follow-up, presented by geographical region: Region III {blacksquare}; Metropolitan region {blacksquare}; and region V {blacksquare}.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, the usefulness of PCR amplification of kDNA minicircle sequences was assessed as a parasitological diagnostic test in 0- to 10-year-old children and adults infected with T. cruzi.

The sensitivity of PCR compared with serology has been found to be between 59% and 100% depending on several factors, such as the geographical area and the age of the subjects studied.2,13,1921 In our study we found different PCR sensitivity in the 0- to 10-year-old children and adults. While the PCR sensitivity against serology was 100% in children, this figure was only 69% among adults. However, this figure could be as high as 75% when larger volumes of blood were processed, as described by Junqueira et al.3 Similar results have been obtained in adult chronic chagasic patients from Brazil and schoolchildren from Bolivia.3,22 This result correlates with the lower sensitivity of xenodiagnosis. Among children, this method detected T. cruzi in 79.1% of cases, but only in 21.3% of adults. These results are in agreement with the notion that parasitaemias can be higher in children than in adults, or that a large fraction of the 0- to 10-year-old children can be considered as indeterminate acute cases.23 These figures also suggest that xenodiagnosis is not the best method to evaluate chemotherapeutic efficacy, especially in adults.

Specific treatment of Chagas' disease has been recommended for the acute phase of the infection, and recently for all chagasic patients.24 Treatment efficacy with nifurtimox ranged between 35% and 80%, and most of the reported trials have been done in adult patients under medical care in the late phase of chronic infection.6,7,10 Cure assessment in chronic infection is nevertheless controversial, mainly because of the lack of sensitive or specific tests to document parasitological cure. The high sensitivity and specificity of the PCR-based diagnosis of T. cruzi infection make it a suitable tool for the follow-up of a chemotherapeutic treatment of chagasic patients, particularly children. Therefore, the method could be used to assess parasite clearance from blood or cardiac tissue as described previously.2,12

Very few studies have been conducted to evaluate antiparasitic treatment by PCR in chronic and acute cases. The previous studies with chronic cases did not include patients followed up, hence it is impossible to compare them with the present results.12,21 In our study, the parasitological cure in 0- to 10-year-old children was variable, and several months were required to obtain a prolonged negative result by PCR, even though xenodiagnosis rapidly turned negative after 3 months in all geographical areas studied, probably owing to the lower sensitivity of the method, as shown here in adult cases.

We detected a late conversion to negative PCR results in the V region compared with the other endemic areas. A probable explanation for this observation is incomplete treatment. We discarded the hypothesis of parasite drug resistance,25 since T. cruzi genotypes circulating in the nearby V and Metropolitan regions were identical.26 Finally, no significance was found between patient age and PCR conversion.

Our observation of a slow rate of PCR conversion in all geographical areas can be explained by the late administration of the drug after infection, contrary to the early efficacy of treatment obtained in congenital cases, ranging from a few days to 3 months.27 We suggest that PCR-based detection of T. cruzi amplifies DNA from viable parasites, but also from lysed parasites from infected cells (polymorphonuclear cells and/or muscle fibres). Our results of early xenodiagnosis conversion after treatment indicate that a large fraction of live parasites are rapidly eliminated from blood (within the first 3–6 months of treatment). However, PCR became negative several months after xenodiagnosis did, suggesting that persistent shedding of parasitic kDNA into the blood from infected cells occurred in treated chagasic patients. This explanation correlates with the finding of T. cruzi DNA detected by PCR in the sera of chagasic patients.28

All serological reactions in our patients remained positive after treatment, except in two very young individuals, as described in chronic cases.10 Again, the persistence of antibody responses in treated and parasitologically cured chagasic patients could be explained by the presence for long periods, in the lymph nodes, of parasite antigens from lysed parasites, as reported previously.29 However, therapeutic effectiveness as assessed by serological cure seems to depend on the delay between infection and the start of treatment.

There are still no ideal anti-chagasic drugs and those in use (nifurtimox and benznidazole) are well known for their undesirable side effects.6 In our study, none of the patients treated were excluded from the study because of adverse drug effects. These results demonstrate the tolerance and efficacy of nifurtimox against T. cruzi in 0- to 10-year-old children when low doses are used, in spite of the pharmacokinetics of the drug.30

Finally, PCR-based detection of T. cruzi appears most useful for monitoring infected subjects undergoing chemotherapy. Long-term follow-up studies of up to 3 years are necessary to evaluate the efficacy of treatment, to initiate a new treatment or to show whether drug resistance has developed. This well-characterized group of children is presently being followed up to confirm treatment efficacy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The manuscript revision of Dr Jorge Fernández is appreciated. This work was supported by the International Atomic Energy Agency RLA 6/026, DID-Enlace 2000, University of Chile, and by FONDECYT-Chile 1970768.


    Notes
 
* Corresponding author. Tel: +56-2-678-6062; Fax: +56-2-735-5580; E-mail: asolari{at}machi.med.uchile.cl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 23 January 2001; returned 13 July 2001; revised 23 July 2001; accepted 2 August 2001