Diagnosis Of Congenital Chagas Disease Research Articles

Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots.

Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field.

Chemiluminescent Microparticle Immunoassay for the Diagnosis of Congenital Chagas Disease: A Prospective Study in Spain.

Congenital Chagas disease (CCD) has become a global health problem. Historically, the diagnosis of CCD has been carried out using parasitological methods and traditional serological techniques, however, new serological techniques such as chemiluminescent microparticle immunoassays (CMIA) have been developed in the last few years with many advantages compared with traditional serological tests. A total of 75 children born to 72 Latin American Chagas-infected mothers were consecutively enrolled and studied by CMIA and indirect immunofluorescence (IIF) at 0-2, 6, 9, and 12 months of age. At the end of the follow-up, 74 out of 75 children were considered uninfected and one child was diagnosed with CCD. Our study emphasizes the need to carry out serological follow-up on every newborn from a mother with Chagas disease and shows that CMIA assay is a great diagnostic tool as a single serological test at 9 months of age to rule out CCD or to identify possible transmission.

Diagnostic Accuracy of Two Molecular Tools for Diagnosis of Congenital Chagas Disease.

Background and ObjectiveThe real prevalence of congenital Chagas disease is undefined because of difficulties in the detection of Trypanosoma cruzi by microscopic examination. The aim of this study was to determine the diagnostic accuracy of two molecular diagnostic tools, qPCR and LAMP, in the diagnosis of congenital Chagas disease in a clinical setting.MethodsTo this end, we conducted a prospective cohort study in a tertiary care center, of infants under 9 months of age, born in Buenos Aires to women with Chagas disease. Blood samples were collected for microscopic examination and molecular diagnosis at baseline. If negative, infants were followed up until 9 months of age to determine a final diagnosis by serology. In-house qPCR and LAMP previously validated were challenged as index tests.ResultsA total of 154 participants were potentially eligible, 120 of whom were enrolled. Finally, 102 (66.2%) of them fulfilled the follow-up. The diagnosis of congenital Chagas disease was confirmed in 13 infants and excluded in 89. Both the sensitivity and specificity of the qPCR were 100.0% (95% confidence interval 75.3–100.0 and 95% confidence interval 95.9–100.0, respectively), whereas the sensitivity and specificity of LAMP were 69.2% (95% confidence interval 38.6–90.9) and 100% (95% confidence interval 95.9–100.0), respectively.ConclusionsThe qPCR agreed with the current diagnostic algorithm, and was a reliable and sensitive tool to detect congenital Chagas disease earlier, providing an appropriate and timely identification of infected infants requiring treatment. LAMP was able to detect congenital Chagas disease in infected infants by naked-eye visualization in accordance with a microscopic examination. The advantages of molecular diagnostic tools should be taken into account by the health system to improve congenital Chagas disease diagnosis.

Congenital Chagas disease: progress toward implementation of pregnancy-based screening.

Lack of recognition of congenital Chagas disease in infants of mothers from endemic regions who are living in countries nonendemic for Trypanosoma cruzi infection suggests a high rate of underdiagnosis. Pregnancy is the optimal access point for identifying Chagas disease in at-risk mothers and their infants. In this review, we update progress toward implementation of pregnancy-based screening for congenital Chagas disease in nonendemic settings. International organizations have updated recommendations for diagnosis, treatment and prevention of congenital Chagas disease. Reports of successful implementation of pregnancy-based screening at some centers provide a model for optimizing diagnosis of congenital Chagas disease. Screening family members of index patients may identify additional T. cruzi-infected persons. Promising tests to augment current diagnostic modalities for maternal and congenital Chagas disease are in development. Universal or risk-based screening would be cost-effective. More healthcare providers are now aware that treatment of congenital Chagas disease is curative and are promoting efforts to make pregnancy-based screening for congenital Chagas disease a standard of care. Ongoing efforts to implement routine pregnancy-based screening for congenital Chagas disease in nonendemic regions will mutually benefit infants, their mothers and family members and can prevent potentially fatal Chagas cardiomyopathy.

Evaluation of the Performance of the Loopamp Trypanosoma cruzi Detection Kit for the Diagnosis of Chagas Disease in an Area Where It Is Not Endemic, Spain.

In Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for Trypanosoma cruzi DNA detection showed good analytical performance and ease of use. We aimed to evaluate the performance of the Loopamp Trypanosoma cruzi detection kit (Eiken Chemical Co. Ltd., Japan) (Tcruzi-LAMP) for congenital and chronic CD diagnosis using well-characterized samples. We included samples from 39 congenital and 174 chronic CD cases and from 48 uninfected children born to infected mothers and 34 nonchagasic individuals. The sensitivity, specificity, and accuracy of Tcruzi-LAMP were estimated using standard case definitions for congenital CD (positive result by parasitological or PCR tests or serology after 9 months of age) and chronic CD (positive serology by at least two tests). The Tcruzi-LAMP results were read by visual examination and a real-time fluorimeter. For congenital CD, Tcruzi-LAMP sensitivity was 97% for both types of reading; specificity was 92% by visual examination and 94% by fluorimeter. For chronic CD, sensitivity was 47% and specificity 100%. The accuracy in congenital CD was >94% versus 56% in chronic CD. The agreement of Tcruzi-LAMP with PCR tests was better in congenital CD (kappa, 0.86 to 0.91) than in chronic CD (kappa, 0.67 to 0.83). The Loopamp Trypanosoma cruzi detection kit showed good performance for the diagnosis of congenital CD. Tcruzi-LAMP, like PCR, can be useful for the screening and early diagnosis of congenital infection.

Development and Evaluation of a Three-Dimensional Printer–Based DNA Extraction Method Coupled to Loop Mediated Isothermal Amplification for Point-of-Care Diagnosis of Congenital Chagas Disease in Endemic Regions

Vertical transmission of Trypanosomacruzi is the cause of congenital Chagas disease, a re-emerging infectious disease that affects endemic and nonendemic regions alike. An early diagnosis is crucial because prompt treatment achieves a high cure rate, precluding evolution to symptomatic chronic Chagas disease. However, early diagnosis involves low-sensitive parasitologic assays, making necessary serologic confirmation after 9 months of life. With the aim of implementing early diagnostic strategies suitable for minimally equipped laboratories, a T. cruzi-loop-mediated isothermal amplification (LAMP) prototype was coupled with an automated DNA-extraction device repurposed from a three-dimensional printer (PrintrLab). The whole process takes <3 hours to yield a result, with an analytical sensitivity of 0.1 to 2 parasite equivalents per milliliter, depending on the T. cruzi strain. Twenty-five blood samples from neonates born to seropositive mothers were tested blindly. In comparison to quantitative real-time PCR, the PrintrLab-LAMP dual strategy showed high agreement, while both molecular-based methodologies yielded optimal sensitivity and specificity with respect to microscopy-based diagnosis of congenital Chagas disease. PrintrLab-LAMP detected all 10 congenitally transmitted T. cruzi infections, showing promise for point-of-care early diagnosis of congenital Chagas disease.

The Immunoglobulin M-Shed Acute Phase Antigen (SAPA)-test for the Early Diagnosis of Congenital Chagas Disease in the Time of the Elimination Goal of Mother-to-Child Transmission.

Diagnosis of congenital Chagas disease (CChD) in most endemic areas is based on low-sensitive microscopy at birth and 9-month immunoglobulin G (IgG), which has poor adherence. We aim to evaluate the accuracy of the Immunoglobulin M (IgM)-Shed Acute Phase Antigen (SAPA) test in the diagnosis of CChD at birth. Two cohort studies (training and validation cohorts) were conducted in 3 hospitals in the department of Santa Cruz, Bolivia. Pregnant women were screened for Chagas disease, and all infants born to seropositive mothers were followed for up to 9 months to diagnose CChD. A composite reference standard was used to determine congenital infection and was based on the parallel use of microscopy, quantitative polymerase chain reaction (qPCR), and IgM-trypomastigote excreted-secreted antigen (TESA) blot at birth and/or 1 month, and/or the detection of anti-Trypanosoma cruzi IgG at 6 or 9 months. The diagnostic accuracy of the IgM-SAPA test was calculated at birth against the composite reference standard. Adherence to the 6- or 9-month follow-up ranged from 25.3% to 59.7%. Most cases of CChD (training and validation cohort: 76.5% and 83.7%, respectively) were detected during the first month of life using the combination of microscopy, qPCR, and/or IgM-TESA blot. Results from the validation cohort showed that when only 1 infant sample obtained at birth was evaluated, the qPCR and the IgM-SAPA test have similar accuracy (sensitivity: range, 79.1%-97.1% and 76.7%-94.3%, respectively, and specificity: 99.5% and 92.6%, respectively). The IgM-SAPA test has the potential to be implemented as an early diagnostic tool in areas that currently rely only on microscopy.

An observational longitudinal study to evaluate tools and strategies available for the diagnosis of Congenital Chagas Disease in a non-endemic country

ObjectivesCongenital Chagas Disease (CCD) has become a global health problem. Early diagnosis and treatment is essential for the cure of the disease. Our aim was to evaluate techniques and samples used for the diagnosis of CCD in order to improve diagnostic strategies. MethodsA total of 181 children born in Spain from Latin American Chagas-infected mothers were consecutively enrolled and studied by microhematocrit, PCR and serology tests at 0–2, 6 and 9–12 months of age and followed up when it was required. Samples of cord blood and peripheral blood were collected for T. cruzi detection by PCR. Parasite culture was performed in patients with a positive PCR. ResultsOf 181 children, 7 children (3.9%) were lost to follow-up. A total of 174 children completed follow-up, 12 were diagnosed with CCD (6.9%) and 162 (93.1%) as uninfected children (negative serology tests at the end of the follow-up). Traditional parasitological diagnosis by microhematocrit had a poor performance (sensitivity was 10%), while PCR in peripheral blood showed high sensitivity (90.9%) and specificity (100%), allowing the early diagnosis of 9 infected children during the first 6-months-old. In the other 3 congenital cases, diagnosis was only possible at 12 months by serological and molecular techniques. However, PCR in cord blood showed low sensitivity (33.3%) and less specificity (96.4%) for the diagnosis. ConclusionPCR in peripheral blood has proven to be the most adequate strategy for the diagnosis of CCD, allowing an early and reliable diagnosis.

2056. Trypanosama cruzi DNA Detection by PCR in Dried Blood Spots Preserved in Filter Paper

BackgroundEarly diagnosis of Congenital Chagas Disease is important to initiate efficient treatment, so it is necessary to develop techniques with lower detection limits and greater specificity like PCR. The lack of validated protocols and the need to send samples to be analyzed in experienced centers makes dried blood spots preserved in filter paper an attractive alternative for the conservation and handling of samples. The aim of this study was to optimize the detection of Trypanosoma cruzi DNA from dried blood spots preserved in filter paper.MethodsFixed sections of Whatman filter paper with different concentrations of T. cruzi were prepared (104/mL at 10–1/mL) and stored at room temperature, 4 and −20°C in the presence or absence of a desiccant. Samples (8 mm2) were taken at 7, 60, 90 and 240 days of preservation. Endpoint PCR, targeting 18S gene, was used for the detection of T. cruzi DNA directly on the filter paper.Results T. cruzi DNA was detected at all sampling times up to the 102/mL concentration independently of conservation. The effect of humidity was observed at 240 days preservation with the observation of faded bands in agarose gels. For the 101/mL concentration, T. cruzi DNA was detected only at 7 days regardless of preservation. When comparing T. cruzi DNA detection using increasing sections of filter paper (8, 16 and 24 mm2), T. cruzi DNA was detected in all areas tested in the concentration of 101 parasites/mL and only when using 24 mm2 for the concentration of 1 parasite/mL.ConclusionDried blood spots preserved in filter paper allowed detection of T. cruzi DNA by endpoint PCR in the different conservation conditions up to 8 months. The detection of parasite DNA was improved by increasing the area of filter paper tested. The conservation of blood on filter paper would provide a safe transport of samples at room temperature to distant specialized laboratories to perform diagnosis using molecular techniques.Disclosures All authors: No reported disclosures.

Lost opportunities in Congenital Chagas disease: retrospective study in a high complexity hospital of La Plata city

Background: Congenital Chagas disease causes high morbimortality in new-borns. Timely diagnosis is of upmost importance, since treatment is highly effective in curing the disease. Methods & Materials: Retrospective, observational study. Clinical records were collected from pregnant patients with positive serology for Chagas disease, and from their children born at the maternity of the General Hospital “General San Martín” between 2011 and 2016. A diagnosis of Chagas disease was considered in the pregnant woman when two methods resulted reactive (being one of them ELISA or IIF). A diagnosis of congenital Chagas disease was considered after the direct detection of the parasite in blood of an infant under 10 months of age, using a parasitological micromethod. In negative cases, and those older than 10 months, it was considered congenital Chagas disease when two serological methods resulted positive (one of them being ELISA or IIF). Results: 55 mother–son binomials were analysed. 54.3% of the mothers were born in Bolivia (30/55), 30.9% were born in Argentina (17/55) and 14.5% were born in Paraguay (8/55). 3.6% (2/55) of the mothers recounted a history of transfusion of blood products. Serological diagnosis in the mother was attained before pregnancy in 47.3% (26/55), during the first trimester in 5.5% (3/55), in the second trimester in 38.2% (21/55) and in the third trimester in 9.1% (5/55%). 96.4% of the new-borns from women with Chagas disease were asymptomatic at birth (53/55) and 3.6% were symptomatic (2/55). The symptom identified was low birth weight. A microstrout assay was performed in 65.5% (36/55) of newborns, with 2.7% (1/36) of positive results. Follow-up with serological tests was performed in merely 18.2% (10/55) of the children, with negative results. Conclusion: With the data analysed we infer that follow-up of children under the year of age is deficient, with lost opportunities in early diagnosis and prompt treatment and thus cure of the disease. Given the small number of patients and lost data from clinical charts, the incidence of congenital Chagas disease could not be estimated.

Comparative Study and Analytical Verification of PCR Methods for the Diagnosis of Congenital Chagas Disease

Congenital infection is currently the first cause of new cases of Chagas disease in Argentina and nonendemic areas worldwide. Its diagnosis is of utmost importance to guarantee curative treatment. To improve such diagnosis, a transfer process of PCR tests to the national laboratory network has been initiated. We performed a comparative study of four PCR assays [two end-point PCR and two duplex real-time quantitative PCR (qPCR) procedures] to detect Trypanosoma cruzi DNA in blood samples. Because satellite DNA and kinetoplastid DNA qPCR methods showed the best performance and the use of two different molecular targets for confirmatory purposes has been recommended, these methods were selected to perform the transfer process and, in consequence, subjected to an analytical verification protocol based on international guidelines. The anticipated reportable range was verified between 0.25 and 105 parasite equivalents per milliliter of blood (par. eq./mL) for both qPCR methods, and the limit of detection was estimated to be 0.87 (95% CI, 0.62-1.24) and 0.43 (95% CI, 0.32-0.59) par. eq./mL for satellite DNA and kinetoplastid DNA qPCR methods, respectively. In addition, both qPCR methods showed trueness and verified precision in the highest and the lowest concentrations tested. This work provides critical knowledge of the technology transfer process planned to cover laboratories of the regional network with known installed facilities.

Toward Improving Early Diagnosis of Congenital Chagas Disease in an Endemic Setting.

Congenital Trypanosoma cruzi transmission is now estimated to account for 22% of new infections, representing a significant public health problem across Latin America and internationally. Treatment during infancy is highly efficacious and well tolerated, but current assays for early detection fail to detect >50% of infected neonates, and 9-month follow-up is low. Women who presented for delivery at 2 urban hospitals in Santa Cruz Department, Bolivia, were screened by rapid test. Specimens from infants of infected women were tested by microscopy (micromethod), quantitative PCR (qPCR), and immunoglobulin (Ig)M trypomastigote excreted-secreted antigen (TESA)-blots at birth and 1 month and by IgG serology at 6 and 9 months. Among 487 infants of 476 seropositive women, congenital T. cruzi infection was detected in 38 infants of 35 mothers (7.8%). In cord blood, qPCR, TESA-blot, and micromethod sensitivities/specificities were 68.6%/99.1%, 58.3%/99.1%, and 16.7%/100%, respectively. When birth and 1-month results were combined, cumulative sensitivities reached 84.2%, 73.7%, and 34.2%, respectively. Low birthweight and/or respiratory distress were reported in 11 (29%) infected infants. Infants with clinical signs had higher parasite loads and were significantly more likely to be detected by micromethod. The proportion of T. cruzi-infected infants with clinical signs has fallen since the 1990s, but symptomatic congenital Chagas disease still represents a significant, albeit challenging to detect, public health problem. Molecular methods could facilitate earlier diagnosis and circumvent loss to follow-up but remain logistically and economically prohibitive for routine screening in resource-limited settings.

Diagnosis of Congenital Chagas Disease Using an Iron Superoxide Dismutase Excreted as Antigen, in Mothers and Their Children During the First Year of Life.

Chagas disease caused by Trypanosoma cruzi is endemic in Latin America. Human infection is mainly spread by Triatominae insects. Other forms of transmission are congenital, blood transfusion and organ transplantation. Anti-T. cruzi antibodies were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) in 155 serum samples from mothers and their babies. Indirect immunofluorescence (IFA) and a commercial test were used to validate efficacy of a specific ELISA-iron-excreted superoxide dismutase assay. Sera from babies were collected at 6 and 12 months, whereas maternal samples were obtained after delivery. Calostrum and umbilical cord samples were simultaneously obtained. Anti-T. cruzi antibodies were detected in 8 (5.16%) mothers by ELISA-WB, in 7 (4.51%) using IFA and in 1 (0.64%) by a commercial kit. Nine (5.80%) 6-month-old children were positive by ELISA-WB and 7 (4.51%) by IFA; negative results were obtained when the commercial kit was used. At 12 month of age, 15 (9.67%) children were positive by ELISA-WB, 13 (8.38%) by IFA and 1 (0.64%) by the commercial test. Antibodies were detected in 4 mothers whose children were serologically negative. Four other mothers and their children were positive, but only one of them had detected antibodies in umbilical cord up to 12 months, thus assuming vertical transmission. The use of iron-excreted superoxide dismutase as antigen in serologic tests for detection of T. cruzi yielded promising results as diagnostic procedure.

Text message interventions for follow up of infants born to mothers positive for Chagas disease in Tucumán, Argentina: a feasibility study.

BackgroundDiagnosis of congenital Chagas disease occurs at 9 months of age, making effective treatment challenging due to loss to follow-up. Mobile health (mHealth) has been utilized to improve communication and treatment adherence in many chronic diseases, although no studies of mHealth in Trypanosoma cruzi-infected individuals have been conducted. Text message interventions, a subset of mHealth, has shown to improve appointment attendance and is relatively simple to set up, thus making it an ideal mechanism to facilitate communication with individuals in low-resource settings.ObjectiveThe aim of this study is to understand the acceptability, utilization, and barriers of an SMS-based appointment reminder to confirm a post-partum home visit to women in Tucumán, Argentina and whether these factors differ in urban and rural populations.MethodsWomen that tested positive for Chagas disease were invited to receive SMS reminders of their follow-up 4-week postpartum home visit. Demographic information and SMS contact preferences were collected at hospital discharge, and variables on mHealth utilization and barriers were recorded at follow-up.Results77 (70.6 %) of women possessed a cell phone for personal use. All eligible women owned phones compatible with SMS messages. The appointment reminder SMS was widely accepted with 64/72 (88.9 %) enrolled women receiving the SMS message and 58/64 (90.6 %) replying. Ninety-two percent of women stated that the text message was a useful reminder for the follow-up home visit. Women living in rural areas were less likely to own a cell phone for personal use and were significantly less likely to have internet access on their phone than women living in urban areas (RR 0.30, 95 % CI 0.10–0.89). Furthermore, women from rural areas faced barriers to mHealth uptake such as change of phone number and response to messages from the hospital team at higher rates than women from urban areas, although these differences were not statistically significant.ConclusionsThere is generally widespread acceptance and utilization of mHealth among this group of women with access to cell phones. However, there are still many barriers to overcome before mHealth interventions attain complete penetration in a population, most notably the issue of cell phone for personal use.

Use of a novel chagas urine nanoparticle test (chunap) for diagnosis of congenital chagas disease.

BackgroundDetection of congenital T. cruzi transmission is considered one of the pillars of control programs of Chagas disease. Congenital transmission accounts for 25% of new infections with an estimated 15,000 infected infants per year. Current programs to detect congenital Chagas disease in Latin America utilize microscopy early in life and serology after 6 months. These programs suffer from low sensitivity by microscopy and high loss to follow-up later in infancy. We developed a Chagas urine nanoparticle test (Chunap) to concentrate, preserve and detect T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease.Methodology/Principal FindingsThis is a proof-of-concept study of Chunap for the early diagnosis of congenital Chagas disease. Poly N-isopropylacrylamide nano-particles functionalized with trypan blue were synthesized by precipitation polymerization and characterized with photon correlation spectroscopy. We evaluated the ability of the nanoparticles to capture, concentrate and preserve T. cruzi antigens. Urine samples from congenitally infected and uninfected infants were then concentrated using these nanoparticles. The antigens were eluted and detected by Western Blot using a monoclonal antibody against T. cruzi lipophosphoglycan. The nanoparticles concentrate T. cruzi antigens by 100 fold (western blot detection limit decreased from 50 ng/ml to 0.5 ng/ml). The sensitivity of Chunap in a single specimen at one month of age was 91.3% (21/23, 95% CI: 71.92%–98.68%), comparable to PCR in two specimens at 0 and 1 month (91.3%) and significantly higher than microscopy in two specimens (34.8%, 95% CI: 16.42%–57.26%). Chunap specificity was 96.5% (71/74 endemic, 12/12 non-endemic specimens). Particle-sequestered T. cruzi antigens were protected from trypsin digestion.Conclusion/SignificanceChunap has the potential to be developed into a simple and sensitive test for the early diagnosis of congenital Chagas disease.

Predictive role of polymerase chain reaction in the early diagnosis of congenital Trypanosoma cruzi infection

The efficacy of specific chemotherapy in congenital Chagas disease before the first year of life ranges between 90 and 100%. Between this age and 15 years of age, the efficacy decreases to around 60%. Therefore, early infection detection is a priority in vertical transmission. The aim of this work was to assess whether polymerase chain reaction (PCR) plays a predictive role in the diagnosis of congenital Chagas disease as compared to conventional parasitological and serological methods. To this end, we studied a total of 468 children born to Trypanosoma cruzi seroreactive mothers came from Argentina, Bolivia and Paraguay, who lived in the city of Buenos Aires and suburban areas (Argentina), a non-endemic area of this country. These children were assessed by PCR from 2004 to 2009 with the specific primers Tcz1 and Tcz2, and 121 and 122. PCR allowed detecting 49 T. cruzi-positive children. Eight of these 49 children were excluded from the analysis: six because they did not complete follow-up and two because the first control was performed after 12 months of age. Parasitological methods allowed detecting 25 positive children, 7 of whom had been earlier diagnosed by PCR (1.53±2.00 vs. 6.71±1.46 months; p=0.0002). Serological methods allowed detecting 16 positive children, 12 of whom had been earlier diagnosed by PCR (1.46±1.48 vs. 11.77±4.40 months; p<0.0001). None of the children negative by PCR was positive by serological or parasitological methods. This study shows that PCR allows early diagnosis in congenital Chagas disease. At present, an early positive PCR is not indicative for treatment. However, a positive PCR would alert the health system to search only those infected infants diagnosed by early PCR and thus generate greater efficiency in the diagnosis and treatment of congenital T. cruzi infection.

Shed acute-phase antigen protein in an ELISA system for unequivocal diagnosis of congenital Chagas disease

(A) Anti-shed acute-phase antigen IgG antibodies of 2074 noninfected infants. (B) Anti-shed acute-phase antigen IgG antibodies of 209 congenitally T. cruzi-infected infants.OD: Optical density.Chag...

In Nonendemic Areas, Is Microscopy Better than Polymerase Chain Reaction for Diagnosis of Congenital Chagas Disease?

In Nonendemic Areas, Is Microscopy Better than Polymerase Chain Reaction for Diagnosis of Congenital Chagas Disease?

The Value of Molecular Studies for the Diagnosis of Congenital Chagas Disease in Northeastern Argentina

Polymerase chain reaction (PCR) as a diagnostic tool for congenital Chagas disease is scarcely used. To assess the usefulness of PCR, a study in neonates was carried out to compare PCR with standard methods; that is bloodstream parasites by microhematocrit, and/or seroconversion by two conventional procedures. Seventeen neonates, born to mothers with positive Trypanosoma cruzi serology were analyzed by serologic, parasitological and molecular procedures, in blood samples taken at birth, and when aged 2-4 and 9 months. For a better comparison of sensitivity between both parasitological methods (microhematocrit and PCR), the number of samples of neonates studied at birth extended to 121. In children followed-up until 9 months of age, a 100% correlation between PCR and routine diagnosis was found, with PCR showing its highest sensitivity from Month 2 onwards. Comparative analysis between both parasitological methods, on samples taken at birth, showed a higher sensitivity of PCR respect the microhematocrit (P < 0.0008).

AMNIOTIC FLUID IS NOT USEFUL FOR DIAGNOSIS OF CONGENITAL TRYPANOSOMA CRUZI INFECTION

Although Trypanosoma cruzi can be transmitted transplacentally and induce congenital infection, no data are available about the presence of this parasite in human amniotic fluid. We examined 8, 19, and 4 amniotic fluid samples (collected at delivery or by aspiration of gastric content of neonates) from control uninfected mothers (M-B-), infected mothers delivering uninfected newborns (M+B-), and mothers of confirmed congenital cases (M+B+), respectively. Polymerase chain reaction (PCR), using nuclear and kinetoplastic DNA primers (Tcz1-Tcz2 and 121-122), were negative for all control M-B- samples, but positive for 5 of 19 M+B- and 2 of 4 M+B+ samples. To determine the number of parasites in the positive samples, real-time PCR using S35/S36 kinetoplastic DNA was performed. Only one M+B+ sample presented a high parasitic DNA amount, whereas the other six PCR-positive samples displayed traces of T. cruzi DNA. In conclusion, the release of parasites in amniotic fluid is probably a rare event that cannot be helpful for the routine diagnosis of congenital Chagas disease.