Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease.

BACKGROUND: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina. METHODS: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines. FINDINGS: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved. INTERPRETATION: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence. FUNDING: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Rapid Diagnostic Tests for Trypanosoma cruzi Infection: Field Evaluation of Two Registered Kits in a Region of Endemicity and a Region of Nonendemicity in Argentina.

Infection by Trypanosoma cruzi (Chagas disease [ChD]) affects around 7 million people in the Americas, most of whom are unaware of their status due to lack of clinical manifestations and poor access to diagnosis. Rapid diagnostic tests (RDTs) are widely used for screening for different infections (HIV, hepatitis B, and syphilis), and their application for ChD would facilitate access to diagnosis, especially in remote areas where health services have scarce resources. We conducted a prospective intervention study in 2018 to evaluate in the field two in vitro RDTs for ChD, authorized by the National Administration of Medicaments, Aliments, and Medical Technologies of Argentina (ANMAT), in areas of endemicity and nonendemicity in Argentina. We recruited 607 volunteers older than 18 years in Salta province and the city of Buenos Aires. The RDTs Ab Standard Diagnostics SD Bioline (SD) and Check Chagas Wiener Lab (WL) were performed in situ with whole-blood samples, and confirmatory serology was done at a reference center. The rate of infection with T. cruzi was 17.8% (108/607). The SD test showed 97.2% sensitivity (95% confidence interval [CI], 93.5 to 100) and 91.7% specificity (95% CI, 96.2 to 99.2%), and the WL test showed 93.4% sensitivity (95% CI, 88.2 to 98.6%) and 99.1% specificity (95% CI, 91.9 to 100%). The sensitivity and specificity for the two RDTs tested were higher than previously reported. These results encourage the use of the tested RDTs in Salta province and for further field studies for the implementation of these RDTs in other epidemiological scenarios. This will be very important to improve access to diagnosis of Chagas and its clinical management as a neglected disease, especially in remote areas with health access barriers.

Trypanosoma cruzi Virulence Factors for the Diagnosis of Chagas' Disease.

trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes.

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease. Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment. Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA-CCR7-CD62L- T cells prior to drug therapy was an independent indicator of successful treatment. Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.

Chagas cardiomyopathy associated with serological cure after trypanocidal treatment during childhood.

Chagas disease is a chronic parasitological disease, which could cause cardiac manifestations in approximately one-third of affected individuals. Benznidazole and nifurtimox are used to treat this parasitological infection caused by Trypanosoma cruzi. Conventionally, the criterion for cure is consistently negative serological tests after treatment. We report a case of a patient who was treated when she was 13 years old and achieved T. cruzi negative seroconversion but developed Chagas disease cardiomyopathy as an adult.

Chagas cardiomyopathy associated with serological cure after trypanocidal treatment during childhood

Abstract Chagas disease is a chronic parasitological disease, which could cause cardiac manifestations in approximately one-third of affected individuals. Benznidazole and nifurtimox are used to treat this parasitological infection caused by Trypanosoma cruzi. Conventionally, the criterion for cure is consistently negative serological tests after treatment. We report a case of a patient who was treated when she was 13 years old and achieved T. cruzi negative seroconversion but developed Chagas disease cardiomyopathy as an adult.

Some Limitations for Early Diagnosis of Congenital Chagas Infection by PCR.

Trypanosoma cruzi, the causing agent of Chagas disease, can be transmitted to the offspring of infected pregnant women, thus being an epidemiologically important way of parasite transmission in humans. In addition, the migration of infected women from endemic areas to nonendemic countries may export this parasite infection. The diagnosis of congenital Chagas disease relies on the detection of the parasite because maternal antibodies are passively transferred to infants during pregnancy. The diagnosis of congenital infection can also be confirmed by detection of infant-specific anti-T cruzi antibodies at 10 months after delivery. Because early detection of T cruzi infection in newborns allows an efficient trypanocidal treatment and cure, more sensitive molecular techniques such as DNA amplification are being used for a prompt parasitological diagnosis of children born to seropositive mothers. In this report, we describe a diagnosis case of a child congenitally infected with T cruzi who tested negative for parasite detection both by microscopic observation and DNA amplification at 20 days and 6 months after delivery. However, at 7 months of age, a hemoculture was made from the infant's blood, and the infective parasite was finally isolated and classified as T cruzi discrete typing unit I. In a retrospective study, real-time polymerase chain reaction also allowed detecting the parasite but failed to detect any parasite load in earlier control samples. This case report stresses that even when molecular techniques are negative, a long-term follow-up is necessary for the diagnosis of infants congenitally infected with T cruzi.

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.

Rapid detection of Trypanosoma cruzi by colorimetric loop-mediated isothermal amplification (LAMP): A potential novel tool for the detection of congenital Chagas infection.

Early diagnosis of congenital Trypanosomacruzi transmission in newborns is essential because babies show high indices of cure. Conventional diagnosis is based on microscopic examination and serology. Molecular diagnosis is a promising alternative to replace conventional diagnosis, although it is not well suited for adoption in laboratories with limited resources. Isothermal DNA amplification methods have the advantage of not requiring expensive equipment. The aim of this work was to apply loop-mediated isothermal amplification (LAMP) to detect congenital infection in babies colorimetrically. This assay was able to detect all T. cruzi discrete typing units and Leishmania braziliensis, but not other pathogens. The assay showed a limit of detection of 50 parasites/mL in spiked artificial samples. This assay was tested in 27 blood samples of babies born to T. cruzi-infected mothers and showed 100% of concordance with conventional diagnosis. This is the first study to detect T. cruzi in clinical samples by LAMP, showing that this assay would be useful in the detection of congenital T. cruzi infection. The advantages of this novel tool include the speed with which the assays can be completed, the no-need of trained personnel, and the fact that it can be performed without complex and expensive laboratory equipment.

Inmunoserología y métodos moleculares para el diagnóstico de Chagas: revisión sistemática rápida / Immunoserology and molecular techniques for diagnosis of Chagas´ disease: rapid systematic review / Imunoserologia e métodos moleculares para o diagnóstico de Chagas: revisão sistemática rápida

Con el objetivo de incrementar la precisión diagnóstica, la Organización Mundial de la Salud recomienda la realización de dos o más pruebas inmunoserológicas para el diagnóstico de la enfermedad de Chagas en etapa crónica. El objetivo de este trabajo fue realizar una revisión sistemática rápida acerca del desempeño de las técnicas inmunoserológicas y métodos moleculares en la población general. Se identificaron 178 estudios de los cuales fueron incluidos nueve. Las técnicas de ELISA mostraron la mayor sensibilidad (82-98%) y especificidad (96-100%). Los métodos rápidos mostraron valores de sensibilidad entre 88-93% y especificidad 97- 100%, mientras que los métodos moleculares (PCR) presentaron niveles muy variables de sensibilidad (22-92%) y especificidad (70-100%). Estos resultados muestran que las técnicas de ELISA cuentan con una sensibilidad y especificidad adecuadas. La PCR, al igual que los métodos rápidos, mostró una gran variabilidad en los resultados, debido principalmente a la heterogeneidad de la técnicas y profusión de métodos elaborados de manera in house.

In order to increase diagnostic accuracy, the World Health Organization recommends performing two or more immunoserological tests for the diagnosis of Chagas disease in chronic stage. The aim of this work was to make a rapid systematic review of the performance of immunoserological techniques and molecular methods in the general population. A total of 178 studies were identified, nine ofwhich were included. ELISA techniques showed the highest sensitivity (82-98%) and specificity (96-100%). Rapid methods presented values of sensitivity between 88-93% and 97-100% of specificity, while the molecular methods (PCR) showed highly variable levels of sensitivity (22-92%) and specificity (70-100%). These results indicate that ELISA techniques have adequate sensitivity and specificity. PCR, as well as rapid methods, showed great variability in the results, mainly due to the heterogeneity of the techniques and abundance of "in-house" methods.

Visando a aumentar a precisão do diagnóstico, a Organização Mundial da Saúde recomenda executar dois ou mais testes imunoserológicos para o diagnóstico na fase crônica da doença de Chagas. O objetivo deste trabalho foi fazer uma revisão sistemática rápida sobre o desempenho de técnicas imunoserológicas e métodos moleculares na população em geral. Foram identificados 178 estudos dos quais se incluíram nove. Técnicas de ELISA mostraram a maior sensibilidade (82-98%) e especificidade (96-100%). Métodos rápidos apresentaram valores de sensibilidade e especificidade de 88-93% e 97-100%, respectivamente enquanto que os métodos moleculares (PCR) tinham níveis extremamente variáveis de sensibilidade (22-92%) e especificidade (70-100%). Estes resultados mostram que as técnicas de ELISA possuem sensibilidade e especificidade adequadas. A PCR, bem como os métodos rápidos, mostrou grande variabilidade nos resultados, principalmente devido à heterogeneidade das técnicas e profusão de métodos desenvolvidos de modo in house.

Diagnosis of congenital Trypanosoma cruzi infection: A serologic test using Shed Acute Phase Antigen (SAPA) in mother-child binomial samples.

Chagas congenital infection is an important health problem in endemic and non-endemic areas in which Trypanosoma cruzi-infected women can transmit the parasite to their offspring. In this study, we evaluated the antibody levels against the T. cruzi Shed Acute Phase Antigen (SAPA) in 91 binomial samples of seropositive pregnant women and their infected and non-infected children by ELISA. In 70 children without congenital T. cruzi transmission, the titers of anti-SAPA antibodies were lower than those of their seropositive mothers. In contrast, 90.5% of 21 congenitally infected children, at around 1 month of age, showed higher anti-SAPA antibody levels than their mothers. Subtracting the SAPA-ELISA mother OD value to the SAPA-ELISA child OD allowed efficient detection of most T. cruzi congenitally infected children immediately after birth, when total anti-parasite antibodies transferred during pregnancy are still present in all children born to seropositive women. A positive correlation was observed between parasitemia levels in mothers and infants evaluated by quantitative DNA amplification and anti-SAPA antibody titers by ELISA. As SAPA serology has proved to be very efficient to detect T. cruzi infection in mother-child binomial samples, it could be of extreme help for early diagnosis of newborns, in maternities and hospitals where DNA amplification is not available. This prompt diagnosis may prevent drop out of the long-term follow-up for future diagnosis and may ensure early trypanocidal treatment, which has proved to be efficient to cure infants with congenital Chagas disease.

Comparative evaluation of 11 commercialized rapid diagnostic tests for detecting Trypanosoma cruzi antibodies in serum banks in areas of endemicity and nonendemicity.

Chagas disease is one of the main public health issues in Latin America. Increasingly during the past few decades, Trypanosoma cruzi infection has been detected in North America, Europe, and the Western Pacific, mainly as a result of population movement. The limited availability of rapid serological diagnostic tests hinders rapid diagnosis and early treatment in areas of endemicity and nonendemicity. In collaboration with 11 national reference laboratories (NRLs) from different geographical areas, we evaluated the performances of commercialized serological rapid diagnostic tests (RDT) for T. cruzi infection. Eleven commercialized T. cruzi infection RDTs were evaluated on a total of 474 samples extensively tested with at least three different techniques for Chagas disease, maintained at controlled low temperatures, and stored in the serum banks of the 11 NRLs. We measured the sensitivity, specificity, and concordance of each RDT and provided an additional questionnaire to evaluate its ease of use. The selected RDTs in this study were performed under controlled laboratory conditions. Out of the 11 RDTs, we found 8 of them to be useful, with the cassette format favored over the strip. We did not observe significant differences in RDT performances in the different regions. Overall, the performance results were lower than those disclosed by the manufacturers. The results of this evaluation validate the possibility of using RDTs to diagnose Chagas disease, thereby decreasing the time to treatment at a primary health care facility for patients who are willing to be treated. Further studies should be conducted in the laboratory and in the field to confirm these data, expressly to evaluate reproducibility in resource-limited settings, or using whole blood in clinical settings in areas of endemicity and nonendemicity.

How to improve the early diagnosis of Trypanosoma cruzi infection: relationship between validated conventional diagnosis and quantitative DNA amplification in congenitally infected children.

BACKGROUND: According to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10-12 months of age. METHODOLOGY AND PRINCIPAL FINDINGS: Parasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI. SIGNIFICANCE: This report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.

[Maternal-fetal transmission of Trypanosoma cruzi in Argentina]. / La transmisión madre-hijo del Trypanosoma cruzi en la Argentina.

In the neonates born to T. cruzi infected mothers, the diagnosis of the congenital transmission relays on the detection of the parasites and/or the specific antibodies non-transferred by their mothers, in the absence of blood transfusion and vectorial transmission. In the early stage, approximately until the 7th month of life, when maternal immunoglobulins could be present, the diagnosis depends on the detection of the parasite. Then, in the late stage, from the 8th month, the detection of specific antibodies by at least 2 of 3 serological tests confirms the infection in the neonates. The diagnostic follow up of the children born to a group of sero-reactive pregnant women was carried out in the INP. The 11% of the mothers (29 out of 267) transmitted the infection to their children. The neonates of 20 of these mothers were diagnosed in the early stage, 14 and 6 in one or two controls, respectively. In the 9 remaining mothers the children were diagnosed in the late stage of the infection, mainly serologicaly. Our analisis of previously published reports stressed that the maternal-fetal transmission rate depends on the time of diagnostic follow up of the child. In this reports, mean values of mother to child transmission reported was 2% and 9% when the diagnosis of the neonates born to sero-reactive mothers was carried out only in the early stage or in the early and also the late stage, respectively.

La transmisión madre-hijo del Trypanosoma cruzi en la Argentina / Maternal fetal-transmission of Trypanosoma cruzi in Argentina

El diagnóstico de la transmisión congénita del T. cruzi en hijos de mujeres infectadas se realiza por detección de la parasitemia y/o de anticuerpos específicos no transferidos por la madre, en ausencia de transfusión sanguínea y transmisión vectorial. En la etapa temprana, aproximadamente hasta el 7° mes de vida, cuando es posible la presencia de inmunoglobulinas maternas, el diagnóstico depende de la detección del parásito. Luego, en la etapa tardía, a partir del 8° mes de vida, la detección de anticuerpos específicos con por lo menos 2 de 3 pruebas serológicas permiten el diagnóstico del niño. En el INP hemos realizado el seguimiento de los niños de un grupo de mujeres embarazadas sero-reactivas que concurrieron para el diagnóstico de la infección. El 11% de ellas (29 de 267) transmitieron la infección a sus niños. Los hijos de 20 de estas mujeres fueron diagnosticados en la etapa temprana, con 1 o 2 controles parasitológicos, en 14 y 6 casos respectivamente. En las 9 madres restantes los niños fueron diagnosticados principalmente por la serología en la etapa tardía de la infección. Nuestro análisis de los datos publicados anteriormente enfatizan que el porcentaje de la transmisión madre-hijo depende principalmente del tiempo de seguimiento diagnóstico del niño. En estos trabajos, cuando el diagnóstico del niño se realizó sólo en la etapa temprana se notificó aproximadamente un 2% de transmisión materno-fetal, mientras que cuando también se estudiaron a los niños en la etapa tardía se encontró un promedio de 9% de casos de transmisión congénita.

In the neonates born to T. cruzi infected mothers, the diagnosis of the congenital transmission relays on the detection of the parasites and/or the specific antibodies non-transferred by their mothers, in the absence of blood transfusion and vectorial transmission. In the early stage, approximately until the 7th month of life, when maternal immunoglobulins could be present, the diagnosis depends on the detection of the parasite. Then, in the late stage, from the 8th month, the detection of specific antibodies by at least 2 of 3 serological tests confirms the infection in the neonates. The diagnostic follow up of the children born to a group of sero-reactive pregnant women was carried out in the INP. The 11% of the mothers (29 out of 267) transmitted the infection to their children. The neonates of 20 of these mothers were diagnosed in the early stage, 14 and 6 in one or two controls, respectively. In the 9 remaining mothers the children were diagnosed in the late stage of the infection, mainly serologicaly. Our analisis of previously published reports stressed that the maternal-fetal transmission rate depends on the time of diagnostic follow up of the child. In this reports, mean values of mother to child transmission reported was 2% and 9% when the diagnosis of the neonates born to sero-reactive mothers was carried out only in the early stage or in the early and also the late stage, respectively.