Quantitative PCR-Based Diagnosis and Follow-up of Chagas Disease Primary Infection After Solid Organ Transplant: A Multicentre Study.

Chagas disease in solid organ transplant recipients may present as a primary infection (PI). Early detection is crucial for timely treatment. This is the largest observational multicentre study evaluating qPCR for early diagnosis and treatment monitoring of PI in seronegative recipients of organs from seropositive donors. Of 34 patients admitted at 5 health centers, PI was detected by qPCR in 8 (23.5%) within a posttransplant period of 40 days (interquartile range [IQR], 31-50 days). No PI was detected by the Strout test or clinical symptoms/signs. All patients had favorable treatment outcome with negative qPCR 31 days (IQR, 18-35 days) after treatment, with no posttreatment relapse episodes.

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

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Genetic Characterization of Trypanosoma cruzi I Populations from an Oral Chagas Disease Outbreak in Venezuela: Natural Resistance to Nitroheterocyclic Drugs.

The oral transmission of Chagas disease (oCD) in Venezuela announced its appearance in 2007. Different from other populations affected by oCD and despite close supervision during treatment with nitroheterocyclic drugs, the result was treatment failure. We studied genetic features of natural bloodstream parasite populations and populations after treatment of nine patients of this outbreak. In total, we studied six hemoculture isolates, eight Pre-Tx blood samples, and 17 samples collected at two or three Post-Tx time-points between 2007 and 2015. Parasitic loads were determined by quantitative polymerase chain reaction (qPCR), and discrete typing units (DTU), minicircle signatures, and Tcntr-1 gene sequences were searched from blood samples and hemocultures. Half-maximal inhibitory concentration (IC50) values were measured from the hemocultures. All patients were infected by TcI. Significant decrease in parasitic loads was observed between Pre-Tx and Post-Tx samples, suggesting the evolution from acute to chronic phase of Chagas disease. 60% of intra-DTU-I variability was observed between Pre-Tx and Post-Tx minicircle signatures in the general population, and 43 single-nucleotide polymorphisms (SNPs) were detected in a total of 12 Tcntr-1 gene sequences, indicative of a polyclonal source of infection. SNPs in three post-Tx samples produced stop codons giving rise to putative truncated proteins or displaced open reading frames, which would render resistance genes. IC50 values varied from 5.301 ± 1.973 to 104.731 ± 4.556 µM, demonstrating a wide range of susceptibility. The poor drug response in the Pre-Tx parasite populations may be associated with the presence of naturally resistant parasite clones. Therefore, any information that can be obtained on drug susceptibility from in vitro assays, in vivo assays, or molecular characterization of natural populations of Trypanosoma cruzi becomes essential when therapeutic guidelines are designed in a given geographical area.

Molecular detection and parasite load of Trypanosoma cruzi in digestive tract tissue of Chagas disease patients affected by megacolon.

Chagas disease, caused by the Trypanosoma cruzi parasite in the Americas affects ∼ 7 million people, 30% with cardiac tissue damage and 10-15% with digestive disorders. In this study, we have developed a protocol to detect the presence of the parasite and estimate its load in resected dysfunctional tissue segments of chronically infected patients with digestive megacolon. We have included samples from 43 individuals, 38/5 with positive/negative serology for Chagas disease and digestive syndromes. Samples of 1.5 to 2.0 cm2 were taken from different points of the dysfunctional digestive tract in specialized centres in Cochabamba, Bolivia. T. cruzi cultures were performed by inoculation with NNN-LIT culture medium, and genomic material was obtained from the samples for multiplex qPCR with TaqMan probes targeting satellite nuclear DNA. Cultures failed to isolate T. cruzi but qPCR reached a sensitivity of 42.1% (16/38) with all three spots and in triplicate. A new quantification methodology using synthetic satellite DNA as quantitation standard revealed parasite loads ranging from 2.2 × 102 to 1.0 × 106 satellite DNA copies/µl. Positive samples from the distal end showed a higher parasite load. The results of the present study strengthen and add further evidence to previous findings in an experimental mouse model of chronic T. cruzi infection, providing a valuable tool to improve scientific knowledge on the relevance of the digestive tract in parasite persistence, and underline the need of a better understanding of host-pathogen interaction in digestive tissues, considering pathophysiology, disease immunology and response to treatment.

Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease.

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

Detection and identification of Kinetoplastids of zoonotic interest by HRM-qPCR analysis in Canis lupus familiaris from Argentinean Mesopotamia.

This work aimed to conduct a first PCR-based approach for differential diagnosis of kinetoplastidean infections in dogs. Diagnosis of Kinetoplastid infections in domestic animals is difficult, since parasitemia is intermittent and signs are nonspecific; it is mainly based on parasitological smears or concentration techniques, which lack sensitivity and depend on operator` expertise. Dogs are relevant reservoirs in transmission of Kinetoplastids; they function as sentinels to detect active transmission cycles before they involve humans. Trypanosoma cruzi, Trypanosoma evansi, and various species of Leishmania genus are multi-host parasites, capable of parasitizing dogs among a vast number of reservoirs. An algorithm based on sequential Real-Time PCR-High Resolution Melting (HRM) (qPCR-HRM) assays directed at 24S alpha ribosomal DNA, ITS1 and Hsp70 designed to distinguish among T. cruzi, T. rangeli, T. evansi and Leishmania spp. was tested in fourteen dogs with suspicion of kinetoplastid diseases. A qPCR control of DNA integrity in the tested sample, targeted to the mammalian interphotoreceptor retinoid-binding protein (IRBP) gene fragment was incorporated to the algorithm. T. evansi was detected in four dogs and L. infantum in one. Two of five qPCR positive cases were smear negative. Smear and T. evansi qPCR positive cases corresponded to animals that died despite being treated, indicating the association of parasitemia with disease severity. This laboratory tool increases the possibility of confirming outbreaks of kinetoplastid diseases with zoonotic potential and identify the etiological agents involved.

Characterization and Follow-Up of Trypanosoma cruzi Natural Populations Refractory to Etiological Chemotherapy in Oral Chagas Disease Patients.

We aimed to characterize the genetic constitution of natural T. cruzi populations involved in an Oral Chagas Disease (OCD) outbreak at a rural school of the community of Chichiriviche de la Costa, Venezuela, which affected patients did not respond to the etiological treatment. Peripheral blood samples and/or hemocultures were obtained from twenty-nine OCD patients at time of diagnosis or along nine years of Post-treatment (Tx) follow-up. The IgG serology, T. cruzi discrete typing units (DTU), satellite DNA-qPCR parasitic loads, and minicircle signatures were determined at Pre-Tx and after Tx. The serological titles and parasitic loads changed after treatment, with a significant decrease of IgG titers (Spearman's r value= -0.961) and median parasite loads from 2.869 [IQR = 2.113 to 3.720] to 0.105 [IQR = -1.147 to 1.761] log10 par eq. /mL at Pre-Tx and Post-Tx, respectively, suggesting infection evolution from acute to chronic phase, without seroconversion or parasitological eradication, which was indicative of treatment failure. All patients were infected with T. cruzi DTU I populations. At Pre-Tx their median Jaccard genetic distances were 0.775 [IQR = 0.708 to 0.882], decreasing in genetic variability towards the end of follow-up (Mann-Whitney U test p= 0.0031). Interestingly, no Post-Tx minicircle signature was identical to its Pre-Tx counterpart population in a same patient, revealing selection of parasite subpopulations between the primary infection and Post-Tx. The parasitic populations isolated from hemocultures showed a lower number of bands in the minicircle signatures with respect to the signatures obtained directly from the patients' blood samples, demonstrating a process of parasitic selection and reduction of the population variability that initially infected the patients. Decrease of parasitic loads after treatment as well as Pre- and Post-Tx intra-TcI diversity might be a consequence of both, natural evolution of the acute infection to the chronic phase and persistence of refractory populations due to Tx selection.

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi.

BACKGROUND: Chagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes. METHODOLOGY/PRINCIPAL FINDINGS: To obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication. CONCLUSION/SIGNIFICANCE: This research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action.

Correction: Mixed infections by different Trypanosoma cruzi discrete typing units among Chagas disease patients in an endemic community in Panama.

[This corrects the article DOI: 10.1371/journal.pone.0241921.].

Toward the Establishment of a Single Standard Curve for Quantification of Trypanosoma cruzi Natural Populations Using a Synthetic Satellite Unit DNA Sequence.

Accurate diagnostic tools and surrogate markers of parasitologic response to treatment are needed for managing Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (TcI) and CL-Brener (TcVI) stocks similar 95% limit of detection values [0.903 (0.745 to 1.497) and 0.667 (CI, 0.113 to 3.927) copy numbers/µL, respectively] when synthetic DNA was the standard for quantification, allowing direct comparison of loads in samples infected with different discrete typing units. This standard curve was evaluated in 205 samples (38 acute oral and 19 chronic Chagas disease patients) from different geographical areas infected with various genotypes, including samples obtained during treatment follow-up; high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase 3 clinical trials, to follow up patients under treatment or at risk of reactivation, and in experimental models using different parasite strains.

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.

Differential tissue distribution of discrete typing units after drug combination therapy in experimental Trypanosoma cruzi mixed infection.

The aim of the present work was to evaluate the distribution of the different clones of the parasite prevailing after treatment with benznidazole (BZ) and clomipramine (CLO), in mice infected with Trypanosoma cruzi, Casibla isolate which consists of a mixture of two discrete typing units (DTUs). Albino Swiss mice were infected and treated with high and low concentrations of BZ (100 or 6.25 mg/kg), CLO (5 or 1.25 mg/kg), or the combination of both low doses (BZ6.25 + CLO1.25), during the acute phase of experimental infection. Treatment efficacy was evaluated by comparing parasitaemia, survival and tissular parasite presence. For DTUs genotyping, blood, skeletal and cardiac muscle samples were analysed by multiplex quantitative polymerase chain reaction. The combined treatment had similar outcomes to BZ6.25; BZ100 was the most effective treatment, but it failed to reach parasite clearance and produced greater histological alterations. Non-treated mice and the ones treated with monotherapies showed both DTUs while BZ6.25 + CLO1.25 treated mice showed only TcVI parasites in all the tissues studied. These findings suggest that the treatment may modify the distribution of infecting DTUs in host tissues. Coinfection with T. cruzi clones belonging to different DTUs reveals a complex scenario for the treatment of Chagas disease and search for new therapies.

Mixed infections by different Trypanosoma cruzi discrete typing units among Chagas disease patients in an endemic community in Panama.

BACKGROUND: Trypanosoma cruzi, the hemoparasite that causes Chagas disease, is divided into six Discrete Typing Units or DTUs: TcI-TcVI plus Tcbat. This genetic diversity is based on ecobiological and clinical characteristics associated with particular populations of the parasite. The main objective of this study was the identification of DTUs in patients with chronic chagasic infections from a mountainous rural community in the eastern region of Panama. METHODS: A total of 106 patients were tested for Chagas disease with three serological tests (ELISA, rapid test, and Western blot). Molecular diagnosis and DTU typing were carried out by conventional PCRs and qPCR targeting different genomic markers, respectively. As a control sample for the typing, 28 patients suspected to be chagasic from the metropolitan area of Panama City were included. RESULTS: Results showed a positivity in the evaluated patients of 42.3% (33/78); high compared to other endemic regions in the country. In the control group, 20/28 (71.43%) patients presented positive serology. The typing of samples from rural patients showed that 78.78% (26/33) corresponded to TcI, while 9.09% (3/33) were mixed infections (TcI plus TcII/V/VI). Seventy-five percent (15/20) of the patients in the control group presented TcI, and in five samples it was not possible to typify the T. cruzi genotype involved. CONCLUSIONS: These results confirm that TcI is the main DTU of T. cruzi present in chronic chagasic patients from Panama. However, the circulation of other genotypes (TcII/V/VI) in this country is described for the first time. The eco-epidemiological characteristics that condition the circulation of TcII/V/VI, as well as the immune and clinical impact of mixed infections in this remote mountainous region should be investigated, which will help local action programs in the surveillance, prevention, and management of Chagas disease.

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.

Short-course Benznidazole treatment to reduce Trypanosoma cruzi parasitic load in women of reproductive age (BETTY): a non-inferiority randomized controlled trial study protocol.

BACKGROUND: Retrospective observational studies suggest that transmission of Trypanosoma cruzi does not occur in treated women when pregnant later in life. The level of parasitemia is a known risk factor for congenital transmission. Benznidazole (BZN) is the drug of choice for preconceptional treatment to reduce parasitic load. The fear of treatment-related side effects limits the implementation of the Argentine guideline recommending BZN 60d/300 mg (or equivalent) treatment of T. cruzi seropositive women during the postpartum period to prevent transmission in a future pregnancy. A short and low dose BZN treatment might reduce major side effects and increase compliance, but its efficacy to reduce T. cruzi parasitic load compared to the standard 60d/300 mg course is not yet established. Clinical trials testing alternative BZN courses among women of reproductive age are urgently needed. METHODS AND DESIGN: We are proposing to perform a double-blinded, non-inferiority randomized controlled trial comparing a short low dose 30-day treatment with BZN 150 mg/day (30d/150 mg) vs. BZN 60d/300 mg. We will recruit not previously treated T. cruzi seropositive women with a live birth during the postpartum period in Argentina, randomize them at 6 months postpartum, and follow them up with the following specific aims: Specific aim 1: to measure the effect of BZN 30d/150 mg compared to 60d/300 mg preconceptional treatment on parasitic load measured by the frequency of positive Polymerase Chain Reaction (PCR) (primary outcome) and by real-time quantitative PCR (qPCR), immediately and 10 months after treatment. Specific aim 2: to measure the frequency of serious adverse events and/or any adverse event leading to treatment interruption. TRIAL REGISTRATION: ClinicalTrials.gov . Identifier: NCT03672487 . Registered 14 September 2018.

Real-time polymerase chain reaction based algorithm for differential diagnosis of Kinetoplastidean species of zoonotic relevance.

Kinetoplastids are a group of flagellated protozoa that infect a vast repertoire of mammals and insect vectors. From a zoonotic point of view, domestic animals are critical reservoirs for transmission of Kinetoplastidean parasites. Due to their proximity to humans, they assume substantial epidemiological importance in the context of these zoonoses and consequently in public health. Their reliable identification is relevant to understand their eco-epidemiological involvement in transmission cycles. This work aimed to develop an algorithm based on sequential Real-Time PCR (qPCR) assays targeted to different loci (24S alpha rDNA, ITS1 and Hsp70) allowing distinction among Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma evansi and Leishmania species in biological samples collected from mammalian reservoirs and triatomine vectors. The algorithm includes a first qPCR test targeted to endogenous genes conserved within mammals and within triatomine vectors as internal controls of DNA sample integrity and/or qPCR inhibition. This algorithm was evaluated in biological samples from domestic cattle (N = 14), dogs (N = 19) and triatomines (N = 19). Analytical sensitivity of 24S alpha rDNA for detection of T. rangeli was 10 fg of DNA, with a linear range between 10 fg and 10 ng. For T. cruzi it varied depending on the Discrete typing unit. The ITS1 qPCR showed an analytical sensitivity of 100 pg/reaction and 100 fg/reaction of Leishmania spp. and T. evansi DNAs. In mammal field samples, four T. cruzi 24S alpha rDNA sequences and fourteen ITS1 amplicons specific for T. evansi were detected. qPCR-HRM analysis directed to the Hsp70 gene diagnosed two dogs with Leishmania infantum infection. Among 19 triatomine field samples, T. cruzi was detected in five; T. rangeli in eight and one specimen showed a mixed infection. This diagnostic algorithm can provide more accurate records of kinetoplastidean infection burden in vectors and reservoirs, relevant to update current eco-epidemiological maps in co-endemic regions.

Development and evaluation of a duplex TaqMan qPCR assay for detection and quantification of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts.

BACKGROUND: A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. RESULTS: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. CONCLUSIONS: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

Role of nucleic acid amplification assays in monitoring treatment response in chagas disease: Usefulness in clinical trials.

Chagas disease has become a global health problem due to migration of infected people out of Latin America to non-endemic countries. For more than 40 years, only the nitroimidazole compounds Benznidazole and Nifurtimox, have been used for specific treatment of Trypanosoma cruzi infection with disappointing results, specially due to the long duration of treatment and adverse events in the chronic phase. In the last years, ergosterol inhibitors have been also proposed for specific treatment. Different randomized clinical trials were performed for evaluating their treatment efficacy and safety. One of the greatest concerns in clinical trials is to provide an early surrogate biomarker of response to trypanocidal chemotherapy. Serological response is slow and the classical parasitological tests have poor sensitivity and are time-consuming. Nowadays, PCR is the most helpful tool for assessing treatment response in a short period of time. Different protocols of PCR have been developed, being quantitative real time PCR based on amplification of repetitive satellite or minicircle DNA sequences plus an internal amplification standard, the mostly employed strategies in clinical trials. Standardized protocols and the use of an external quality assessment ensure adequate technical procedures and reliable data. Clinical trials have shown a significant reduction in parasite loads, reaching undetectable DNA levels in bloodstream after specific treatment, however events of treatment failure have also been reported. Treatment failure could be due to inadequate penetrance of the drugs into the affected tissues, to the presence of primary or secondary drug resistance of the infecting strains as well as to the existence of dormant parasite variants reluctant to drug action. The early diagnosis of drug resistance would improve clinical management of Chagas disease patients, allowing dictating alternative therapies with a combination of existing drugs or new anti-T. cruzi agents. The aim of this review was to describe the usefulness of detecting T.cruzi DNA by means of real time PCR assays, as surrogate biomarker in clinical trials for evaluating new drugs for CD or new regimens of available drugs and the possibility to detect treatment failure.