Alterations in Placental Gene Expression of Pregnant Women with Chronic Chagas Disease.

Trypanosoma cruzi infection in women of reproductive age is associated with congenital transmission and adverse pregnancy outcomes. The placenta is a key barrier to infection. Gene expression profiles of term placental environment from T. cruzi-seropositive (SP) and -seronegative (SN) mothers were characterized by RNA-Seq. Nine pools of placental RNA paired samples were used: three from SN and six from SP tissues. Each pool consisted of female/male newborns and vaginal/cesarean delivery binomials. No newborn was congenitally infected. T. cruzi satellite DNA quantitative PCR in placental tissues and maternal and neonatal blood, and parasite 18S quantitative RT-PCR from placental RNA were negative, except in three SP women's bloodstream. To identify pathways associated with maternal T. cruzi infection, a gene-set association analysis was implemented: SP placental samples showed overexpression of inflammatory response and lymphocytic activation, whereas numerous biosynthetic processes were down-regulated. About 42 genes showed a significant fold-change between SP and SN groups. KISS1 and CGB5 were down-regulated, whereas KIF12, HLA-G, PRG2, TAC3, FN1, and ATXN3L were up-regulated. Several expressed genes in SP placentas encode proteins associated with preeclampsia and miscarriage. This first transcriptomics study in human term placental environment shows a placental response that may affect the fetus while protecting it from parasite infection; this host response could be responsible for the low rate of congenital transmission in chronic Chagas disease.

Human Polymorphisms in Placentally Expressed Genes and Their Association With Susceptibility to Congenital Trypanosoma cruzi Infection.

BACKGROUND: It is currently unclear why only a proportion of children born to Trypanosoma cruzi-infected mothers acquire the infection. We have examined the association of 11 single-nucleotide polymorphisms (SNPs) located in genes coding for placental expression enzymes as genetic markers of susceptibility to congenital T. cruzi infection (hereafter, "congenital infection"): rs2014683 and rs1048988 in ALPP; rs11244787 and rs1871054 in ADAM12; rs243866, rs243865, rs17859821, rs243864, and rs2285053 in MMP2; and rs3918242 and rs2234681 in MMP9. METHODS: Two groups of children born to mothers seropositive for T. cruzi were compared: 101 had congenital infection, and 116 were uninfected. Novel high-resolution melting and capillary electrophoresis genotyping techniques were designed and used. RESULTS: Logistic regression analysis showed that mutations in rs11244787 and rs1871054 (in ADAM12) and rs243866, rs17859821, and rs2285053 (in MMP2) were associated with susceptibility to congenital infection. Multifactor dimensionality reduction revealed that genotyping results for rs11244787, rs1871054, rs243866, rs17859821 and rs243864 sites would be a good predictor of congenital infection. CONCLUSIONS: Our results suggest an important role of human polymorphisms in proteins involved in extracellular matrix remodeling and the immune response during congenital infection. To our knowledge, this is the first study demonstrating the association between mutations in placentally expressed genes and susceptibility to congenital infection.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

BACKGROUND: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). METHODS/PRINCIPAL FINDINGS: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. CONCLUSIONS/SIGNIFICANCE: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.

BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.