Congenital Chagas disease: an update.

Congenital infection with Trypanosoma cruzi is a global problem, occurring on average in 5% of children born from chronically infected mothers in endemic areas, with variations depending on the region. This presentation aims to focus on and update epidemiological data, research methods, involved factors, control strategy and possible prevention of congenital infection with T. cruzi. Considering that etiological treatment of the child is always effective if performed before one year of age, the diagnosis of infection in pregnant women and their newborns has to become the standard of care and integrated into the surveillance programs of syphilis and human immunodeficiency virus. In addition to the standard tests, polymerase chain reaction performed on blood of neonates of infected mothers one month after birth might improve the diagnosis of congenital infection. Recent data bring out that its transmission can be prevented through treatment of infected women before they become pregnant. The role of parasite genotypes and host genetic factors in parasite transmission and development of infection in foetuses/neonates has to be more investigated in order to better estimate the risk factors and impact on health of congenital infection with T. cruzi.

In situ microscopy analysis reveals local innate immune response developed around Brucella infected cells in resistant and susceptible mice.

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.

Trypanosoma cruzi Infection in Pregnancies without Congenital Transmission Is Associated with Reduced Fetal Growth: A Cross-Sectional Study in Argentina, Honduras, and Mexico.

We aimed to measure the association between Trypanosoma cruzi infection in pregnancy and reduced fetal growth in the absence of T. cruzi congenital transmission. We conducted a cross-sectional study of secondary data of all singleton live births between 2011 and 2013 in five hospitals from Argentina, Honduras, and Mexico. We excluded newborns with T. cruzi infection. Noninfected pregnant people were those without any positive rapid tests. The main study outcomes were birth weight, head circumference, and length for gestational age and sex. Logistic regression models were adjusted for country, age, education level, and obstetric history. Of the 26,544 deliveries, 459 (1.7%) pregnant people were found by rapid tests to be positive for T. cruzi. Of these, 320 were positive by enzyme-linked immunosorbent assay and 231 had a positive polymerase chain reaction (PCR) test. Uninfected newborns from T. cruzi-infected pregnant people were more likely to have birth weights below the 5th and 10th percentiles and head circumferences below the 3rd and 10th percentiles. Among T. cruzi-infected pregnant people diagnosed by PCR, the odds ratios were 1.58 for birth weight below the 10th percentile (95% CI, 1.12-2.23) and 1.57 for birth weight below the 5th percentile (95% CI, 1.02-2.42). Higher T. cruzi parasitic loads in pregnancy had a stronger association with reduced fetal growth (both in birth weight and head circumference), with an odds ratio of 2.31 (95% CI, 1.36-3.91) for a birth weight below the 5th percentile. The association shows, irrespective of causality, that newborns of pregnancies with T. cruzi have an increased risk of reduced fetal growth. We recommend further studies to assess other potential confounders and the causality of these associations.

Fatal alveolar echinococcosis of the lumbar spine.

For the last 10 years, the southern part of Belgium has been recognized as a low-risk area of endemicity for alveolar echinococcosis. This infection, caused by Echinococcus multilocularis, usually induces a severe liver condition and can sometimes spread to other organs. However, alveolar echinococcosis involving bones has been described only very rarely. Here, a fatal case of spondylodiscitis due to E. multilocularis contracted in southern Belgium is reported.

Congenital transmission of Trypanosoma cruzi in Argentina, Honduras, and Mexico: study protocol.

BACKGROUND: Trypanosoma cruzi has been divided into Discrete Typing Units I and non-I (II-VI). T. cruzi I is predominant in Mexico and Central America, while non-I is predominant in most of South America, including Argentina. Little is known about congenital transmission of T. cruzi I. The specific aim of this study is to determine the rate of congenital transmission of T. cruzi I compared to non-I. METHODS/DESIGN: We are conducting a prospective study to enroll at delivery, 10,000 women in Argentina, 7,500 women in Honduras, and 13,000 women in Mexico. We are measuring transmitted maternal T. cruzi antibodies by performing two rapid tests in cord blood (Stat-Pak, Chembio, Medford, New York, and Trypanosoma Detect, InBios, Seattle, Washington). If at least one of the results is positive, we are identifying infants who are congenitally infected by performing parasitological examinations on cord blood and at 4-8 weeks, and serological follow-up at 10 months. Serological confirmation by ELISA (Wiener, Rosario, Argentina) is performed in cord and maternal blood, and at 10 months. We also are performing T. cruzi standard PCR, real-time quantitative PCR and genotyping on maternal venous blood and on cord blood, and serological examinations on siblings. Data are managed by a Data Center in Montevideo, Uruguay. Data are entered online at the sites in an OpenClinica data management system, and digital pictures of data forms are sent to the Data Center for quality control. Weekly reports allow for rapid feedback to the sites.

Monocytes from Uninfected Neonates Born to Trypanosoma cruzi-Infected Mothers Display Upregulated Capacity to Produce TNF-α and to Control Infection in Association with Maternally Transferred Antibodies.

Activated monocytes/macrophages that produce inflammatory cytokines and nitric oxide are crucial for controlling Trypanosoma cruzi infection. We previously showed that uninfected newborns from T. cruzi infected mothers (M+B- newborns) were sensitized to produce higher levels of inflammatory cytokines than newborns from uninfected mothers (M-B- newborns), suggesting that their monocytes were more activated. Thus, we wondered whether these cells might help limit congenital infection. We investigated this possibility by studying the activation status of M+B- cord blood monocytes and their ability to control T. cruzi in vitro infection. We showed that M+B- monocytes have an upregulated capacity to produce the inflammatory cytokine TNF-α and a better ability to control T. cruzi infection than M-B- monocytes. Our study also showed that T. cruzi-specific Abs transferred from the mother play a dual role by favoring trypomastigote entry into M+B- monocytes and inhibiting intracellular amastigote multiplication. These results support the possibility that some M+B- fetuses may eliminate the parasite transmitted in utero from their mothers, thus being uninfected at birth.

Macrophage-infectivity potentiator of Trypanosoma cruzi (TcMIP) is a new pro-type 1 immuno-stimulating protein for neonatal human cells and vaccines in mice.

This work identifies the protein "macrophage infectivity potentiator" of Trypanosoma cruzi trypomastigotes, as supporting a new property, namely a pro-type 1 immunostimulatory activity on neonatal cells. In its recombinant form (rTcMIP), this protein triggers the secretion of the chemokines CCL2 and CCL3 by human umbilical cord blood cells from healthy newborns, after 24h in vitro culture. Further stimulation for 72h results in secretion of IFN-γ, provided cultures are supplemented with IL-2 and IL-18. rTcMIP activity is totally abolished by protease treatment and is not associated with its peptidyl-prolyl cis-trans isomerase enzymatic activity. The ability of rTcMIP to act as adjuvant was studied in vivo in neonatal mouse immunization models, using acellular diphtheria-tetanus-pertussis-vaccine (DTPa) or ovalbumin, and compared to the classical alum adjuvant. As compared to the latter, rTcMIP increases the IgG antibody response towards several antigens meanwhile skewing antibody production towards the Th-1 dependent IgG2a isotype. The amplitude of the rTcMIP adjuvant effect varied depending on the antigen and the co-presence of alum. rTcMIP did by contrast not increase the IgE response to OVA combined with alum. The discovery of the rTcMIP immunostimulatory effect on neonatal cells opens new possibilities for potential use as pro-type 1 adjuvant for neonatal vaccines. This, in turn, may facilitate the development of more efficient vaccines that can be given at birth, reducing infection associated morbidity and mortality which are the highest in the first weeks after birth.

Crucial role of gamma interferon-producing CD4+ Th1 cells but dispensable function of CD8+ T cell, B cell, Th2, and Th17 responses in the control of Brucella melitensis infection in mice.

Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4(+) T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8(+) T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.

Activation of cord blood myeloid dendritic cells by Trypanosoma cruzi and parasite-specific antibodies, proliferation of CD8+ T cells, and production of IFN-γ.

We previously reported that Trypanosoma cruzi, the agent of Chagas disease, induces in congenitally infected fetuses a strong, adult-like parasite-specific CD8(+) T cell response producing IFN-γ (Hermann et al. in Blood 100:2153-2158, 2002). This suggests that the parasite is able to overcome the immaturity of neonatal antigen presenting cells, an issue which has not been previously addressed. We therefore investigated in vitro the ability of T. cruzi to activate cord blood DCs and compared its effect to that on adult cells. We show that T. cruzi induces phenotypic maturation of cord blood CD11c(+) myeloid DCs (mDCs), by enhancing surface expression of CD40, CD80, and CD83, and that parasite-specific IgG purified from cord blood of neonates born to T. cruzi-infected mothers amplify such expression. CD83, considered as the best marker of mature DCs, reaches higher level on cord blood than on adult mDCs. Allo-stimulation experiments showed that T. cruzi-activated cord blood mononuclear cells enriched in DCs (eDCs) stimulate proliferation of cord blood and adult CD3(+) T cells to a similar extent. Of note, T. cruzi-activated eDCs from cord blood trigger more potent proliferation of CD8(+) than CD8(-) (mainly CD4(+)) adult T cells, a feature not observed with adult eDCs. T cell proliferation is associated with IFN-γ release and down-regulation of IL-13 production. These data show that T. cruzi potently activates human cord blood mDCs and endows eDCs to trigger CD8(+) T cell proliferation and favor type 1 immune response. Interestingly, maternal antibodies can strengthen the development of mature DCs that might contribute to overcome the immunological immaturity associated with early life.

Trypanosoma cruzi activates cord blood myeloid dendritic cells independently of cell infection.

We recently showed that T. cruzi parasites enhance expression of co-stimulatory surface molecules on cord blood myeloid dendritic cells (mDCs). This study aims to gain insight into the role of live parasites and intracellular infection in mDC activation using CSFE-labelled parasites. First, we observed that only a low proportion of mDCs was infected by T. cruzi after overnight culture of whole blood samples and trypomastigotes, as compared with monocytes and granulocytes. Cord blood mDCs were also less infected than their adult counterpart. Second, expression levels of HLA-DR and co-stimulatory molecules CD80, CD83 and CD86 were similar on infected and uninfected mDCs. Parasite lysate also triggered mDCs phenotypic maturation of both cord and adult blood cells, though in a lower extent than live parasites. These results strongly support a central role for extracellular trypomastigotes in activation of mDCs when parasites are incubated with whole blood cells. However, viability of trypomastigotes was not absolutely required for mDC activation.

Maternal fetal transmission of Trypanosoma cruzi: a problem of public health little studied in Mexico.

The first case of neonatal Chagas was reported in Mexico in 1998, but there have been no studies since then. Therefore, we investigated the rates of congenital infection of Trypanosoma cruzi by examining the seroprevalence among 1448 pregnant women in Oaxaca, Jalisco and Mexico City. We performed ELISAs to screen for recombinant and total antigens in mothers, and examined the frequency of congenital T. cruzi transmission by PCR with cord blood and antibody testing in children when they reached two years old. Our results showed that the prevalence of infection in pregnant women was 7.32% (106/1448) overall, and 4.4% (35/794) in Oaxaca, 12.02% (67/557) in Jalisco and 4.12% (4/97) in the Mexico City. In Oaxaca, T. cruzi infection was detected by PCR in 20% (7/35) of infants born to seroreactive mothers and 11.9% (8/67) in Jalisco. No infections were identified in infants from the Mexico City. From these only eleven serological follow up their children are agree to take blood. Therefore, the maternal-fetal overall transmission rate was 4.08% (4/98) in Oaxaca and 9.1% (3/33) in Jalisco 1.5% (1/65) children with positive serology were given specific treatment Chagas. In conclusion, these are the first reports of the rates of congenital Chagas disease in Mexico. The seroprevalence was higher in mothers from Jalisco, and could be related to that there is not the periodic fumigation of the transmitting vector performed in that state. The high rates of maternal-fetal transmission found in Oaxaca could be related to the differences of pathogenicity of trypanosome. No association between both the rate of congenital transmission and the gynecologic anthropometric data was observed.

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

Is Antibody-Dependent Enhancement of Trypanosoma cruzi Infection Contributing to Congenital/Neonatal Chagas Disease?

The newborns of women infected with the parasite Trypanosoma cruzi (the agent of Chagas disease) can be infected either before birth (congenitally), or after birth (as e.g., by vector route). Congenital Chagas disease can induce high levels of neonatal morbidity and mortality. Parasite-infected pregnant women transmit antibodies to their fetus. Antibodies, by opsonizing parasites, can promote phagocytosis and killing of T. cruzi by cells expressing FcγR, on the mandatory condition that such cells are sufficiently activated in an inflammatory context. Antibody-dependent enhancement (ADE) is a mechanism well described in viral infections, by which antibodies enhance entry of infectious agents into host cells by exploiting the phagocytic FcγR pathway. Previously reported Chagas disease studies highlighted a severe reduction of the maternal-fetal/neonatal inflammatory context in parasite-transmitting pregnant women and their congenitally infected newborns. Otherwise, experimental observations brought to light ADE of T. cruzi infection (involving FcγR) in mouse pups displaying maternally transferred antibodies, out of an inflammatory context. Herein, based on such data, we discuss the previously unconsidered possibility of a role of ADE in the trans-placental parasite transmission, and/or the development of severe and mortal clinical forms of congenital/neonatal Chagas disease in newborns of T. cruzi-infected mothers.

Killer cell immunoglobulin-like receptor expression induction on neonatal CD8(+) T cells in vitro and following congenital infection with Trypanosoma cruzi.

Major histocompatibility complex (MHC) class I-specific inhibitory natural killer receptors (iNKRs) are expressed by subsets of T cells but the mechanisms inducing their expression are poorly understood, particularly for killer-cell immunoglobulin-like receptors (KIRs). The iNKRs are virtually absent from the surface of cord blood T cells but we found that KIR expression could be induced upon interleukin-2 stimulation in vitro. In addition, KIR expression was enhanced after treatment with 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation. In vivo induction of KIR expression on cord blood T cells was also observed during a human congenital infection with Trypanosoma cruzi which triggers activation of fetal CD8(+) T cells. These KIR(+) T cells had an effector and effector/memory phenotype suggesting that KIR expression was consecutive to the antigenic stimulation; however, KIR was not preferentially found on parasite-specific CD8(+) T cells secreting interferon-gamma upon in vitro restimulation with live T. cruzi. These findings show that KIR expression is likely regulated by epigenetic mechanisms that occur during the maturation process of cord blood T cells. Our data provide a molecular basis for the appearance of KIRs on T cells with age and they have implications for T-cell homeostasis and the regulation of T-cell-mediated immune responses.

Vertical transmission of Trypanosoma cruzi in the Province of Choapa, IV Region, Chile: Preliminary Report (2005-2008).

Congenital Chagas disease acquired special importance in Chile after the certification of the control of Triatoma infestans and transmission by blood donors affected with Trypanosoma cruzi. In order to establish adequate protocols for intervention and control in infected mother-neonate pairs in endemic zones of Chagas disease, we present partial results (2005-2008) of a pilot project which is being carried out in the Province of Choapa, IV Region, Chile, whose objectives are: determine the current prevalence of the disease in pregnant women, estimate the incidence of vertical transmission of T. cruzi to newborns, determine the lineages of the parasite present in mothers who do and do not transmit the disease, determine the prevalence of Chagas disease in maternal grandmothers of neonates and study placental histopathology. Preliminary results indicated that in this study period, 3.7% of the women who gave birth in the Province have Chagas disease and 2.5% of their newborns were infected. The most frequent T. cruzi genotypes found in mothers studied during pregnancy were TCI and TCIId, either alone or in mixed infections. A high percentage (74.3%) of the grandmothers studied was infected with the parasite. In 29 placentas from mothers with Chagas disease we observed edema, necrosis, fibrinoid deposits and slight lymphoplasmocyte infiltration. In three placentas we found erythroblastosis and in one of them amastigote forms of T. cruzi; this was one of the cases of congenital infection. The evaluation of the diagnostic and control protocols generated will allow us to determine if it has been possible to modify the natural history of vertical transmission of T. cruzi in Chile.

Comparison of methodologies for detecting Trypanosoma cruzi parasites by microscopic observation of microhematocrit capillary tubes.

INTRODUCTION: The microscopic examination of microhematocrit tubes (mHCT) has been proposed as the gold standard for acute and congenital Chagas disease diagnosis. We compared different mHCT methodologies detecting T. cruzi parasites in the blood. METHODS: The rotating method, water mount, and immersion oil methods were compared for their suitability, sensitivity, and specificity. RESULTS: The rotating method was easier, faster, and more sensitive than the others with 100% specificity. CONCLUSIONS: The rotating method is feasible for laboratory technicians with standard training in microscopic techniques and is recommended for the diagnosis of acute Chagas disease in primary health care facilities.

Phylogenetic Analysis of Trypanosoma cruzi from Pregnant Women and Newborns from Argentina, Honduras, and Mexico Suggests an Association of Parasite Haplotypes with Congenital Transmission of the Parasite.

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a high genetic variability and has been classified into six discrete typing units (DTUs) named TcI through TcVI. This genetic diversity is believed to be associated with clinical characteristics and outcomes, but evidence supporting such associations has been limited. Herein, we performed a phylogenetic analysis of T. cruzi sequences of the mini-exon intergenic region obtained from a large cohort of pregnant women and newborns from Argentina, Honduras, and Mexico, to assess parasite genetic diversity and possible associations with congenital transmission. Analysis of 105 samples (including five paired samples) from maternal and umbilical cord blood indicated that T. cruzi DTU distribution was similar among pregnant women and newborns from these three countries, with a high frequency of TcII-TcV-TcVI DTUs, including mixed infections with TcI. However, phylogenetic analysis revealed that although the same parasite haplotypes circulated in these three countries, they were present at different frequencies, leading to significant geographic differences. Of importance, a strong association was observed between parasite haplotypes and congenital infection of newborns. Thus, the identification of parasite haplotypes in pregnant women, but not of parasite DTUs, may help predict congenital transmission of T. cruzi.

Chagas disease, France.

Chagas disease (CD) is endemic to Latin America; its prevalence is highest in Bolivia. CD is sometimes seen in the United States and Canada among migrants from Latin America, whereas it is rare in Europe. We report 9 cases of imported CD in France from 2004 to 2006.

Congenital Transmission of Trypanosoma cruzi in Argentina, Honduras, and Mexico: An Observational Prospective Study.

Compared with South America, there is a lack of epidemiologic studies about the risk of congenital transmission of Trypanosoma cruzi in Central America and Mexico. It has been suggested that T. cruzi genotypes might differ by region and that congenital transmission might vary according to the parasite's genotype. Our objective was to compare T. cruzi congenital transmission rates in three countries. We performed an observational prospective study in 2011-2014 enrolling women at delivery in one hospital in Argentina, two hospitals in Honduras, and two hospitals in Mexico. Congenital T. cruzi infection was defined as the presence of one or more of the following criteria: presence of parasites in cord blood (direct parasitological microscopic examination) with positive polymerase chain reaction (PCR) in cord blood, presence of parasites in infant's blood at 4-8 weeks (direct parasitological microscopic examination), and persistence of T. cruzi-specific antibodies at 10 months, as measured by at least two tests. Among 28,145 enrolled women, 347 had at least one antibody rapid test positive in cord blood and a positive enzyme-linked immunosorbent assay in maternal blood. PCR in maternal blood was positive in 73.2% of the cases, and genotyping identified a majority of non-TcI in the three countries. We found no (0.0%; 95% confidence interval [CI]: 0.0, 2.0) confirmed congenital case in Honduras. Congenital transmission was 6.6% (95% CI: 3.1, 12.2) in Argentina and 6.3% (95% CI: 0.8, 20.8) in Mexico. Trypanosoma cruzi non-TcI predominated and risks of congenital transmission were similar in Argentina and Mexico.