Pupillary Light Reflexes are Associated with Autonomic Dysfunction in Bolivian Diabetics But Not Chagas Disease Patients

Autonomic dysfunction is common in Chagas disease and diabetes. Patients with either condition complicated by cardiac autonomic dysfunction face increased mortality, but no clinical predictors of autonomic dysfunction exist. Pupillary light reflexes (PLRs) may identify such patients early, allowing for intensified treatment. To evaluate the significance of PLRs, adults were recruited from the outpatient endocrine, cardiology, and surgical clinics at a Bolivian teaching hospital. After testing for Chagas disease and diabetes, participants completed conventional autonomic testing (CAT) evaluating their cardiovascular responses to Valsalva, deep breathing, and orthostatic changes. PLRs were measured using specially designed goggles, then CAT and PLRs were compared as measures of autonomic dysfunction. This study analyzed 163 adults, including 96 with Chagas disease, 35 patients with diabetes, and 32 controls. PLRs were not significantly different between Chagas disease patients and controls. Patients with diabetes had longer latency to onset of pupil constriction, slower maximum constriction velocities, and smaller orthostatic ratios than nonpatients with diabetes. PLRs correlated poorly with CAT results. A PLR-based clinical risk score demonstrated a 2.27-fold increased likelihood of diabetes complicated by autonomic dysfunction compared with the combination of blood tests, CAT, and PLRs (sensitivity 87.9%, specificity 61.3%). PLRs represent a promising tool for evaluating subclinical neuropathy in patients with diabetes without symptomatic autonomic dysfunction. Pupillometry does not have a role in the evaluation of Chagas disease patients

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

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

Sustained Domestic Vector Exposure Is Associated With Increased Chagas Cardiomyopathy Risk but Decreased Parasitemia and Congenital Transmission Risk Among Young Women in Bolivia

Vector exposure showed a direct association with Chagas cardiomyopathy, but an inverse relationship with maternal parasitemia and congenital transmission. We hypothesize that repeated antigen exposure maintains an inflammatory response, increasing cardiomyopathy, but this upregulation improves control of parasitemia during pregnanc

Chagas Cardiomyopathy in the Context of the Chronic Disease Transition

Latin America is undergoing a transition from disease patterns characteristic of developing countries with high rates of infectious disease and premature deaths to a pattern more like industrialized countries, in which chronic conditions such as obesity, hypertension and diabetes are more common. Many rural residents with Chagas disease have now migrated to cities, taken on new habits and may suffer from both types of disease. We studied heart disease among 394 adults seen by cardiologists in a public hospital in the city of Santa Cruz, Bolivia; 64% were infected with T. cruzi, the parasite that causes Chagas disease. Both T. cruzi infected and uninfected patients had a high rate of hypertension (64%) and overweight (67%), with no difference by infection status. Nearly 60% of symptomatic congestive heart failure was due to Chagas disease; mortality was also higher for infected than uninfected patients. Males and older patients had more severe Chagas heart disease. Chagas heart disease remains an important cause of congestive heart failure in this hospital population, but often occurs in patients who also have obesity, hypertension and/or other cardiac risk factors

ENDOTHELIAL, IMMUNOLOGICAL AND STRUCTURAL ASSESSMENT OF PLACENTAL SPECIMENS FROM T. cruzi VERTICAL TRANSMISSION CASES IN SANTA CRUZ DE LA SIERRA – BOLIVIA

The role of the placenta in the vertical transmission of T. cruzi, as well as the risk factors that contribute to vertical transmission, are not well defined.. Few studies have evaluated the use of the placenta for diagnosis of Chagas disease in newborns. Accurate identification of infected newborns is a high priority in control programs since the cure rates are high. Using immunohistochemistry, we evaluated endothelial and structural impairment of placental cells, as well as features of the immune response that may be contributors in congenital Chagas disease. Immunohistochemistry markers of disturbed placental barrier were standardized and matched to obstetrical and neonatal data. No differences were found in placental measurements of neonatal and obstetrical characteristics in cases of vertical transmission. Conventional testing, achieved diagnosis after 6 months of age. In contrast, TESA-blot and quantitative polymerase-chain reaction (q-PCR) (on cord blood or umbilical tissue) identified the majority of cases at birth. Successful immunohistochemistry techniques were identified for further evaluation. We also assessed placental barrier impairment linked to endothelial cells and dysfunctional endothelial tight junction’ proteins using markers of angiogenesis. Expression of endothelial tight junction proteins was increased at fetal blood vessels and decreased at syncytiotrophoblast. Markers of angiogenesis showed decreased expression at the placental barrier. Dysregulated angiogenesis and impaired cell-to-cell contact at the syncytiotrophoblast layer are related to placental barrier impairment and may be associated with congenital infection. The expression of structural proteins was increased in fetal blood vessels and the syncytiotrophoblast, while the expression of tight junction proteins was decreased in the cytotrophoblast. Expression of mediators of innate immune response was increased in the syncytiotrophoblast and fetal blood vessels. Expression of markers of macrophage activation was decreased in the syncytiotrophoblast. Increased expression of structural proteins at the syncytiotrophoblast is related to impaired placental barrier and may be associated with T. cruzi vertical transmission. In conclusion, molecular tests for diagnosis of congenital Chagas Disease should be implemented in poor resource settings. Signs of impaired placental barrier and immune response are documented in this study. Future research identifying placental biomarkers at maternal serum would be very useful for perinatal diagnosis

Use of a Novel Chagas Urine Nanoparticle Test (Chunap) for Diagnosis of Congenital Chagas Disease

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

Trypanosoma cruzi-infected pregnant women without vector exposure have higher parasitemia levels: implications for congenital transmission risk.

Congenital transmission is a major source of new Trypanosoma cruzi infections, and as vector and blood bank control continue to improve, the proportion due to congenital infection will grow. A major unanswered question is why reported transmission rates from T. cruzi-infected mothers vary so widely among study populations. Women with high parasite loads during pregnancy are more likely to transmit to their infants, but the factors that govern maternal parasite load are largely unknown. Better understanding of these factors could enable prioritization of screening programs to target women most at risk of transmission to their infants.We screened pregnant women presenting for delivery in a large urban hospital in Bolivia and followed infants of infected women for congenital Chagas disease. Of 596 women screened, 128 (21.5%) had confirmed T. cruzi infection; transmission occurred from 15 (11.7%) infected women to their infants. Parasite loads were significantly higher among women who transmitted compared to those who did not. Congenital transmission occurred from 31.3% (9/29), 15.4% (4/26) and 0% (0/62) of women with high, moderate and low parasite load, respectively (χx2 for trend 18.2; p<0.0001). Twin births were associated with higher transmission risk and higher maternal parasite loads. Infected women without reported vector exposure had significantly higher parasite loads than those who had lived in an infested house (median 26.4 vs 0 parasites/mL; p<0.001) with an inverse relationship between years of living in an infested house and parasite load.We hypothesize that sustained vector-borne parasite exposure and repeated superinfection by T. cruzi may act as an immune booster, allowing women to maintain effective control of the parasite despite the down-regulation of late pregnancy

Levels of parasitemia in infants congenitally infected with <i>T. cruzi</i>.

<p>PCR targeting <i>T. cruzi</i> kinetoplast DPT was done from blood from umbilical cord at birth or blood samples. Number of individuals per group of age: 0 months: n = 9; 1 month: n = 10; 6 months: n = 6; 9 months: n = 4.</p

Sequestration and concentration of <i>T. cruzi</i> antigens by bait functionalized poly(NIPAm) particles.

<p>Silver stain analysis: A. H49 antigen (50 ng) was spiked in 250 µl of normal urine samples, and incubated with different NIPAm particles for 15 min at room temperature. Particles were separated by centrifugation, and a SDS-PAGE analysis was performed of the particles (containing bound proteins) and the supernatant (20 µl, containing unbound proteins). NIPAm based particles functionalized with different affinity baits successfully captured and concentrated <i>T. cruzi</i> antigens (H49 recombinant protein). Poly(NIPAm/trypan blue) (TB) particles completely captured H49 antigen and deplete the supernatant. IS: Initial Solution. S: Supernatant (unbound proteins). P: Particles (containing bound proteins). B. 250 µl of H49 antigen (20 ng) was incubated with poly (NIPAm/trypan blue) particles. After incubation and centrifugation the supernatant (S, unbound proteins) was saved and particles were washed with 250 µl of miliQ water. After centrifugation, the wash solution (W) was saved and the elution of antigens from particles was performed using acetonitrile-based elution buffer (E). A complete elution of H49 antigen from poly(NIPAm/TB) particles was obtained, and <i>T. cruzi</i> antigens were not lost during the washing step. E: Elute, W: washing solution (20 µl), P: Particle content after elution indicating not presence of H49 antigen, S: supernatant (20 µl, unbound proteins), IS: Initial Solution (20 µl, corresponds to 1.6 ng). C. Poly(NIPAm/TB) particles completely capture different types of <i>T. cruzi</i> antigens. TESA: Trypomastigote excretory-secretory antigen and 1F8: recombinant antigen 1F8. E: Elute from particles. S: Supernatant (unbound proteins). Western Blot analysis using a mouse monoclonal antibody to LPG of <i>T. cruzi</i>: D. Concentration of trypomastigote lysate antigen (TLA) in urine using poly(NIPAm/TB) particles. Poly(NIPAm/TB) particles capture and concentrate TLA antigen in the presence of excess competing proteins in urine. The limit of detection of TLA antigen by Western Blot substantially improves when urine samples were treated with particles, from 50 ng/ml without particle treatment to 0.5 ng/ml with particle treatment.</p