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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.

Chagas disease affects 8-11 million people throughout the Americas. Early detection is crucial for timely treatment and to prevent non-vectorial transmission. Recombinant antigen-based rapid tests had high sensitivity and specificity in laboratory evaluations, but no Peruvian specimens were included in previous studies. We evaluated Stat-Pak and Trypanosoma Detect rapid tests in specimens from Bolivia and Peru. Specimens positive by three conventional assays were confirmed positives; specimens negative by two or more assays were confirmed negatives. In Bolivian specimens, Stat-Pak and Trypanosoma Detect tests were 87.5% and 90.7% sensitive, respectively; both showed 100% specificity. Sensitivity in Peruvian specimens was much lower: 26.6-33.0% (Stat-Pak) and 54.3-55.2% (Trypanosoma Detect); both had specificities > 98%. Even in Bolivian specimens, these sensitivities are inadequate for stand-alone screening. The low sensitivity in Peru may be related to parasite strain differences. Chagas disease rapid tests should be field tested in each geographic site before widespread implementation for screening. Copyright

BackgroundCongenital 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.Methodology/principal findingsWe 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; pConclusions/significanceWe 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.

Trypanosoma cruzicauses Chagas disease, which affects an estimated 7 million to 8 million people. Chagas disease is endemic throughout Latin America, with the highest prevalence in Bolivia. Conventional diagnosis requires a well-equipped laboratory with experienced personnel. We evaluated the Chagas Detect Plus (CDP) (InBios, Seattle, WA), a rapid immunochromatographic assay for IgG antibodies toT. cruzi. CDP performance was compared to infection status based on results obtained by indirect hemagglutination assay, immunofluorescent-antibody test, and enzyme-linked immunosorbent assay. Confirmed infection required positive results by at least 2 conventional assays. We used specimens from adults of both sexes in a general hospital in the city of Santa Cruz and from pregnant women in a hospital and children in villages in the Bolivian Chaco, an area of hyperendemicity. CDP was performed in paired whole-blood and serum specimens from 385 individuals in the two hospital studies and in 200 serum specimens from the community study. CDP showed sensitivities/specificities of 96.2% (95% confidence interval, 92.7 to 98.4)/98.8% (95.9 to 99.9) in whole blood and 99.3% (97.5 to 99.9)/96.9% (94.2 to 98.6) in serum, with no differences by sex, age group, or study site. CDP showed excellent sensitivity and specificity in our study population, comparable to those of conventional serology. The test is reliable for field surveys, requires no laboratory equipment, and performed well in serum and whole blood. The CDP could also be used for accurate maternal screening to identify neonates at risk of congenital transmission. CDP performance data in diverse geographic areas are needed to strengthen the evidence base for its use.

Background Detection of congenital T. cruzi 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 T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease. Methodology/Principal Findings 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 T. cruzi 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 T. cruzi lipophosphoglycan. The nanoparticles concentrate T. cruzi 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 T. cruzi antigens were protected from trypsin digestion. Conclusion/Significance Chunap has the potential to be developed into a simple and sensitive test for the early diagnosis of congenital Chagas disease., Author Summary Congenital Chagas disease is one of the main pillars for the control of Chagas disease because 25% of new infections occur by this route. Conventional diagnosis of congenital Chagas disease is based on microscopy at birth and serology at 9 months. However microscopy misses many infections and many at-risk infants fail to complete serology at six to nine months. We have developed a Chagas urine nanoparticle test (Chunap) for concentration and detection of T. cruzi antigens. Chunap was evaluated in urine samples of 1-month old children. At this age children have the highest levels of parasitemia and therefore also excrete the highest levels of antigen. Parents prefer a urine test to having their baby's blood drawn. Chunap diagnosed congenital infection in a single urine sample as well as PCR in two blood samples. This study also shows that hydrogel/trypan blue particles used in our test efficiently capture, concentrate and protect urinary T. cruzi antigens from enzymatic degradation. Chunap allows for the early diagnosis of congenital Chagas disease, and with appropriate adaptation, may allow early point-of-care intervention.

Trypanosoma cruzi polymerase chain reaction (PCR) is widely used, but sensitivity varies widely. We compared PCR using 121/122 primers targeting kinetoplast minicircle DNA in whole blood, buffy coat, and clot from Bolivian women. Sensitivity was significantly higher in clot (60.1%) than buffy coat (46.5%) or whole blood (40%). The use of clot could simplify specimen collection while improving sensitivity.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major cause of morbidity and mortality in Central and South America. Geographic variations in the sensitivity of serologic diagnostic assays to T. cruzi may reflect differences in T. cruzi exposure. We measured parasite-specific T-cell responses among seropositive individuals in two populations from South America with widely varying antibody titers against T. cruzi. Antibody titers among seropositive individuals were significantly lower in Arequipa, Peru compared with Santa Cruz, Bolivia. Similarly, the proportion of seropositive individuals with positive T-cell responses was lower in Peru than Bolivia, resulting in overall lower frequencies of interferon-gamma (IFNgamma)-secreting cells from Peruvian samples. However, the magnitude of the IFNgamma response was similar among the IFNgamma responders in both locations. These data indicate that immunological discrepancies based on geographic region are reflected in T-cell responses as well as antibody responses.

Background Chagas disease control campaigns relying upon residual insecticide spraying have been successful in many Southern American countries. However, in some areas, rapid reinfestation and recrudescence of transmission have occurred. Methodology/Principal Findings We conducted a cross-sectional survey in the Bolivian Chaco to evaluate prevalence of and risk factors for T. cruzi infection 11 years after two rounds of blanket insecticide application. We used a cubic B-spline model to estimate change in force of infection over time based on age-specific seroprevalence data. Overall T. cruzi seroprevalence was 51.7%. The prevalence was 19.8% among children 2–15, 72.7% among those 15–30 and 97.1% among participants older than 30 years. Based on the model, the estimated annual force of infection was 4.3% over the two years before the first blanket spray in 2000 and fell to 0.4% for 2001–2002. The estimated annual force of infection for 2004–2005, the 2 year period following the second blanket spray, was 4.6%. However, the 95% bootstrap confidence intervals overlap for all of these estimates. In a multivariable model, only sleeping in a structure with cracks in the walls (aOR = 2.35; 95% CI = 1.15–4.78), age and village of residence were associated with infection. Conclusions/Significance As in other areas in the Chaco, we found an extremely high prevalence of Chagas disease. Despite evidence that blanket insecticide application in 2000 may have decreased the force of infection, active transmission is ongoing. Continued spraying vigilance, infestation surveillance, and systematic household improvements are necessary to disrupt and sustain interruption of infection transmission., Author Summary Despite significant gains in the reduction of the burden of Chagas disease in many South American countries, active transmission and significant burden remain in areas such as the Gran Chaco. High initial vector density, poor housing material, peridomestic infestation, insecticide resistance, and a lack of systematic insecticide spraying and vector surveillance have previously been incriminated for failure to interrupt and sustain interruption of transmission. We conducted a census, seroprevalence, and epidemiologic study in a rural area in the Bolivian Chaco. The prevalence of infection was almost 20% in children and over 80% in adults. We estimated the intensity of transmission over time based on infection prevalence by age. We found that after the first spray program, transmission appeared to fall transiently but then increased again quickly. Sleeping in a structure with cracks in the walls, age and village of residence were associated with increased likelihood of infection. These findings suggest that consistently repeated systematic spraying campaigns accompanied by housing improvements are necessary to interrupt and sustain interruption of vector-borne transmission.

Background Patients with Chagas disease have migrated to cities, where obesity, hypertension and other cardiac risk factors are common. Methodology/Principal Findings The study included adult patients evaluated by the cardiology service in a public hospital in Santa Cruz, Bolivia. Data included risk factors for T. cruzi infection, medical history, physical examination, electrocardiogram, echocardiogram, and contact 9 months after initial data collection to ascertain mortality. Serology and PCR for Trypanosoma cruzi were performed. Of 394 participants, 251 (64%) had confirmed T. cruzi infection by serology. Among seropositive participants, 109 (43%) had positive results by conventional PCR; of these, 89 (82%) also had positive results by real time PCR. There was a high prevalence of hypertension (64%) and overweight (body mass index [BMI] >25; 67%), with no difference by T. cruzi infection status. Nearly 60% of symptomatic congestive heart failure was attributed to Chagas cardiomyopathy; mortality was also higher for seropositive than seronegative patients (p = 0.05). In multivariable models, longer residence in an endemic province, residence in a rural area and poor housing conditions were associated with T. cruzi infection. Male sex, increasing age and poor housing were independent predictors of Chagas cardiomyopathy severity. Males and participants with BMI ≤25 had significantly higher likelihood of positive PCR results compared to females or overweight participants. Conclusions Chagas cardiomyopathy remains an important cause of congestive heart failure in this hospital population, and should be evaluated in the context of the epidemiological transition that has increased risk of obesity, hypertension and chronic cardiovascular disease., Author Summary 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.

Background. We conducted a study of congenital Trypanosoma cruzi infection in Santa Cruz, Bolivia. Our objective was to apply new tools to identify weak points in current screening algorithms, and find ways to improve them. Methods. Women presenting for delivery were screened by rapid and conventional serological tests. For infants of infected mothers, blood specimens obtained on days 0, 7, 21, 30, 90, 180, and 270 were concentrated and examined microscopically; serological tests were performed for the day 90, 180, and 270 specimens. Maternal and infant specimens, including umbilical tissue, were tested by polymerase chain reaction (PCR) targeting the kinetoplast minicircle and by quantitative PCR. Results. Of 530 women, 154 (29%) were seropositive. Ten infants had congenital T. cruzi infection. Only 4 infants had positive results of microscopy evaluation in the first month, and none had positive cord blood microscopy results. PCR results were positive for 6 (67%) of 9 cord blood and 7 (87.5%) of 8 umbilical tissue specimens. PCR-positive women were more likely to transmit T. cruzi than were seropositive women with negative PCR results ( ). Parasite loads determined by quantitative PCR were higher for mothers of infected infants P ! .05 than for seropositive mothers of uninfected infants ( ). Despite intensive efforts, only 58% of at-risk infants P ! .01 had a month 9 specimen collected. Conclusions. On the basis of the low sensitivity of microscopy in cord blood and high rate of loss to followup, we estimate that current screening programs miss one-half of all infected infants. Molecular techniques may improve early detection.

Chagas disease affects 8-11 million people throughout the Americas. Early detection is crucial for timely treatment and to prevent non-vectorial transmission. Recombinant antigen-based rapid tests had high sensitivity and specificity in laboratory evaluations, but no Peruvian specimens were included in previous studies. We evaluated Stat-Pak and Trypanosoma Detect rapid tests in specimens from Bolivia and Peru. Specimens positive by three conventional assays were confirmed positives; specimens negative by two or more assays were confirmed negatives. In Bolivian specimens, Stat-Pak and Trypanosoma Detect tests were 87.5% and 90.7% sensitive, respectively; both showed 100% specificity. Sensitivity in Peruvian specimens was much lower: 26.6-33.0% (Stat-Pak) and 54.3-55.2% (Trypanosoma Detect); both had specificities98%. Even in Bolivian specimens, these sensitivities are inadequate for stand-alone screening. The low sensitivity in Peru may be related to parasite strain differences. Chagas disease rapid tests should be field tested in each geographic site before widespread implementation for screening.

Trypanosoma cruzi polymerase chain reaction (PCR) is widely used, but sensitivity varies widely. We compared PCR using 121/122 primers targeting kinetoplast minicircle DNA in whole blood, buffy coat, and clot from Bolivian women. Sensitivity was significantly higher in clot (60.1%) than buffy coat (46.5%) or whole blood (40%). The use of clot could simplify specimen collection while improving sensitivity.