Impaired spatial working memory and reduced hippocampal neuronal density in a rat model of neurocysticercosis.

Neurocysticercosis (NCC) is the most common parasitic disease affecting the nervous system and is a leading cause of acquired epilepsy worldwide, as well as cognitive impairment, especially affecting memory. The aim of this study was to evaluate the effect of NCC on spatial working memory and its correlation with hippocampal neuronal density, in a rat model of NCC. This experimental study was conducted on female (n = 60) and male (n = 73) Holtzman rats. NCC was induced by intracranial inoculation of T. solium oncospheres in 14 day-old-rats. Spatial working memory was assessed using the T-maze test at 3, 6, 9, and 12 months post-inoculation, and sensorimotor evaluation was performed at 12 months post-inoculation. Hippocampal neuronal density was evaluated by immunostaining of NeuN-positive cells of the CA1 region. Of the rats inoculated with T. solium oncospheres, 87.2% (82/94) developed NCC. The study showed a significant decline in spatial working memory over a 1-year follow-up period in rats experimentally infected with NCC. Males showed an early decline that started at 3 months, while females demonstrated it at 9 months. Additionally, a decrease in neuronal density was observed in the hippocampus of NCC-infected rats, with a more significant reduction in rats with cysts in the hippocampus than in rats with cysts in other brain areas and control rats. This rat model of NCC provides valuable support for the relationship between neurocysticercosis and spatial working memory deficits. Further investigations are required to determine the mechanisms involved in cognitive impairment and establish the basis for future treatments.

Experimental brain infection with cysticercosis in sheep. / Infección experimental cerebral con cisticercosis en ovejas.

OBJECTIVE.: To explore the feasibility of developing a sheep model of neurocysticercosis (NCC) by intracranial infection with T. solium oncospheres. MATERIALS AND METHODS.: We carried out an experimental infection model of NCC in sheep. Approximately 10 T. solium oncospheres previously cultured for 30 days were inoculated intracranially into ten sheep. The oncospheres, in 0.1 mL of physiological saline, were injected into the parietal lobe through an 18-gauge needle. RESULTS.: After three months, granulomas were found in two sheep. In a third sheep we identified a 5 mm diameter cyst in the right lateral ventricle and histological evaluation confirmed that the cyst corresponded to a T. solium larva. Immunohistochemistry with monoclonal antibodies directed against membrane components and excretory/secretory antigens of the T. solium cyst was also used to confirm the etiology of the found granulomas. One of them showed reactivity to the monoclonal antibodies used, thus confirming that it was a cysticercus. CONCLUSION.: This experiment is the proof of concept that it is possible to infect sheep with cysticercosis by intracranial inoculation.

OBJETIVO: . Explorar la viabilidad de desarrollar un modelo de neurocisticercosis (NCC) de oveja mediante infección intracraneal de oncosferas de T. solium. MATERIALES Y MÉTODOS.: Se realizó un modelo de infección experimental de NCC en ovejas. Se inocularon aproximadamente 10 posoncósferas de T. solium cultivadas previamente por 30 días por vía intracraneal en diez ovejas. Las oncósferas, en 0,1 mL de solución salina fisiológica, se inyectaron en el lóbulo parietal a través de una aguja de calibre 18. RESULTADOS.: Después de tres meses, en dos ovejas se encontraron granulomas y en una tercera identificó un quiste de 5 mm de diámetro en el ventrículo lateral derecho y la evaluación histológica confirmó que el quiste corresponde a una larva de T. solium. También se utilizó inmunohistoquímica con anticuerpos monoclonales dirigidos contra componentes de membrana y antígenos excretorios/secretorios del quiste de T. solium para confirmar la etiología de los granulomas encontrados. Uno de ellos mostro reactividad ante los anticuerpos monoclonales utilizados, confirmando así que se trató de un cisticerco. CONCLUSIÓN.: Este experimento es la prueba de concepto de que es posible infectar ovejas con cisticercosis por inoculación intracraneal.

Infección experimental cerebral con cisticercosis en ovejas / Experimental brain infection with cysticercosis in sheep

RESUMEN Objetivo . Explorar la viabilidad de desarrollar un modelo de neurocisticercosis (NCC) de oveja mediante infección intracraneal de oncosferas de T. solium. Materiales y métodos. Se realizó un modelo de infección experimental de NCC en ovejas. Se inocularon aproximadamente 10 posoncósferas de T. solium cultivadas previamente por 30 días por vía intracraneal en diez ovejas. Las oncósferas, en 0,1 mL de solución salina fisiológica, se inyectaron en el lóbulo parietal a través de una aguja de calibre 18. Resultados. Después de tres meses, en dos ovejas se encontraron granulomas y en una tercera identificó un quiste de 5 mm de diámetro en el ventrículo lateral derecho y la evaluación histológica confirmó que el quiste corresponde a una larva de T. solium. También se utilizó inmunohistoquímica con anticuerpos monoclonales dirigidos contra componentes de membrana y antígenos excretorios/secretorios del quiste de T. solium para confirmar la etiología de los granulomas encontrados. Uno de ellos mostro reactividad ante los anticuerpos monoclonales utilizados, confirmando así que se trató de un cisticerco. Conclusión. Este experimento es la prueba de concepto de que es posible infectar ovejas con cisticercosis por inoculación intracraneal.

ABSTRACT Objective. To explore the feasibility of developing a sheep model of neurocysticercosis (NCC) by intracranial infection with T. solium oncospheres. Materials and methods. We carried out an experimental infection model of NCC in sheep. Approximately 10 T. solium oncospheres previously cultured for 30 days were inoculated intracranially into ten sheep. The oncospheres, in 0.1 mL of physiological saline, were injected into the parietal lobe through an 18-gauge needle. Results. After three months, granulomas were found in two sheep. In a third sheep we identified a 5 mm diameter cyst in the right lateral ventricle and histological evaluation confirmed that the cyst corresponded to a T. solium larva. Immunohistochemistry with monoclonal antibodies directed against membrane components and excretory/secretory antigens of the T. solium cyst was also used to confirm the etiology of the found granulomas. One of them showed reactivity to the monoclonal antibodies used, thus confirming that it was a cysticercus. Conclusion. This experiment is the proof of concept that it is possible to infect sheep with cysticercosis by intracranial inoculation.

Porcine model of neurocysticercosis by intracarotid injection of Taenia solium oncospheres: Dose assessment, infection outcomes and serological responses.

BACKGROUND: Neurocysticercosis (NCC) is the infection of the human central nervous system (CNS) by Taenia solium larvae that cause significant neurological morbidity. Studies on NCC pathophysiology, host-parasite interactions or therapeutic agents are limited by the lack of suitable animal models. We have previously reported that carotid injection of activated T. solium oncospheres directs parasites into the CNS and consistently reproduces NCC. This study assessed the minimal dose required to consistently obtain NCC by intracarotid oncosphere injection and compared antigen and antibody response profiles by dose-group. METHODS/PRINCIPAL FINDINGS: Three groups of pigs were infected with either 2500 (n = 10), 5000 (n = 11), or 10000 (n = 10) oncospheres. Two pigs died during the study. Necropsy exam at day 150 post-infection (PI) demonstrated viable NCC in 21/29 pigs (72.4%), with higher NCC rates with increasing oncosphere doses (4/9 [44.4%], 9/11 [81.8%] and 8/9 [88.9%] for 2500, 5000, and 10000 oncospheres respectively, P for trend = 0.035). CNS cyst burden was also higher in pigs with increasing doses (P for trend = 0.008). Viable and degenerated muscle cysticerci were also found in all pigs, with degenerated cysticerci more frequent in the 2500 oncosphere dose-group. All pigs were positive for circulating parasite antigens on ELISA (Ag-ELISA) from day 14 PI; circulating antigens markedly increased at day 30 PI and remained high with plateau levels in pigs infected with either 5000 or 10000 oncospheres, but not in pigs infected with 2500 oncospheres. Specific antibodies appeared at day 30 PI and were not different between dose-groups. CONCLUSION/SIGNIFICANCE: Intracarotid injection of 5000 or more oncospheres produces high NCC rates in pigs with CNS cyst burdens like those usually found in human NCC, making this model appropriate for studies on the pathogenesis of NCC and the effects of antiparasitic treatment.

Proliferative cells in racemose neurocysticercosis have an active MAPK signalling pathway and respond to metformin treatment.

Racemose neurocysticercosis is an aggressive infection caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord, resulting in the displacement of the surrounding host tissue and chronic inflammation. We previously demonstrated that the continued growth of the racemose bladder wall is associated with the presence of mitotically active cells but the nature and control of these proliferative cells are not well understood. Here, we demonstrated by immunofluorescence that the racemose cyst has an active mitogen-activated protein kinases (MAPK) signalling pathway that is inhibited after treatment with metformin, which reduces racemose cell proliferation in vitro, and reduces parasite growth in the murine model of Taenia crassiceps cysticercosis. Our findings indicate the importance of insulin receptor-mediated activation of the MAPK signalling pathway in the proliferation and growth of the bladder wall of the racemose cyst and its susceptibility to metformin action. The antiproliferative action of metformin may provide a new therapeutic approach against racemose neurocysticercosis.

IgG Subclasses and Congenital Transmission of Chagas Disease.

The mechanism of vertical transmission of Trypanosoma cruzi is poorly understood. In this study, we evaluated the role of IgG subclasses in the congenital transmission of Chagas disease. We conducted a case-control study in a public maternity hospital in Santa Cruz, Bolivia, enrolling women at delivery. Thirty women who transmitted T. cruzi to their newborns (cases), and 51 women who did not (controls) were randomly selected from 676 total seropositive women. Trypanosoma cruzi-specific IgG1, IgG2, and IgG3 levels were measured by in-house ELISA. The IgG4 levels were unmeasurable as a result of low levels in all participants. Quantitative polymerase chain reaction results and demographic factors were also analyzed. One-unit increases in normalized absorbance ratio of IgG1 or IgG2 levels increased the odds of congenital T. cruzi transmission in Chagas-seropositive women by 2.0 (95% CI: 1.1-3.6) and 2.27 (95% CI: 0.9-5.7), adjusted for age and previous blood transfusion. Odds of congenital transmission were 7.0 times higher in parasitemic mothers (95% CI: 2.3-21.3, P < 0.01) compared with nonparasitemic mothers. We observed that all mothers with IgG1 ≥ 4 were transmitters (sensitivity = 20%, specificity = 100%). Additionally, no mothers with IgG2 < 1.13 were transmitters (sensitivity = 100%, specificity = 21.6%). We demonstrated that IgG subclasses and parasite presence in blood are associated with vertical transmission of T. cruzi and could identify women at increased risk for congenital transmission by measuring IgG subclasses. These measures have potential as objective screening tests to predict the congenital transmission of Chagas.

Changes in inflammatory gene expression in brain tissue adjacent and distant to a viable cyst in a rat model for neurocysticercosis.

BACKGROUND: The parasite Taenia solium causes neurocysticercosis (NCC) in humans and is a common cause of adult-onset epilepsy in the developing world. Hippocampal atrophy, which occurs far from the cyst, is an emerging new complication of NCC. Evaluation of molecular pathways in brain regions close to and distant from the cyst could offer insight into this pathology. METHODS: Rats were inoculated intracranially with T. solium oncospheres. After 4 months, RNA was extracted from brain tissue samples in rats with NCC and uninfected controls, and cDNA was generated. Expression of 38 genes related to different molecular pathways involved in the inflammatory response and healing was assessed by RT-PCR array. RESULTS: Inflammatory cytokines IFN-γ, TNF-α, and IL-1, together with TGF-ß and ARG-1, were overexpressed in tissue close to the parasite compared to non-infected tissue. Genes for IL-1A, CSF-1, FN-1, COL-3A1, and MMP-2 were overexpressed in contralateral tissue compared to non-infected tissue. CONCLUSIONS: The viable cysticerci in the rat model for NCC is characterized by increased expression of genes associated with a proinflammatory response and fibrosis-related proteins, which may mediate the chronic state of infection. These pathways appear to influence regions far from the cyst, which may explain the emerging association between NCC and hippocampal atrophy.

Identification and culture of proliferative cells in abnormal Taenia solium larvae: Role in the development of racemose neurocysticercosis.

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment.

In vitro model of postoncosphere development, and in vivo infection abilities of Taenia solium and Taenia saginata.

Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1ß, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue.

Trypomastigote Excretory Secretory Antigen Blot Is Associated With Trypanosoma cruzi Load and Detects Congenital T. cruzi Infection in Neonates, Using Anti-Shed Acute Phase Antigen Immunoglobulin M.

Background: Congenital Trypanosoma cruzi infection accounts for an estimated 22% of new cases of Chagas disease in Latin America. However, neonatal diagnosis is challenging, as 9-month follow-up for immunoglobulin G testing is poor, quantitative polymerase chain reaction (qPCR) analysis is not routinely performed, and the micromethod misses ≥40% of congenital infections. Methods: Biorepository samples from new mothers and their infants from Piura, Peru, (an area of nonendemicity), and Santa Cruz, Bolivia (an area of endemicity) were accessed. Infant specimens were assessed using the micromethod, qPCR analysis, and a trypomastigote excretory secretory antigen (TESA) blot for detection of immunoglobulin M (IgM)-specific shed acute phase antigen (SAPA) bands, using qPCR as the gold standard. Results: When compared to qPCR, IgM TESA blot was both sensitive and specific for congenital Chagas disease diagnosis. Cumulative sensitivity (whether only 4 bands or all 6 bands were present) was 80% (95% confidence interval [CI], 59%-92%). Specificity was 94% (95% CI, 92%-96%) in the area of endemicity and 100% in the area of nonendemicity. SAPA bands occurred sequentially and in pairs, and parasite loads correlated highly with the number of SAPA bands present. The micromethod detected infection in fewer than half of infected infants. Conclusions: The IgM TESA blot for detection of SAPA bands is rapid, relatively inexpensive, and more sensitive than the micromethod and may be a useful point-of-care test for detection of congenital T. cruzi infection.

Axonal swellings and spheroids: a new insight into the pathology of neurocysticercosis.

Neurocysticercosis is a parasitic brain disease caused by the larval form (Cysticercus cellulosae) of Taenia solium and is the leading cause of preventable epilepsy worldwide. However, the pathophysiology and relation to the wide range of clinical features remains poorly understood. Axonal swelling is emerging as an important early pathological finding in multiple neurodegenerative diseases and as a cause of brain injury, but has not been well described in neurocysticercosis. Histological analysis was performed on human, rat and porcine NCC brain specimens to identify axonal pathology. Rat infection was successfully carried out via two routes of inoculation: direct intracranial injection and oral feeding. Extensive axonal swellings, in the form of spheroids, were observed in both humans and rats and to a lesser extent in pigs with NCC. Spheroids demonstrated increased immunoreactivity to amyloid precursor protein and neurofilament indicating probable impairment of axonal transport. These novel findings demonstrate that spheroids are present in NCC which is conserved across species. Not only is this an important contribution toward understanding the pathogenesis of NCC, but it also provides a model to analyze the association of spheroids with specific clinical features and to investigate the reversibility of spheroid formation with antihelminthic treatment.

Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

BACKGROUND: Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody) and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA. CONCLUSIONS: Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

Novel rat model for neurocysticercosis using Taenia solium.

Neurocysticercosis is caused by Taenia solium infecting the central nervous system and is the leading cause of acquired epilepsy and convulsive conditions worldwide. Research into the pathophysiology of the disease and appropriate treatment is hindered by lack of cost-effective and physiologically similar animal models. We generated a novel rat neurocysticercosis model using intracranial infection with activated T. solium oncospheres. Holtzman rats were infected in two separate groups: the first group was inoculated extraparenchymally and the second intraparenchymally, with different doses of activated oncospheres. The groups were evaluated at three different ages. Histologic examination of the tissue surrounding T. solium cysticerci was performed. Results indicate that generally infected rats developed cysticerci in the brain tissue after 4 months, and the cysticerci were observed in the parenchymal, ventricle, or submeningeal brain tissue. The route of infection did not have a statistically significant effect on the proportion of rats that developed cysticerci, and there was no dependence on infection dose. However, rat age was crucial to the success of the infection. Epilepsy was observed in 9% of rats with neurocysticercosis. In histologic examination, a layer of collagen tissue, inflammatory infiltrate cells, perivascular infiltrate, angiogenesis, spongy change, and mass effect were observed in the tissue surrounding the cysts. This study presents a suitable animal model for the study of human neurocysticercosis.

Taenia solium infection in Peru: a collaboration between Peace Corps Volunteers and researchers in a community based study.

BACKGROUND: Neurocysticercosis is a leading cause of seizures and epilepsy in most of the world, and it occurs when Taenia solium larval cysts infect the central nervous system. T. solium tapeworm infection is endemic in much of Peru, but there are scarce data on the prevalence in many rural highland communities where it is likely to be hyper-endemic. Peace Corps Volunteers live and work in these communities; however, to our knowledge, they have not been used to facilitate public health research. MATERIALS AND METHODS: We utilized Peace Corps Volunteers to estimate the prevalence of T. solium tapeworm infection in seven rural communities in northern Peru. A convenience non-random sampling frame was used. Peace Corps Volunteers facilitated the collection of stool samples (N = 2,328), which were analyzed by sedimentation and microscopy. Niclosamide treatment and purgation preceded species identification, which was done by PCR-REA. RESULTS: Taenia sp. egg-positive stool samples were found in three of the seven communities we surveyed. The overall prevalence of Taenia sp. egg positivity was 2.1% (49/2,328) (95% CI = 1.6-2.8%) with prevalence up to 4.3% (42/977) (95% CI = 3.1-5.8%) by community. All 34 of the specimens tested by PCR-REA were T. solium. The overall prevalence of T. solium tapeworm infection was 1.5% (34/2,328) (95% CI = 1.0-2.0%). Prevalence up to 2.9% (28/977) (95% CI = 1.9-4.1%) by community was observed. CONCLUSION/SIGNIFICANCE: This study recorded high T. solium tapeworm prevalence, and identified hyper-endemic rural communities. It demonstrates that synergy between researchers and Peace Corps Volunteers can be an effective means to conducting large-scale, community-based studies in remote areas of Peru.

Relationship between serum antibodies and Taenia solium larvae burden in pigs raised in field conditions.

BACKGROUND: Serological tests have been used for the diagnosis of Taenia solium infection in pigs. However, those serological results do not necessarily correlate with the actual infection burden after performing pig necropsy. This study aimed to evaluate the Electro Immuno Transfer Blot (EITB) seropositivity with infection burden in naturally infected pigs. METHODOLOGY/PRINCIPAL FINDINGS: In an endemic area of Peru, 476 pigs were sampled. Seroprevalence was 60.5 ± 4.5% with a statistically higher proportion of positive older pigs (>8 months) than young pigs. The logistic model showed that pigs >8 month of age were 2.5 times more likely to be EITB-positive than ≤ 8 months. A subset of 84 seropositive pigs were necropsied, with 45.2% (38/84) positive to 1-2 bands, 46.4% (39/84) to 3 bands, and 8.3% (7/84) to 4+ bands. 41 out of 84 positive pigs were negative to necropsy (48.8%) and 43 (51%) had one or more cysts (positive predictive value). Older pigs showed more moderate and heavy infection burdens compared to younger pigs. In general, regardless of the age of the pig, the probability of having more cysts (parasite burden) increases proportionally with the number of EITB bands. CONCLUSIONS/SIGNIFICANCE: The probability of being necropsy-positive increased with the number of bands, and age. Therefore, the EITB is a measure of exposure rather than a test to determine the real prevalence of cysticercosis infection.

A field trial of alternative targeted screening strategies for Chagas disease in Arequipa, Peru.

BACKGROUND: Chagas disease is endemic in the rural areas of southern Peru and a growing urban problem in the regional capital of Arequipa, population ∼860,000. It is unclear how to implement cost-effective screening programs across a large urban and periurban environment. METHODS: We compared four alternative screening strategies in 18 periurban communities, testing individuals in houses with 1) infected vectors; 2) high vector densities; 3) low vector densities; and 4) no vectors. Vector data were obtained from routine Ministry of Health insecticide application campaigns. We performed ring case detection (radius of 15 m) around seropositive individuals, and collected data on costs of implementation for each strategy. RESULTS: Infection was detected in 21 of 923 (2.28%) participants. Cases had lived more time on average in rural places than non-cases (7.20 years versus 3.31 years, respectively). Significant risk factors on univariate logistic regression for infection were age (OR 1.02; p = 0.041), time lived in a rural location (OR 1.04; p = 0.022), and time lived in an infested area (OR 1.04; p = 0.008). No multivariate model with these variables fit the data better than a simple model including only the time lived in an area with triatomine bugs. There was no significant difference in prevalence across the screening strategies; however a self-assessment of disease risk may have biased participation, inflating prevalence among residents of houses where no infestation was detected. Testing houses with infected-vectors was least expensive. Ring case detection yielded four secondary cases in only one community, possibly due to vector-borne transmission in this community, apparently absent in the others. CONCLUSIONS: Targeted screening for urban Chagas disease is promising in areas with ongoing vector-borne transmission; however, these pockets of epidemic transmission remain difficult to detect a priori. The flexibility to adapt to the epidemiology that emerges during screening is key to an efficient case detection intervention. In heterogeneous urban environments, self-assessments of risk and simple residence history questionnaires may be useful to identify those at highest risk for Chagas disease to guide diagnostic efforts.

Use of Individual-level Covariates to Improve Latent Class Analysis of Trypanosoma Cruzi Diagnostic Tests.

Statistical methods such as latent class analysis can estimate the sensitivity and specificity of diagnostic tests when no perfect reference test exists. Traditional latent class methods assume a constant disease prevalence in one or more tested populations. When the risk of disease varies in a known way, these models fail to take advantage of additional information that can be obtained by measuring risk factors at the level of the individual. We show that by incorporating complex field-based epidemiologic data, in which the disease prevalence varies as a continuous function of individual-level covariates, our model produces more accurate sensitivity and specificity estimates than previous methods. We apply this technique to a simulated population and to actual Chagas disease test data from a community near Arequipa, Peru. Results from our model estimate that the first-line enzyme-linked immunosorbent assay has a sensitivity of 78% (95% CI: 62-100%) and a specificity of 100% (95% CI: 99-100%). The confirmatory immunofluorescence assay is estimated to be 73% sensitive (95% CI: 65-81%) and 99% specific (95% CI: 96-100%).