Performance Assessment of Treponemal and Nontreponemal Tests for the Diagnosis of Acquired Syphilis.

There are a variety of nontreponemal test (NTT) and treponemal test (TT) kits for the serologic diagnosis of syphilis. Because of the complexity of the infection (multiple clinical stages) and the different antigens used in these kits, a systematic evaluation of the accuracy of the currently available commercial tests is warranted. Our objective was to evaluate the performance of commercially available tests for the diagnosis of syphilis infection. In this study, we analyzed one NTT (Venereal Disease Research Laboratory [VDRL] test, Wiener Laboratories, Rosario, Argentina) and two TTs (fluorescent treponemal antibody absorption [FTA-ABS] test, Euroimmun, Lübeck, Germany, and syphilis recombinant ELISA v. 4.0 test [ELISA], Wiener Laboratories, Rosario, Argentina) using a panel of 187 samples, including serum samples from 31 individuals with primary syphilis, 77 with secondary syphilis, and 79 with latent syphilis. An additional 192 samples from uninfected individuals and 323 serum samples from individuals with other diseases were included. The sensitivities of the VDRL, ELISA, and FTA-ABS tests were 97.9%, 100%, and 96.3%, respectively. The VDRL and ELISA tests showed a specificity of 100%, and the FTA-ABS test showed a specificity of 99.5%. Accuracy was 98.9% for the VDRL test, 100% for the ELISA, and 97.9% for the FTA-ABS test. For primary, secondary, and latent syphilis, the ELISA achieved a diagnostic performance of 100%, whereas the sensitivity for the VDRL and FTA-ABS tests ranged from 96.8% to 98.7% and 93.7% to 98.7%, respectively. No difference was observed when the tests were used as traditional or reverse algorithms. In general, all three tests are able to discriminate positive and negative samples for syphilis, regardless of the diagnostic algorithm.

Feline leishmaniasis in an animal shelter in northeastern Brazil: Clinical aspects, coinfections, molecular detection, and serological study of a new recombinant protein.

Infection and clinical cases of leishmaniasis caused by Leishmania infantum in cats have been increasingly reported in several countries, including Brazil. In this study, we used an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) based on a recombinant antigen (rKDDR-plus) to detect anti-Leishmania antibodies in cats from an animal shelter in northeastern Brazil. We compared the results with an ELISA using L. infantum crude antigen (ELISA-CA). We also investigated the presence of Leishmania DNA in blood or ocular conjunctival samples as well as the association between Leishmania PCR positivity and serological positivity to feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and Toxoplasma gondii. Concerning serological assays, a higher positivity was detected using the ICT-rKDDR-plus (7.5%; 7/93) as compared to ELISA-rKDDR-plus (5.4%; 5/93) and ELISA-CA (4.3%; 4/93). Upon PCR testing, 52.7% (49/93) of the ocular conjunctival swabs and 48.3% (44/91) of the blood samples were positive. Together, PCR and serological testing revealed overall positivities of 73.1% (68/93) and 12.9% (12/93), respectively. Among PCR-positive samples, 45.5% (31/68) showed co-infection with FIV, 17.6% (12/68) with FeLV, and 82.3% (56/68) with T. gondii. More than half of the PCR-positive cats showed at least one clinical sign suggestive of leishmaniasis (58.8%; 40/68) and dermatological signs were the most frequent ones (45.5%; 31/68). Both tests employing the recombinant antigen rKDDR-plus (i.e., ICT-rKDDR-plus and ELISA-rKDDR-plus) detected more positive cats than the ELISA-CA but presented low overall accuracy. PCR testing using either blood or ocular conjunctival samples detected much more positive cats than serological tests.

Evaluating the diagnostic accuracy of TpN17 and TmpA recombinant proteins in syphilis detection: a phase II study.

Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen's performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values.

IgG Isotypes Targeting a Recombinant Chimeric Protein of Trypanosoma cruzi in Different Clinical Presentations of Chronic Chagas Disease.

Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, which leads to a spectrum of clinical presentations that range from asymptomatic to severe cardiac involvement. The host immune response plays a pivotal role in disease progression. Ig isotypes may contribute to disease pathogenesis. Investigating these components can provide insights into the immunopathogenic mechanisms underlying CD. This cross-sectional study aims to establish a correlation between the Ig profile of individuals infected with T. cruzi with the clinical forms of chronic CD. Serum samples were collected from partner institutions in different states of Brazil. Individuals diagnosed with chronic CD were categorized based on the clinical form of the disease. The indirect ELISA method using the recombinant chimeric Molecular Biology Institute of Paraná membrane protein 8.4 as the antigen was used to determine the Ig profile, including total IgG, IgG1, IgG2, IgG3, and IgG4. Ninety-seven serum samples from patients classified as negative (NEG, n = 38), indeterminate (IND, n = 24), mild cardiac (MC, n = 20), and severe cardiac (SC, n = 15) forms were analyzed. IgG1 exhibited greater levels compared with the other isotypes, showing a significant difference between the MC and IND groups. IgG3 levels were greater in individuals from the MC group compared with the SC group. IgG1 and IgG3 isotypes can serve as biomarkers to evaluate the progression of CD because they exhibit variations across clinical groups. Additional longitudinal studies are necessary to explore the relationship between antibody kinetics and the development of tissue damage.

Usefulness of receptor binding domain protein-based serodiagnosis of COVID-19.

Objectives: This study evaluated the performance of recombinant receptor binding domain (RBD) protein-based enzyme-linked immunosorbent assays (RBD-ELISAs) for detecting anti-SARS-CoV-2 immunoglobulin (Ig) G and IgM antibodies. Methods: In this study, 705 sera from SARS-CoV-2-infected individuals and 315 sera from healthy individuals were analyzed. Results: The RBD-ELISA IgG exhibited high specificity (99.1%) and moderate sensitivity (48.0%), with an overall diagnostic accuracy of 73.5%. RBD-ELISA IgM demonstrated specificity at 94.6% and sensitivity at 51.1%, with an accuracy of 72.8%. Both assays displayed improved performance when analyzing samples collected 15-21 days post-symptom onset, achieving sensitivity and accuracy exceeding 88% and 90%, respectively. Combining RBD-ELISA IgG and IgM in parallel analysis enhanced sensitivity to 98.6% and accuracy to 96.2%. Comparing these RBD-ELISAs with commercially available tests, the study found overlapping sensitivity and similar specificity values. Notably, the combined RBD-ELISA IgG and IgM showed superior performance. Cross-reactivity analysis revealed low false-positive rates (4.4% for IgG, 3.7% for IgM), primarily with viral infections. Conclusion: This research underscores the potential of RBD-based ELISAs for COVID-19 diagnosis, especially when assessing samples collected 15-21 days post-symptom onset and utilizing a parallel testing approach. The RBD protein's immunogenicity and specificity make it a valuable tool for serodiagnosis, offering an alternative to polymerase chain reaction-based methods, particularly in resource-limited settings.

Seroepidemiological Survey of Chronic Chagas Disease in a Rural Community in Southern Bahia, Brazil, Using Recombinant Chimeric Antigens.

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected tropical disease with life-threatening implications. In this study, we conducted a seroepidemiological survey to determine the prevalence and clinical profiles of CD in 217 individuals from an impoverished rural community in Southern Bahia, Brazil. The overall prevalence of CD in the studied community was 0.92%, detected through latent class analysis (LCA). Two individuals tested positive for anti-T. cruzi IgG, both being male farmers. One case was a 22-year-old man born in Camamu, with no evidence of congenital transmission, suggesting other routes of transmission such as vector-borne transmission due to migratory activities. The other case was a 69-year-old man born in São Felipe, who had lived in an adobe/brick house and had a pacemaker due to cardiac involvement caused by CD. The prevalence in this community was lower than expected, given the socioeconomic conditions and environmental factors that contribute to T. cruzi transmission. This could be attributed to the implementation of preventive measures and vector control programs by the Brazilian Government. However, continuous monitoring and surveillance are essential to sustain control efforts and detect any potential re-emergence of the disease. While the overall prevalence was low, the detection of positive cases underscores the need for continued surveillance and control measures in vulnerable populations, such as rural communities. Active surveillance, early diagnosis, and timely treatment are crucial in preventing disease progression and complications, thereby enhancing the effectiveness of screening and treatment programs.

Seroepidemiology of Chagas disease in at-risk individuals in Caraíbas, a city with high endemicity in Bahia State, Brazil.

Introduction: In Brazil, an estimated 1.1 million people are infected with Trypanosoma cruzi, the causative agent of Chagas disease (CD). Despite the high number of cases, the estimated prevalence of infection per 100 inhabitants is low (0.03). However, the actual number of chronically infected individuals is still unknown. Therefore, we sought to determine the prevalence of chronic CD in at-risk individuals in Caraíbas (Bahia, Brazil) through active case finding. Methods: A total of 572 individuals living in rural or urban areas of Caraíbas were eligible for the study. A serum sample was collected from 226 individuals, and the diagnosis performed according to international guidelines. Results: The overall prevalence of anti-T. cruzi IgG was 4.42%. The median age of anti-T. cruzi IgG-positive individuals was 54.5 years, and the female-to-male ratio was 1.5:1. The prevalence of anti-T. cruzi IgG was similar in rural (4.29%) and urban areas (4.65%). Discussion: Compared with national estimates, we concluded that Caraíbas had a high prevalence for chronic CD and a high risk for persistent transmission. Through our study, it was possible to monitor individuals who were unaware of their clinical condition, thus improving their quality of life.

A multicenter comparative study of the performance of four rapid immunochromatographic tests for the detection of anti-Trypanosoma cruzi antibodies in Brazil.

Diagnosis of Trypanosoma cruzi (T. cruzi) infection in the chronic phase of Chagas disease (CD) is performed by serologic testing. Conventional tests are currently used with very good results but require time, laboratory infrastructure, and expertise. Rapid diagnostic tests (RDTs) are an alternative as the results are immediate and do not require specialized knowledge, making them suitable for epidemiologic studies and promising as a screening tool. Nevertheless, few studies conducted comparative evaluations of RDTs to validate the results and assess their performance. In this study, we analyzed four trades of rapid tests (OnSite Chagas Ab Combo Rapid Test-United States, SD Bioline Chagas AB-United States, WL Check Chagas-Argentina, and TR Chagas Bio-Manguinhos-Brazil) using a panel of 190 samples, including sera from 111 infected individuals, most of whom had low T. cruzi antibody levels. An additional 59 samples from uninfected individuals and 20 sera from individuals with other diseases, mainly visceral leishmaniasis, were included. All tests were performed by three independent laboratories in a blinded manner. Results showed differences in sensitivity from 92.8 to 100%, specificity from 78.5 to 92.4%, and accuracy from 90.5 to 95.3% among the four assays. The results presented here show that all four RDTs have high overall diagnostic ability. However, WL Check Chagas and TR Chagas Bio-Manguinhos were considered most suitable for use in screening studies due to their high sensitivity combined with good performance. Although these two RDTs have high sensitivity, a positive result should be confirmed with other tests to confirm or rule out reactivity/positivity, especially considering possible cross-reactivity with individuals with leishmaniasis or toxoplasmosis.

Epidemiological indicators of Chagas disease in the metropolitan region of Salvador, Bahia, Brazil.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi and transmitted by triatomines. Historical information from the 20th century demonstrates T. cruzi records in the metropolitan region of Salvador (MRS), the third largest urban agglomeration in the Brazilian Northeast and the eighth largest in Brazil, an area with intense migratory activity from CD-endemic regions. Therefore, this study aimed to evaluate CD indicators (prevalence and mortality) in the MRS. METHODS: A mixed ecological and descriptive study was conducted using secondary data. We analyzed data from 2008 to 2015: deaths due to CD, self-reported cases of CD, and blood donors that were non-negative for T. cruzi infection. RESULTS: São Francisco do Conde was one of the municipalities with the highest mortality rates due to CD. The seroprevalence rates varied by year and municipality; those with the highest values were 2008: Vera Cruz, 2009: Mata de São João, 2010: Dias D'Ávila, 2011 and 2015: São Francisco do Conde, 2012: São Sebastião do Passé, and 2013 and 2014: Pojuca. Spatial correlations between the municipalities were not detected. CONCLUSIONS: We conclude that CD is present in the MRS. The indicators analyzed in the MRS are below-state-level data. Given the importance of indicator analysis for the surveillance and control of CD at the state and national levels, it is important to strengthen the surveillance program at the municipal level, including the regions classified as low risk for T. cruzi vector transmission.

Epidemiological indicators of Chagas disease in the metropolitan region of Salvador, Bahia, Brazil

ABSTRACT Background: Chagas disease (CD) is caused by Trypanosoma cruzi and transmitted by triatomines. Historical information from the 20th century demonstrates T. cruzi records in the metropolitan region of Salvador (MRS), the third largest urban agglomeration in the Brazilian Northeast and the eighth largest in Brazil, an area with intense migratory activity from CD-endemic regions. Therefore, this study aimed to evaluate CD indicators (prevalence and mortality) in the MRS. Methods: A mixed ecological and descriptive study was conducted using secondary data. We analyzed data from 2008 to 2015: deaths due to CD, self-reported cases of CD, and blood donors that were non-negative for T. cruzi infection. Results: São Francisco do Conde was one of the municipalities with the highest mortality rates due to CD. The seroprevalence rates varied by year and municipality; those with the highest values were 2008: Vera Cruz, 2009: Mata de São João, 2010: Dias D'Ávila, 2011 and 2015: São Francisco do Conde, 2012: São Sebastião do Passé, and 2013 and 2014: Pojuca. Spatial correlations between the municipalities were not detected. Conclusions: We conclude that CD is present in the MRS. The indicators analyzed in the MRS are below-state-level data. Given the importance of indicator analysis for the surveillance and control of CD at the state and national levels, it is important to strengthen the surveillance program at the municipal level, including the regions classified as low risk for T. cruzi vector transmission.

Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi. The chronic phase of CD is characterized by the presence of IgG anti-T. cruzi antibodies; and diagnosis is performed by serological methods. Because there is no reliable test that can be used as a reference test, WHO recommends the parallel use of two different tests for CD serodiagnosis. If results are inconclusive, samples should be subjected to a confirmatory test, e.g., Western blot (WB) or PCR. PCR offers low sensitivity in the chronic phase, whereas few confirmatory tests based on the WB method are commercially available worldwide. Therefore, new diagnostic tools should be evaluated to fill the gap in CD confirmatory tests. In recent years, four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) have been evaluated in phase I, II and III studies using ELISA, liquid microarray and immunochromatography with 95-100% accuracy. Given the high diagnostic performance of these antigens, the present study investigated the ability of these molecules to diagnose chronic CD using a WB testing platform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed the diagnostic potential of four chimeric antigens using 40 T. cruzi-positive, 24-negative, and three additional positive samples for visceral leishmaniasis (i.e., potentially cross-reactive) using WB as the diagnostic platform. Checkerboard titration with different dilutions of antigens, conjugated antigens, and serum samples was performed to standardize all assays. All IBMP antigens achieved 100% sensitivity, specificity, and accuracy, with the exception of IBMP-8.3, which had 100% specificity despite lack of significance, but lower sensitivity (95%) and accuracy (96.9%). No cross-reactivity was observed in samples positive for leishmaniasis. CONCLUSIONS/SIGNIFICANCE: The present phase I (proof-of-concept) study demonstrated the high diagnostic potential of these four IBMP antigens to discriminate between T. cruzi-positive and -negative samples, making them candidates for phase II and confirmatory testing with WB.

Spatiotemporal distribution analysis of syphilis in Brazil: Cases of congenital and syphilis in pregnant women from 2001-2017.

In Brazil, the notification of congenital (CS) and syphilis in pregnant women (SiP) is compulsory. Notification data provided by the Ministry of Health in combination with the mapping of vulnerable geographic areas is essential to forecasting possible outbreaks and more effectively combating infection through monitoring. We aim to evaluate the spatiotemporal distribution and epidemiological aspects of reported cases of CS and SiP in Brazil. A retrospective ecological study was carried out using secondary surveillance data obtained from the Brazilian National Notifiable Diseases Information System (SINAN) database, considering all reported cases of CS and SiP between 2001 to 2017. Epidemiological characteristics and time trends were analyzed using joinpoint regression models and spatial distribution, considering microregions or states/macroregions as units of analysis. A total of 188,630 (359/100,000 birth lives) CS and 235,895 of SiP (6.3/100,000 inhabitants) were reported during the period studied. In general, the epidemiologic profile of Brazil indicates most reported CS cases occurred in "mixed-race" newborns who were diagnosed within seven days of birth and whose mothers had received prenatal care, but the epidemiologic profile varies by Brazilian macroregion. Regarding SiP, most cases were among women who self-reported 'mixed-race', were aged 20-39 years, had up to eight years of formal education and were diagnosed with primary or latent syphilis. Approximately 549 (98.4%) and 558 (100%) microregions reported at least one case of CS and SiP, respectively. From 2012 to 2016, CS cases increased significantly in almost all Brazilian states, most notably in the South, Southeast, and Central-West macroregions, from 2001-2017 and the relative risk (RR) of SiP increased around 400% (RR: 1,00 to 445,50). Considering the epidemiological scenario of the infection in Brazil, it is necessary to enhance preventive, control and eradication measures.

Technological advances in the serological diagnosis of Chagas disease in dogs and cats: a systematic review.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi, which is transmitted mainly through the feces/urine of infected triatomine bugs. The acute phase lasts 2-3 months and is characterized by high parasitemia and nonspecific symptoms, whereas the lifelong chronic phase features symptoms affecting the heart and/or digestive tract occurring in 30-40% of infected individuals. As in humans, cardiac abnormalities are observed in T. cruzi-infected dogs and cats. We reviewed the technological advances in the serological diagnosis of CD in dogs and cats. METHODS: A review of the published literature during the last 54 years (1968-2022) on the epidemiology, clinical features, diagnosis, treatment and prevention of CD in dogs and cats was conducted. RESULTS: Using predefined eligibility criteria for a search of the published literature, we retrieved and screened 436 publications. Of these, 84 original studies were considered for inclusion in this review. Dogs and cats are considered as sentinels, potentially indicating an active T. cruzi transmission and thus the risk for human infection. Although dogs and cats are reputed to be important for maintaining the T. cruzi domestic transmission cycle, there are no commercial tests to detect past or active infections in these animals. Most published research on CD in dogs and cats have used in-house serological tests prepared with native and/or full-length recombinant antigens, resulting in variable diagnostic performance. In recent years, chimeric antigens have been used to improve the diagnosis of chronic CD in humans with encouraging results. Some of them have high performance values (> 95%) and extremely low cross-reactivity rates for Leishmania spp., especially the antigens IBMP-8.1 to IBMP-8.4. The diagnostic performance of IBMP antigens was also investigated in dogs, showing high diagnostic performance with negligible cross-reactivity with anti-Leishmania infantum antibodies. CONCLUSIONS: The development of a commercial immunodiagnostic tool to identify past or active T. cruzi infections in dogs and cats is urgently needed. The use of chimeric recombinant T. cruzi antigens may help to fill this gap and is discussed in this review.

Integrative and Multidisciplinary Care for People Living With Human T-Cell Lymphotropic Virus in Bahia, Brazil: 20 Years of Experience.

Brazil is home to the highest absolute number of human T-cell lymphotropic virus type-1 (HTLV-1)-infected individuals worldwide; the city of Salvador, Bahia, has the highest prevalence of HTLV-1 infection in Brazil. Due to the complex nature of several diseases associated with this retrovirus, a multidisciplinary health care approach is necessary to care for people living with HTLV-1. The Bahia School of Medicine and Public Health's Integrative Multidisciplinary HTLV Center (CHTLV) has been providing support to people living with HTLV and their families since 2002, striving to ensure physical and mental well-being by addressing biopsychosocial aspects, providing clinical care and follow-up, including to pregnant/postpartum women, as well as comprehensive laboratory diagnostics, psychological therapy, and counseling to family members. To date, CHTLV has served a total of 2,169 HTLV-infected patients. The average patient age is 49.8 (SD 15.9) years, 70.3% are female, most are considered low-income and have low levels of education. The majority (98.9%) are HTLV-1 cases, and approximately 10% have been diagnosed with tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM), while 2.2% have infective dermatitis and 1.1% have adult T-cell lymphoma. In all, 178 pregnant/postpartum women [mean age: 32.7 (±6.5) years] have received care at CHTLV. Regarding vertical transmission, 53% of breastfed infants screened for HTLV tested positive in their second year of life, nearly 18 times the rate found in non-breastfed infants. This article documents 20 years of experience in implementing an integrative and multidisciplinary care center for people living with HTLV in Bahia, Brazil. Still, significant challenges remain regarding infection control, and HTLV-infected individuals continue to struggle with the obtainment of equitable and efficient healthcare.

Performance of Chimeric Trypanosoma cruzi Antigens in Serological Screening for Chagas Disease in Blood Banks.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2-95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4-100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera.

BACKGROUND: Enzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform. METHODOLOGY/PRINCIPAL FINDINGS: DAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV. CONCLUSIONS/SIGNIFICANCE: DAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.

Eco-epidemiology of vectorial Trypanosoma cruzi transmission in a region of northeast Brazil.

Chagas disease (CD) is a parasitic zoonosis endemic in Brazil. Despite virtual control of Triatoma infestans, the main domesticated vector of Trypanosoma cruzi, vectorial transmission by other triatomine species persists in some rural communities. This study aims to characterize triatomines role in transmitting T. cruzi to dogs and humans in the district of Santo Inácio, located in the northwest region of the state of Bahia, Brazil. It also describes environmental factors in housings associated with insect occurrence and assesses the perception, knowledge, and preventive practices adopted by the population regarding CD. Blood samples of humans and dogs, and biological samples of triatomines, were collected between November 2018 and February 2019 and subjected to the detection of T. cruzi by serological and molecular biology tests. Also, we applied a questionnaire to research the perception, knowledge, and local practices of people related to CD. The capture of triatomines in households was associated with exploratory variables of the questionnaires using multivariate logistic regression (p < 0.05). The 155 triatomines captured in the wild and domestic environment were of the species Triatoma sherlocki (n = 151), Panstrongylus sherlocki (n = 1) and Triatoma sordida (n = 3), and had a natural infection rate for T. cruzi by PCR of 18.5%, 100% and 0%, respectively. District residents (n = 126) were seronegative for T. cruzi, while 17.5% (7/40) of the dogs were seropositive. The fact that residents are aware that triatomines can "cause" CD was configured as a protection factor for residents according to the fitted logistic regression model (p = 0.04). However, respondents have limited perception and knowledge about the CD, prevention and control practices for triatomines in a household. The results suggest the existence of a domestic cycle of transmission of T. cruzi between triatomines and dogs, configuring a latent risk of infection to the human population of Santo Inácio. Studies that clarify the potential for the establishing of intrusive triatomines in households, surveillance actions for triatomines, and health education in rural communities are indispensable to prevent the reemergence of CD in vulnerable regions of Brazil and other American countries with similar epidemiological characteristics.

Seroprevalence and detection of Trypanosoma cruzi in dogs living in a non-endemic area for Chagas disease in the legal Amazon region, Brazil.

Trypanosoma cruzi, the etiological agent for Chagas disease, is widely distributed in the Americas. Its hosts are humans and wild and domestic mammals, and its vectors are triatomine insects. Studies have indicated that domestic dogs are sentinel animals in the epidemiology of Chagas disease in endemic regions, including states in the Legal Amazon region of Brazil. In São Luís, the capital of Maranhão, a non-endemic state, the existence of a domestic cycle involving domestic rats has been proven, along with a wild cycle maintained by didelphids. However, no studies on T. cruzi infection in domestic animals in this locality have been conducted. The aim of this study was to investigate occurrence of T. cruzi in dogs living in the Itaqui Bacanga district of São Luís, Maranhão, by means of serological and molecular tests. Blood samples were obtained from 330 dogs and structured epidemiological questionnaires were applied to their keepers. These samples were used in the indirect immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Fisher's exact test was used for statistical calculations with the aim of identifying risk factors. Out of the 330 animals, 105 (31.8%) were reactive in IFAT, 46 (13.0%) in ELISA and 20 (6.0%) in both serological tests. The results were not significant (p > 0.05) when submitted to statistical analysis for the studied variables. From PCR, 58 samples (17.5%) were found to be positive and, of these, one (0.3%) showed similarity to T. cruzi after sequencing. These data demonstrate that dogs were exposed to and infected by T. cruzi. Thus, they can be considered sentinel animals for Chagas disease in the locality studied, which signals that there is a need for epidemiological surveillance actions.