Coupling ELISA to smartphones for POCT of chronic and congenital Chagas disease.

Chagas disease (CD) affects about 7 million people worldwide, presents a large prevalence in Latin America, and is growing in the rest of the world, where congenital CD is the main mode of transmission. Point-of-care testing (POCT) methods are increasingly required to ease early diagnostics and increase treatment success. This work presents the development and validation of a smartphone-integrated ELISA-based POCT system for the detection of both chronic and congenital CD. Expensive and bulky equipment used for ELISA in conventional laboratories was replaced as follows. A miniaturized device was fabricated for incubation of commercial ELISA plates, achieving ∼±1 °C uniformity and stability. The ELISA plate reader was replaced by smartphone camera and image processing, comprising algorithms to account for variability sources and spatial light non-uniformity; thus, additional hardware like a dark-box is not required. The agreement between samples classified with this novel reading method vs. ELISA plate reader was found to be 99.7% and 95.4% for chronic and congenital CD, respectively. Furthermore, a smartphone application was designed and implemented to guide the user during the assay, provide connectivity, and access databases, facilitating patient monitoring and health-policy making. The whole system is aimed to be used as a practical diagnostic tool in primary health care settings, as well as to facilitate patients' follow-up to provide better treatment. Concerning the technology itself, the proposed POCT platform is versatile enough to be readily adapted for the detection of other infectious diseases.

Seroprevalence of anti-SARS-CoV-2 IgG in asymptomatic and pauci-symptomatic people over a 5 month survey in Argentina.

OBJECTIVE: To evaluate the seroprevalence of COVID-19 infection in pauci-symptomatic and asymptomatic people, the associated epidemiological factors, and IgG antibody kinetic over a 5-month period to get a better knowledge of the disease transmissibility and the rate of susceptible persons that might be infected. METHODS: Seroprevalence was evaluated by a cross-sectional study based on the general population of Santa Fe, Argentina (non-probabilistic sample) carried out between July and November 2020. A subgroup of 20 seropositive individuals was followed-up to analyze IgG persistence. For the IgG anti-SARS-CoV-2 antibodies detection, the COVID-AR IgG® ELISA kit was used. RESULTS: 3 000 individuals were included conforming asymptomatic and pauci-symptomatic groups (n=1 500 each). From the total sample, only 8.83% (n=265) presented reactivity for IgG anti-SARS-CoV-2. A significant association was observed between positive anti-SARS-CoV-2 IgG and a history of contact with a confirmed case; the transmission rate within households was approximately 30%. In the pauci-symptomatic group, among the seropositive ones, anosmia and fever presented an OR of 16.8 (95% CI 9.5-29.8) and 2.7 (95% CI 1.6-4.6), respectively (p <0.001). In asymptomatic patients, IgG levels were lower compared to pauci-symptomatic patients, tending to decline after 4 months since the symptoms onset. CONCLUSION: We observed a low seroprevalence, suggestive of a large population susceptible to the infection. Anosmia and fever were independent significant predictors for seropositivity. Asymptomatic patients showed lower levels of antibodies during the 5-month follow-up. IgG antibodies tended to decrease over the end of this period regardless of symptoms.

Functional Autoreactive Anti-ß2 Adrenergic Antibodies May Contribute to Insulin Resistance Profile in Patients with Chronic Chagas Disease.

Potential activation of ß2 adrenergic receptors (ß2AR) by specific autoreactive antibodies (Abs) that arise during the host reaction to Trypanosoma cruzi, could contribute to the elevated prevalence of metabolic disturbances described in patients with chronic Chagas disease (CCD). This study aimed to determine the prevalence of anti-ß2AR Abs in patients with CCD, as well as the correlation of these Abs with the presence of glucose and lipid metabolism disturbances, in order to explore their association with an insulin resistance profile. Additionally, we tested the functional effects of anti-ß2AR Abs employing an in vitro bioassay with neuroendocrine cells expressing ß2AR. A clinical and metabolic evaluation including an OGTT was performed in 80 CCD patients and 40 controls. Anti-ß2AR Abs were measured by an in-house-developed ELISA, and the ß2 adrenergic activity of affinity-purified IgG fractions from patient' sera were assayed in CRE-Luc and POMCLuc transfected AtT-20 cells. A higher proportion of dysglycemia (72.5% vs. 37.5%; p = 0.001) was observed in the CCD group, accompanied by increased HOMA2-IR (p = 0.019), especially in subjects with Abs (+). Anti-ß2AR Abs reactivity (7.01 (2.39-20.5); p = 0.0004) and age >50 years (3.83 (1.30-11.25); p = 0.014) resulted as relevant for IR prediction (AUC: 0.786). Concordantly, Abs (+) CCD patients showed elevated metabolic risk scores and an increased prevalence of atherogenic dyslipidemia (p = 0.040), as compared to Abs (-) patients and controls. On functional bioassays, Abs exerted specific and dose-dependent ß2-agonist effects. Our findings suggest that anti-ß2AR Abs may induce the activation of ß2AR in other tissues besides the heart; furthermore, we show that in patients with CCD these Abs are associated with an insulin resistance profile and atherogenic dyslipidemia, providing biological plausibility to the hypothesis that adrenergic activation by anti-ß2AR Abs could contribute to the pathogenesis of metabolic disturbances described in CCD patients, increasing their cardiovascular risk.

Increased epicardial adipose tissue thickness associated with increased metabolic risk and the presence of heart failure in patients with Chronic Chagas disease.

BACKGROUND: It has been described that Trypanosoma cruzi is capable of promoting metabolic disturbances currently considered as cardiovascular risk factors. Moreover, it has been observed that the protozoa can remain in adipose tissue and alter its immune endocrine functions. The aim of this study was to characterize the thickness of epicardial adipose tissue (EAT) in patients with chronic Chagas disease (CCD) concerning their cardiovascular metabolic risk profile compared with those without CCD. METHODS: A cross-sectional study was performed including T. cruzi seropositive individuals categorized according to a standard CCD classification and a matched seronegative control group. Complete clinical examination, metabolic laboratory tests and transthoracic echocardiography to assess cardiac function and to quantify EAT were performed. RESULTS: Fifty-five individuals aged 46.7±11.9 y, 34 with CCD and 21 in the control group, were included. The CCD group presented higher EAT thickness in relation to controls (4.54±1.28 vs 3.22±0.99 mm; p=0.001), which was significantly associated with the presence of insulin resistance (OR=3, 95% CI 1.58 to 5.73; p<0.001). This group presented lower levels of plasmatic adiponectin than controls, especially in those patients with EAT ≥4.5 mm (p=0.005) who also presented with heart failure more frequently (p=0.01). CONCLUSION: In patients with CCD, a higher EAT thickness is observed and is associated with an increased metabolic risk profile indicated mainly by insulin resistance.

Congenital chagas disease: Development and assessment of a specific IgM capture-based assay for diagnosis of transmission.

Transplacental transmission by Trypanosoma cruzi (T. cruzi) infection can be effectively treated if parasiticide drugs are administered as early as possible during childhood. Furthermore, an ideal situation would be to diagnose the infection near birth in order to avoid the loss of patients during the subsequent follow-up. These situation are desirable due to the maximum benefit of drugs in early stages which, consequently, implies a relevant contribution to eliminate mother-to-child transmission. However, available techniques for that purpose have limitations as being operator-dependent (microhematocrit), require several months follow-up (IgG detection) or specialized laboratories (PCR). In this study we propose to detect specific IgM antibodies (Ab) by developing a capture-based ELISA employing an improved antigen (Ag) to diagnose the transplacental transmission of T. cruzi, and in consequence, to enhance access to effective treatment. Firstly, a new chimera Ag (CP4) was obtained from the fusion of CP1 and CP3 protein, carrying FRA, SAPA, MAP, TSSAII/V/VI and TcD Ag from T. cruzi. Then, we optimized the assay by capturing IgM Ab with a polyclonal anti-IgM Ab and evaluating three Ag formulations to detect specific IgM bound. The formulations were formed as follows: i) F1: CP1 and CP3; ii) F2: CP1, CP3, B13 and P2ß; iii) F3: by CP4. Detection of Ab-binding Ag was carried out using an anti-His Ab since all Ag were expressed with a His-tag. The evaluation panel consisted of sera from vertically infected children under 1-year-old (6 younger than 15 days, 7 older) and samples from non-infected children of women with chronic Chagas Disease. The ELISA assay employing CP4 showed better performance with notable high sensitivity and specificity (92.3% and 93.9%, respectively). Positive and negative likelihood ratios of the test (15.2 and 0.082) suggest its potential clinical relevance in term of post-test probability of infection. In conclution, we developed a standardized and non-operator dependent test to detect specific anti-T. cruzi IgM Ab. Although increased sample size is needed for its validation, our results indicate that this capture-based technique employing CP4 Ag can certainly improve the diagnosis of connatal infection.

Seroprevalence of anti-SARS-CoV-2 IgG in asymptomatic and pauci-symptomatic people over a 5 month survey in Argentina / Seroprevalencia de anticuerpos IgG contra el SARS-CoV-2 en personas asintomáticas y paucisintomáticas en un estudio de 5 meses en Argentina / Soroprevalência de IgG contra SARS-CoV-2 em indivíduos assintomáticos e paucissintomáticos em um inquérito de 5 meses na Argentina

ABSTRACT Objective. To evaluate the seroprevalence of COVID-19 infection in pauci-symptomatic and asymptomatic people, the associated epidemiological factors, and IgG antibody kinetic over a 5-month period to get a better knowledge of the disease transmissibility and the rate of susceptible persons that might be infected. Methods. Seroprevalence was evaluated by a cross-sectional study based on the general population of Santa Fe, Argentina (non-probabilistic sample) carried out between July and November 2020. A subgroup of 20 seropositive individuals was followed-up to analyze IgG persistence. For the IgG anti-SARS-CoV-2 antibodies detection, the COVID-AR IgG® ELISA kit was used. Results. 3 000 individuals were included conforming asymptomatic and pauci-symptomatic groups (n=1 500 each). From the total sample, only 8.83% (n=265) presented reactivity for IgG anti-SARS-CoV-2. A significant association was observed between positive anti-SARS-CoV-2 IgG and a history of contact with a confirmed case; the transmission rate within households was approximately 30%. In the pauci-symptomatic group, among the seropositive ones, anosmia and fever presented an OR of 16.8 (95% CI 9.5-29.8) and 2.7 (95% CI 1.6-4.6), respectively (p <0.001). In asymptomatic patients, IgG levels were lower compared to pauci-symptomatic patients, tending to decline after 4 months since the symptoms onset. Conclusion. We observed a low seroprevalence, suggestive of a large population susceptible to the infection. Anosmia and fever were independent significant predictors for seropositivity. Asymptomatic patients showed lower levels of antibodies during the 5-month follow-up. IgG antibodies tended to decrease over the end of this period regardless of symptoms.

RESUMEN Objetivo. Evaluar la seroprevalencia de la infección por el virus causante de la COVID-19 en personas paucisintomáticas y asintomáticas, los factores epidemiológicos asociados y la cinética de los anticuerpos IgG durante un período de cinco meses para conocer mejor la transmisibilidad de la enfermedad y la tasa de personas susceptibles a estar infectadas. Métodos. Se evaluó la seroprevalencia mediante un estudio transversal basado en la población general de Santa Fe, Argentina (muestra no probabilística) llevado a cabo entre julio y noviembre del 2020. Se realizó un seguimiento de un subgrupo de 20 personas seropositivas para analizar la persistencia de los anticuerpos IgG. Para la detección de los anticuerpos IgG contra SARS-COV-2, se empleó el kit ELISA COVID-AR IgG®. Resultados. Hubo 3 000 participantes divididos en un grupo asintomático y un grupo paucisintomático (n=1 500 cada grupo). De la muestra total, solo 8,83% (n=265) presentó una reactividad de IgG contra el SARS-CoV-2. Se observó una asociación significativa entre anticuerpos IgG positivos contra el SARS-CoV-2 y antecedente de contacto con un caso confirmado. La tasa de transmisión en el hogar fue de 30% aproximadamente. En el grupo paucisintomático, entre las personas seropositivas, la anosmia y la fiebre presentaron un OR de 16,8 (IC 95% 9,5-29,8) y 2,7 (IC 95% 1,6-4,6), respectivamente (p <0,001). En los pacientes asintomáticos, los niveles de IgG fueron inferiores en comparación con los pacientes paucisintomáticos, con tendencia a la baja pasados cuatro meses desde la aparición de los síntomas. Conclusiones. Se observó una seroprevalencia baja, indicadora de una gran población susceptible a la infección. La anosmia y la fiebre fueron factores predictivos independientes de relevancia para la seropositividad. Los pacientes asintomáticos mostraron niveles inferiores de anticuerpos durante el seguimiento de cinco meses. Los anticuerpos IgG tendieron a disminuir hacia el final del período con independencia de los síntomas.

RESUMO Objetivo. Avaliar a soroprevalência de anticorpos contra a COVID-19 em indivíduos paucissintomáticos e assintomáticos, os fatores epidemiológicos associados e a cinética dos anticorpos da classe IgG em um período de 5 meses, visando aprimorar o conhecimento sobre a transmissibilidade da doença e a taxa de suscetíveis à infecção. Métodos. Inquérito transversal de soroprevalência realizado na população geral (amostra não probabilística) de Santa Fé, na Argentina, entre julho e novembro de 2020. Um subgrupo de 20 indivíduos soropositivos foi acompanhado para analisar a persistência de anticorpos IgG. O kit de ensaio imunoenzimático (ELISA) COVID-AR IgG® foi usado para a detecção de anticorpos IgG contra SARS-CoV-2. Resultados. A amostra compreendeu 3 000 indivíduos, divididos entre assintomáticos e paucissintomáticos (n = 1.500 por grupo). Deste total, somente 8,83% (n = 265) apresentaram reatividade, com a detecção de anticorpos IgG contra SARS-CoV-2. Observou-se uma associação significativa entre a presença de anticorpos IgG contra SARS-CoV-2 e histórico de contato com caso confirmado. A taxa de transmissão intradomiciliar foi de aproximadamente 30%. No grupo paucissintomático, entre os soropositivos, o odds ratio (OR) para anosmia foi de 16,8 (IC 95% 9,5-29,8), e para febre, 2,7 (IC 95% 1,6-4,6) (p <0,001). Os indivíduos assintomáticos apresentaram níveis de IgG mais baixos que os paucissintomáticos, com uma tendência de declínio após 4 meses do início dos sintomas. Conclusões. Observou-se uma soroprevalência baixa de anticorpos contra a COVID-19 na população estudada, o que indica um grande número de pessoas suscetíveis à infecção. Anosmia e febre foram preditores importantes independentes de soropositividade. Os assintomáticos apresentaram níveis mais baixos de anticorpos aos 5 meses de acompanhamento. Houve uma tendência de redução dos anticorpos IgG ao final deste período, independentemente da presença de sintomas.

Development and assessment of an improved recombinant multiepitope antigen-based immunoassay to diagnose chronic Chagas disease.

The use of chimeric molecules fusing several antigenic determinants is a promising strategy for the development of low-cost, standardized and reliable kits to determine specific antibodies. In this study, we designed and assessed a novel recombinant chimera that complements the performance of our previously developed chimera, CP1 [FRA and SAPA antigens (Ags)], to diagnose chronic Chagas disease. The new chimeric protein, named CP3, is composed of MAP, TcD and TSSAII/V/VI antigenic determinants. We compared the performance of both chimeric Ags using a panel of 67 Trypanosoma cruzi-reactive sera and 67 non-reactive ones. The sensitivity of CP3 vs CP1 was 100 and 90.2%, and specificity was 92.5 and 100%, respectively. The mixture of CP1 + CP3 achieved 100% of sensitivity and specificity. More importantly, an additional subset of 17 sera from patients with discordant results of conventional serological methods was analysed; the CP1 + CP3 mixture allowed us to accurately classify 14 of them with respect to IIF, the usual technique used in most of the reference centres. These results show an improved performance of the CP1 + CP3 mixture in comparison with enzyme-linked immunosorbent assay and indirect haemagglutination commercial assays.