Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography.

BACKGROUND: Leptospirosis is an emerging zoonosis that affects humans and animals. Immunochromatography rapid test is widely used for early diagnosis of leptospirosis, but with low sensitivity and specificity. OBJECTIVES: To evaluate Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography. METHODS: Insoluble fraction derived from the crude bacterial extract was obtained by serial centrifugation. The polypeptide profile was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immune reactivity of this fraction was assessed by Western Blotting and lateral flow immunochromatography (LFI). It was tested 160 microagglutination test (MAT)-positive sera from patients in the acute phase, 100 MAT-negative sera from patients with acute febrile illness, and 45 patients with other infectious diseases. FINDINGS: There was a predominance of low molecular mass-polypeptide bands, ranging from 2 to 37 kDa. The antibody reactivity of theses polypeptides was found to range from 13-50%, especially between 10 and 38 kDa. Among MAT-positive sera of patients with leptospirosis in the acute phase, 97% were also positive in LFI, indicating high sensitivity. Among MAT-negative sera, all were negative in LFI, indicating high specificity. Only 2% of cross-reactivity was detected. CONCLUSION: The insoluble fraction can be a valuable antigen source for development of point-of-care diagnosis test for leptospirosis.

Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography

BACKGROUND Leptospirosis is an emerging zoonosis that affects humans and animals. Immunochromatography rapid test is widely used for early diagnosis of leptospirosis, but with low sensitivity and specificity. OBJECTIVES To evaluate Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography. METHODS Insoluble fraction derived from the crude bacterial extract was obtained by serial centrifugation. The polypeptide profile was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immune reactivity of this fraction was assessed by Western Blotting and lateral flow immunochromatography (LFI). It was tested 160 microagglutination test (MAT)-positive sera from patients in the acute phase, 100 MAT-negative sera from patients with acute febrile illness, and 45 patients with other infectious diseases. FINDINGS There was a predominance of low molecular mass-polypeptide bands, ranging from 2 to 37 kDa. The antibody reactivity of theses polypeptides was found to range from 13-50%, especially between 10 and 38 kDa. Among MAT-positive sera of patients with leptospirosis in the acute phase, 97% were also positive in LFI, indicating high sensitivity. Among MAT-negative sera, all were negative in LFI, indicating high specificity. Only 2% of cross-reactivity was detected. CONCLUSION The insoluble fraction can be a valuable antigen source for development of point-of-care diagnosis test for leptospirosis.

Protective Immunity of COVID-19 Vaccination with ChAdOx1 nCoV-19 Following Previous SARS-CoV-2 Infection: A Humoral and Cellular Investigation.

Infections caused by SARS-CoV-2 induce a severe acute respiratory syndrome called COVID-19 and have led to more than six million deaths worldwide. Vaccination is the most effective preventative measure, and cellular and humoral immunity is crucial to developing individual protection. Here, we aim to investigate hybrid immunity against SARS-CoV-2 triggered by the ChAadOx1 nCoV-19 vaccine in a Brazilian cohort. We investigated the immune response from ChAadOx1 nCoV-19 vaccination in naïve (noCOVID-19) and previously infected individuals (COVID-19) by analyzing levels of D-dimers, total IgG, neutralizing antibodies (Nabs), IFN-γ (interferon-γ) secretion, and immunophenotyping of memory lymphocytes. No significant differences in D-dimer levels were observed 7 or 15 days after vaccination (DAV). All vaccinated individuals presented higher levels of total IgG or Nabs with a positive correlation (R = 0.88). Individuals in the COVID-19 group showed higher levels of antibody and memory B cells, with a faster antibody response starting at 7 DAV compared to noCOVID-19 at 15 DAV. Further, ChAadOx1 nCoV-19 vaccination led to enhanced IFN-γ production (15 DAV) and an increase in activated T CD4+ naïve cells in noCOVID-19 individuals in contrast with COVID-19 individuals. Hence, our data support that hybrid immunity triggered by ChAadOx1 nCoV-19 vaccination is associated with enhanced humoral response, together with a balanced cellular response.

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.

Performance assessment of a new indirect rapid diagnostic test for plague detection in humans and other mammalian hosts.

Plague is a flea-borne zoonosis that affects a wide range of mammals and still causes outbreaks in human populations yearly across several countries. While crucial for proper treatment, early diagnosis is still a major challenge in low- and middle-income countries due to poor access to laboratory infrastructure in rural areas. To tackle this issue, we developed and evaluated a new Fraction 1 capsular antigen (F1)-based rapid diagnostic test (RDT) as an alternative method for plague serological diagnosis and surveillance in humans and other mammals. In this study, 187 serum samples from humans, dogs, rodents and rabbits were retrospectively assessed using the plague RDT method. To calculate its performance, results were compared to those obtained by traditional hemagglutination (HA) and ELISA, which are well-established methods in the plague routine serodiagnosis. Remarkably, the results from RDT were in full agreement with those from the ELISA and HA assays, resulting in 100% (CI 95% = 95.5-100%) of sensitivity and 100% (CI 95% = 96.6-100%) of specificity. Accordingly, the Cohen's Kappa test coefficient was 1.0 (almost perfect agreement). Moreover, the RDT showed no cross-reaction when tested with sera from individuals positive to other pathogens, such as Y. pseudotuberculosis, Yersinia enterocolitica, Anaplasma platys, Ehrlichia canis and Leishmania infantum. Although preliminary, this study brings consistent proof-of-concept results with high performance of the Plague RDT when compared to HA and ELISA. Although further human and animal population-based studies will be necessary to validate these findings, the data presented here show that the plague RDT is highly sensitive and specific, polyvalent to several mammal species and simple to use in field surveillance or point-of-care situations with instant results.

Correction: High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil-A Phase II study.

[This corrects the article DOI: 10.1371/journal.pntd.0006871.].

High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil - A Phase II study.

BACKGROUND: Canine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85-99%) vs. 83% (IC 95%; 69-92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49-97%) vs. 54% (IC 95%; 33-74%) for asymptomatic dogs and 94% (IC 95%; 83-99%) vs, 88% (IC 95%; 76-95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81-91%) of sensitivity, 94% (IC 95%; 90-97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59-74%), specificity of 87% (IC 95%; 81-92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52-66%), specificity of 98% (IC 95%; 95-99%) and accuracy of 80% (IC 95%; 76-84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63-77%), specificity of 99% (IC 95%; 97-100%) and accuracy of 86% (IC 95%; 83-89%). CONCLUSION: Thus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil.

Highly Accurate Chimeric Proteins for the Serological Diagnosis of Chronic Chagas Disease: A Latent Class Analysis.

The existence of an imperfect reference standard presents complications when evaluating the unbiased performance of novel diagnostic techniques. This is especially true in the absence of a gold standard, as is the case in chronic Chagas disease (CD) diagnosis. To circumvent this constraint, we elected to use latent class analysis (LCA). Previously, our group demonstrated the high performance of four Trypanosoma cruzi-chimeric proteins (Molecular Biology Institute of Paraná [IBMP]-8.1, -8.2, -8.3, and -8.4) for CD diagnosis using several distinct immunoassays. Although commercial tests had previously been established as a reference standard, the diagnostic performance of these chimeric antigens could present bias because these tests fail to produce 100% accurate results. Thus, we used LCA to assess the performance of these IBMP chimeric antigens in chronic CD diagnosis. Using the LCA model as a gold standard, sensitivity and specificity values ranged from 93.5% to 99.4% and 99.6% to 100%, respectively. The accuracy values were 96.2% for IBMP-8.2, approximately 98% for IBMP-8.1 and IBMP-8.3, and nearly 100% for IBMP-8.4. For IBMP-8.1 and IBMP-8.2, higher positive predictive values were associated with increases in hypothetical prevalence. Similarly, higher hypothetical prevalence resulted in lower negative predictive values for IBMP-8.1, IBMP-8.2, and IBMP-8.3. In addition, samples with serodiscordant results from commercial serological tests were analyzed using LCA. Molecular Biology Institute of Paraná -8.1 demonstrated potential for use in confirmatory testing with regard to samples with inconsistent results. Moreover, our findings further confirmed the remarkable performance of the IBMP-8.4 antigen to diagnose chronic CD in both endemic and non-endemic areas.

Accuracy of chimeric proteins in the serological diagnosis of chronic chagas disease - a Phase II study.

BACKGROUND: The performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results. METHODOLOGY/PRINCIPAL FINDINGS: Here, four chimeric proteins (IBMP-8.1 to -8.4) comprising immunodominant regions of different Trypanosoma cruzi antigens were tested by enzyme-linked immunosorbent assay. The proteins were used to detect specific anti-T. cruzi antibodies in the sera of 857 chagasic and 689 non-chagasic individuals to evaluate their accuracy for chronic CD diagnosis. The antigens were recombinantly expressed in Escherichia coli and purified by chromatographic methods. The sensitivity and specificity values ranged from 94.3% to 99.3% and 99.4% to 100%, respectively. The diagnostic odds ratio (DOR) values were 6,462 for IBMP-8.1, 3,807 for IBMP-8.2, 32,095 for IBMP-8.3, and 283,714 for IBMP-8.4. These chimeric antigens presented DORs that were higher than the commercial test Pathozyme Chagas. The antigens IBMP-8.3 and -8.4 also showed DORs higher than the Gold ELISA Chagas test. Mixtures with equimolar concentrations were tested in order to improve the diagnosis accuracy of negative samples with high signal and positive samples with low signal. However, no gain in accuracy was observed relative to the individual antigens. A total of 1,079 additional sera were used to test cross-reactivity to unrelated diseases. The cross-reactivity rates ranged from 0.37% to 0.74% even for Leishmania spp., a pathogen showing relatively high genome sequence identity to T. cruzi. Imprecision analyses showed that IBMP chimeras are very stable and the results are highly reproducible. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the IBMP-8.4 antigen can be safely used in serological tests for T. cruzi screening in blood banks and for chronic CD laboratory diagnosis.

Increase in the expression of CD4 + CD25+ lymphocytic T cells in the indeterminate clinical form of human Chagas disease after stimulation with recombinant antigens of Trypanosoma cruzi.

PURPOSE: Regulatory T cells are involved in the clinical course of chronic Chagas disease, possibly because they exercise a control in the patient's inflammatory response to Trypanosoma cruzi. This study analyzed the levels of CD4 + CD25+ T cells in chronic Chagas disease patients after in vitro stimulation of the peripheral blood mononuclear cells with CRA (Cytoplasmic Repetitive Antigen) or FRA (Flagellar Repetitive Antigen) T. cruzi antigens. METHODS: Groups of patients with the cardiac form and indeterminate form; and non-infected individuals, were selected. The CD4 + CD25+ T lymphocyte population, as well as the FoxP3 expression and the IL10 production, were evaluated by flow cytometry after stimulation with CRA or FRA. RESULT: The IND group presented higher levels of CD4 + CD25+ T cells than the CARD group. However, there was no evidence of a relationship between FoxP3 and IL10 with any of the chronic forms. CONCLUSIONS: Our results suggest the possible involvement of CD4 + CD25+ T cells specific to CRA and FRA in controlling the progression of clinical outcomes. Though, further studies are needed to define which mechanisms activate regulatory T cells and lead to pathology control in chronic human Chagas disease.

Evidence of Leishmania (Leishmania) infantum infection in dogs from Juiz de Fora, Minas Gerais State, Brazil, based on immunochromatographic dual-path platform (DPP®) and PCR assays.

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS / Evidência de infecção por Leishmania (Leishmania) infantum em cães de Juiz de Fora, Minas Gerais, Brasil, detectada pelo teste imunocromatográfico rápido DPP e PCR

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

No Brasil, cães domésticos são considerados como os principais reservatórios da leishmaniose visceral zoonótica, devido à clara correlação positiva existente entre as curvas de infecção humana e canina. Este estudo objetivou a realização de um inquérito sorológico da leishmaniose visceral canina (LVC) em cães abrigados em um canil público de Juiz de Fora, Minas Gerais, Brasil, através do teste rápido imunocromatográfico TR DPP®. Adicionalmente, a PCR convencional e/ou em tempo real foi usada para detectar/confirmar a infecção por L. infantum apenas nos animais DPP positivos. Dos 400 cães estudados, a maioria não apresentou sinais clínicos para a LVC (p < 0,05) e quinze (3,8%) foram sororreativos ao DPP. Não houve diferença estatisticamente significativa entre os cães com DPP positivo e os sinais clínicos para a doença (p > 0,05). PCR convencional e em tempo real confirmaram a infecção por L. infantum em nove (75,0%) dos doze animais DPP positivos que permaneceram vivos durante o estudo. Este é o primeiro estudo soroepidemiológico sobre LVC realizado no município de Juiz de Fora, e os resultados reforçam a idéia de que esta doença está em processo de expansão e urbanização no Brasil. Além disto, este estudo destaca o uso do DPP como uma alternativa para o diagnóstico da LVC em cidades de médio e grande porte, devido à facilidade de execução.

Avaliação hematológica e histopatológica de camundongos BALB/c e C57BL/6 expostos aos antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi / Hematological and histopathological evaluation of BALB/c and C57BL/6 mice exposed to Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi

Os antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi foram inoculados em camundongos BALB/c e C57BL/6 e o seu efeito avaliado a nível hematológico e histopatológico. Os resultados mostraram que o padrão histológico normal dos órgãos e o perfil hematológico dos camundongos não foram modificados sugerindo que esses antígenos não parecem causar dano ao animal.

[Hematological and histopathological evaluation of BALB/c and C57BL/6 mice exposed to Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi]. / Avaliação hematológica e histopatológica de camundongos BALB/c e C57BL/6 expostos aos antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi.

The Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi were inoculated into BALB/c and C57BL/6 mice and its effects evaluated at hematological and histopathological levels. The results showed that the histological pattern of the organs and the hematological profile of mice were not modified suggesting that these antigens are not harmful for the animal.