The present work described a novel sandwich-type electrochemical aptasensor for rapid and sensitive determination of Mycobacterium tuberculosis MPT64 antigen. Herein, a novel carbon nanocomposite ...composed of fullerene nanoparticles, nitrogen-doped carbon nanotubes and graphene oxide (C60NPs-N-CNTs/GO) was facilely synthesized for the first time, which not only possessed a large specific surface area and excellent conductivity, but also exhibited outstanding inherent electroactive property, and therefore served as nanocarrier and redox nanoprobe simultaneously. Gold nanoparticles (AuNPs) was then uniformly anchored onto the surface of such nanocomposite via Au–N bonds to bind with MPT64 antigen aptamer Ⅱ (MAA Ⅱ), forming the tracer label to realize generation and amplification of electrochemical signal. Additionally, conductive polyethyleneimine (PEI)-functionalized Fe-based metal-organic framework (P-MOF) was used as a sensing platform to absorb bimetallic core-shell Au–Pt nanoparticles (Au@Pt), which could accelerate electron transfer and increase the immobilization of MPT64 antigen aptamer Ⅰ (MAA Ⅰ). After the typical sandwich-type protein-aptamer recognition, the inherent electroactivity of the tracer label was provoked by tetraoctylammonium bromide (TOAB), leading to a well-defined current response. Under the optimum condition, the proposed aptasensor showed a wide linear range for MPT64 detection from 1 fg/mL to 1 ng/mL with a limit of detection (LOD) as low as 0.33 fg/mL. More importantly, it was successfully used for MPT64 antigen detection in human serum, exhibiting a promising prospect for TB diagnosis in clinical practice.
Background and purpose: Serological diagnosis of Strongyloides stercoralis (S. stercoralis) is often challenging due to cross-reactivity with other parasitic nematodes. Therefore, it is imperative to ...introduce new high-performance serological tests to flawlessly diagnose this neglected parasitic infection that enters through the digestive system and can even affect infertility. The purpose of the present study was to design a multi-epitope structure for the detection of S. stercoralis. Materials and methods: The present study is a computational immunoinformatics study. To this end, the highly antigenic parts and potential immunodominant epitopes of S. stercoralis were identified from the two antigenic proteins first and then all the selected parts were connected by a suitable linker. Next, the physicochemical characteristics of the designed structure were analyzed. Finally, tertiary structures were built and evaluated to determine the best ones. Results: Bioinformatics evaluation indicated that the designed protein structure can be hydrophilic, heat stable and acidic. In addition, the 3D model made was similar to the structure of natural proteins. Conclusion: Based on the findings of the study, the designed structure can be used as an efficient antigen in the ELISA system for the serological diagnosis of human strongyloidiasis
Prevalence of human T. cruzi DTUs and Chagas disease manifestations in countries of North, Central and South America. CCC, Chronic Chagas Cardiomyopathy; DIG, Digestive form.
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...•Trypanosoma cruzi is partitioned into a minimum of seven discrete typing units (DTUs), TcI–TcVI and Tcbat.•DTUs have distinct, but not exclusive ecological and epidemiological associations.•Hybridization between parasite strains is frequent and challenges the paradigm of prevailing clonal evolution in T. cruzi.•The potential association between DTUs and Chagas disease manifestations, serology and chemotherapy is discussed.•Research priorities for these themes are pointed out and recommendations are issued.
The genetic diversity of Trypanosoma cruzi, the protozoan agent of Chagas disease, is widely recognized. At present, T. cruzi is partitioned into seven discrete typing units (DTUs), TcI–TcVI and Tcbat. This article reviews the present knowledge on the parasite population structure, the evolutionary relationships among DTUs and their distinct, but not exclusive ecological and epidemiological associations. Different models for the origin of hybrid DTUs are examined, which agree that genetic exchange among T. cruzi populations is frequent and has contributed to the present parasite population structure. The geographic distribution of the prevalent DTUs in humans from the southern United States to Argentina is here presented and the circumstantial evidence of a possible association between T. cruzi genotype and Chagas disease manifestations is discussed. The available information suggests that parasite strains detected in patients, regardless of the clinical presentation, reflect the principal DTU circulating in the domestic transmission cycles of a particular region. In contrast, in several orally transmitted outbreaks, sylvatic strains are implicated. As a consequence of the genotypic and phenotypic differences of T. cruzi strains and the differential geographic distribution of DTUs in humans, regional variations in the sensitivity of the serological tests are verified. The natural resistance to benznidazole and nifurtimox, verified in vivo and in vitro for some parasite stocks, is not associated with any particular DTU, and does not explain the marked difference in the anti-parasitic efficacy of both drugs in the acute and chronic phases of Chagas disease. Throughout this review, it is emphasized that the interplay between parasite and host genetics should have an important role in the definition of Chagas disease pathogenesis, anti-T. cruzi immune response and chemotherapy outcome and should be considered in future investigations.
Leptospirosis has worldwide occurrence and shows higher incidence in tropical areas. Equines can present several serovars according to geographic region and it is important analyze de possible ...contribution of animal displacement on the Leptospira spp. strains occurrence. This research aimed to conduct a serological investigation in an equine regiment belonging to the Brazilian Army Guard Cavalry, resident in an urban area of Rio de Janeiro municipality, but with history of displacement between different regions in Brazil. A microscopic agglutination test was performed in a serum bank of 197 horses using 29 Leptospira spp. strains. The test showed 94.92% positivity, with 90.36% of the animals presenting a positive result for two or more serovars. Higher titers (?1:400) could be detected in 54.31% of horses for at least one serovar, whereas seropositivity for more than one bacterial type occurred mostly at lower titers (<1:400). All animals were clinically healthy. Occurrence of serogroup Icterohaemorraghiae in urban areas was confirmed; however, equine serovars Bratislava and Australis presented low frequencies. The serovars Hardjo, Wolffi, and Canicola were detected, and indicated the presence of ruminants and dogs as reservoirs in proximity in some of the places frequented by the animals. It is suggested that travel history has contributed to the high frequency found and a possible greater contact with different types of Leptospira spp. Therefore, veterinarians should consider the history of geographic displacement and other animal species coexistence with horses when conducting their clinical investigation.
Toxoplasmosis manifests no clinical signs in 80% of cases in immunocompetent patient, causing immunization characterized by the persistence of cysts, particularly in brain, muscles, and retina. ...Assessing the serological status, based on testing for serum toxoplasma IgG and IgM antibodies, is essential in cases that are increasingly at risk for the more severe disease forms, such as congenital or ocular toxoplasmosis. This disease also exposes immunosuppressed patients to reactivation, which can lead to more widespread forms and increased mortality. By interpreting the serological results, we can estimate the risk of contamination or reactivation and define appropriate prophylactic and preventive measures, such as hygienic and dietetic, therapeutic, biological, and clinical follow-up, according to the clinical context. We hereby propose practical approaches based on serological data, resulting from a consensus of a group of experts from the French National Reference Center Network for Toxoplasmosis, according to both routine and specific clinical situations.
Bovine paratuberculosis or Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which affects domestic and wild ruminants around the world. The clinical presentation of MAP ...infection is characterized by chronic diarrhea unresponsive to treatment. The objective of the present study was to investigate the seroprevalence and risk factors associated with bovine paratuberculosis in cattle herds of Tuta, Boyacá, Colombia. This descriptive cross-sectional study with simple random sampling was performed on 882 blood samples taken from cattle of different racial and age groups. Blood samples were processed using an indirect enzyme-linked immunosorbent assay (PARACHEK® 2 Kit, Prionics AG, Switzerland). The obtained data were analyzed by the statistical software Epi Info. In this study, a general seroprevalence of 3.1% was found, and seropositivity in females was 3.6%. The highest prevalence of antibodies against MAP was in individuals > 4 years (5%) and the Jersey breed (4.8%). Therefore, the age of over 4 years was identified as a risk factor associated with MAP. Moreover, a statistical association was found between management and biosafety variables, such as pen management (p = 0.012), feeding with concentrate (p = 0.012), and the presence of diarrhea on the farm (p = 0.048). It could be concluded that the disease is present in Tuta, however, considering factors, such as the chronicity of the disease and the diagnostic method used, it is expected that the number of infected animals is much higher than presented in this research.
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•Innovative peptides SPR imunossensor for canine leishmaniasis diagnosis.•Comparison of two immunogenic synthetic peptides as bioreceptors.•This simple label-free methodology allowed ...fast, sensitive, and selective analysis.•Via PCA was able to observe that even smaller times of detection can be explored.•Excellent ability to discriminate between positive and negative canine real samples.
A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor’s analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis (CVL), an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol L-1), and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs’ samples. For this reason, the PEP13 immunosensor was used to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.
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This study aimed to evaluate different serological strategies for the postnatal diagnosis of congenital toxoplasmosis (CT) and establish a biological algorithm for CT diagnosis. The study ...analyzed serological data of immunoglobulins M, A, and G (IgM, IgA, IgG) performed by immunoenzymatic and compared immunological profile (CIP) assays in 668 newborns with CT diagnosis across four testing periods: P1 (D0– D10), P2 (D11–D35), P3 (D36–D45), and P4 (>D45). Forty-nine percent of the 668 CT cases were diagnosed during P1 and 34%, 4%, and 12% during P2, P3, and P4, respectively. CIP assays detected neosynthetized IgMs/IgGs in 98% of CT cases diagnosed during P1, while IgMs and IgAs were detected in 90% and 57% of CT cases diagnosed during P2 and in 88% and 67% of diagnoses made during P3, respectively. Detection of neosynthesized IgMs/IgGs, IgMs, and IgAs by immunoassay contributed to CT diagnosis in 81%, 77%, and 60% of cases, respectively. In total, 46% of serum samples were positive for all three parameters, 27% for two, and 27% for one of the three. The study recommends using the CIP assay as standard during P1 for CT diagnosis and IgM and IgA immunoassays after P1. A clinical and biological follow-up in a specialized center with a close collaboration between biologists and clinicians is highly recommended to increase the chances of early diagnosis. Overall, this study provides useful information for the development of a biological algorithm for CT diagnosis, which can aid in early detection and appropriate treatment of this disease.
•Serum of D. repens experimentally-infected dogs tested using heartworm antigen test.•No cross-reactivity was observed in D. repens serum samples prior to heat- treatment.•Heat-treatment induces ...false-positive antigen results in D. repens infected serum.•Cross-reactivity was observed before patency and remained throughout infection.
Antigen enzyme-linked immunosorbent assay tests are widely used for the diagnosis of heartworm infection in dogs. While commercially-available heartworm antigen tests have high sensitivity and specificity, false-negative test results can occur in dogs with low worm burdens, female-only infections, or prior to patency. The use of immune complex dissociation (ICD) methods have demonstrated increased sensitivity in the detection of Dirofilaria immitis antigens and the reversal of false-negative antigen results. However, there are concerns pertaining to false-positive antigen results due to infections of other nematode parasites, especially post-ICD. Therefore, this study evaluated the effect of heat-treatment on serum samples of dogs experimentally-infected with Dirofilaria repens during the course of infection, to assess for potential cross-reactivity on heartworm antigen tests. Archived serum samples from three dogs experimentally-infected with D. repens were utilized. All samples were tested for cross-reactivity pre- and post-heat-treatment using the DiroCHEK® Heartworm Antigen test kit throughout infection (day -9 through 404 days post-infection; dpi). All heat-treated samples tested false-positive starting at 164 dpi and continuing through 404 dpi, thereby testing positive prior to patency. No cross-reactivity was observed for any dog at any time point prior to heat-treatment. Our results suggest that the ICD method decreased the specificity of heartworm antigen tests and caused cross-reactivity of serum from dogs experimentally infected with D. repens. In conclusion, heat-treatment of serum in areas co-endemic for D. repens and D. immitis has limited clinical value, and should be used with caution.
The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection ...in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti-T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63–0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30–0.91). Cutoff readjustments increased the Se and Sp values to equal to or above 97% for all tests, except for TgSALUVET WB, which can be used as a reference for initial validation of tests, but it is not recommended for routine diagnosis. Seroconversion was recorded from two weeks post-infection by most of the tests, with significantly higher IgG levels in sera from pigs infected with the T. gondii type III vs. type II isolate. Again, differences regarding the test employed were observed. Differences in the diagnostic performance among tests evidenced the need to harmonize serological techniques to obtain comparable and reliable results.
•Western blot is an appropriate reference test for initial technique validation.•A comparative study of commercial and in-house tests led to harmonized serological results.•Pigtype ELISA, TgSALUVET ELISA 2.0 and TgSALUVET IFAT showed excellent diagnostic performance.•The PrioCHECK and IDScreen ELISA tests required further readjustments to increase Se and Sp.•Higher IgG levels were detected in pigs infected with type III vs. type II isolates by most of the tests.
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