The use of peptides for immunodiagnosis of human Chagas disease.

Chagas disease, caused by the protozoa Trypanosoma cruzi, continues to be a serious public health problem in Latin America, worsened by the limitations in its detection. Given the importance of developing new diagnostic methods for this disease, the present review aimed to verify the number of publications dedicated to research on peptides that demonstrate their usefulness in serodiagnosis. To this end, a bibliographic survey was conducted on the PubMed platform using the keyword "peptide" or "epitope" combined with "Chagas disease" or "Trypanosoma cruzi"; "diagno*" or "serodiagnosis" or "immunodiagnosis", without period restriction. An increasing number of publications on studies employing peptides in ELISA and rapid tests assays was verified, which confirms the expansion of research in this field. It is possible to observe that many of the peptides tested so far originate from proteins widely used in the diagnosis of Chagas, and many of them are part of commercial tests developed. In this sense, as expected, promising results were obtained for several peptides when tested in ELISA, as many of them exhibited sensitivity and specificity values above 90%. Furthermore, some peptides have been tested in several studies, confirming their diagnostic potential. Despite the promising results observed, it is possible to emphasize the need for extensive testing of peptides, using different serological panels, in order to confirm their potential. The importance of producing an effective assay capable of detecting the clinical stages of the disease, as well as new immunogenic antigens that enable new serological diagnostic tools for Chagas disease, is evident.

Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

Molecular methods for diagnosis of monkeypox: A mini-review.

BACKGROUND: Monkeypox is a global public health issue caused by the monkeypox virus (MPXV). As of October 28, 2022, a total of 77,115 laboratory-confirmed cases and 3,610 probable cases, including 36 deaths, were reported, with 9,070 cases reported in Brazil, the second most affected country. The need to develop national technologies for the rapid diagnosis of emerging diseases for mass testing of the population is evident, as observed in the SARS-CoV-2 pandemic. OBJECTIVE: With that in mind, this article provides an overview of current methods, techniques, and their applications in the molecular detection of monkeypox, focusing the search on real-time polymerase chain reaction (qPCR), polymerase chain reaction (PCR), and polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). METHODS: The relevant documents or papers covered in this study were selected by a search in international bibliographic databases. The search terms used in the databases were aimed at summarizing existing knowledge on molecular diagnostic methods, such as monkeypox; MPX, MPXV, qPCR, PCR, PCR-ELISA, diagnosis and detection searched separately or together using the Boolean operator "AND" either in the title or abstract. The searches took place in September 2022, and the corresponding articles were selected between 2012 and 2022. RESULTS: We found 256 documents in total and twelve studies addressing the molecular diagnosis of monkeypox were classified as possible sources for this review. CONCLUSION: It is evident there is a pressing need to develop national technologies for rapid diagnosis of emerging diseases for mass testing of the population. It is also extremely important to have national detection kits with greater diagnostic capacity to assist in developing effective public policies in countries affected by this disease.

Rational Design and Evaluation of the Recombinant Multiepitope Protein for Serodiagnosis of Rubella.

BACKGROUND: Rubella is an infection caused by rubella virus (RV) and is generally regarded as a mild childhood disease. The disease continues to be of public health importance mainly because when the infection is acquired during early pregnancy, it often results in fetal abnormalities, which are classified as congenital rubella syndrome (CRS). An accurate diagnosis of rubella is thus of pivotal importance for proper treatment. OBJECTIVES: The aim of the study was to produce a recombinant multiepitope protein (rMERUB) for the diagnosis of rubella, based on conserved immunodominant epitopes of glycoprotein E1 and E2. METHODS: A synthetic gene was designed and cloned into vector pET21a with a 6xHis tag at the Cterminal for affinity purification and overexpressed in Escherichia coli cells. Biophysical analysis of rMERUB was performed by circular dichroism. Biological activity was assessed using an in-house ELISA assay. RESULTS: Expression in Escherichia coli showed a ~22 kDa protein that was purified and used to perform structural assays and an IgG ELISA. Structural analyses reveal that rMERUB has a ß leaf pattern that promotes the exposure of epitopes, thus allowing antibody recognition. Evaluation of 33 samples (22=positive; 11=negative) was performed using in-house ELISA and this was compared with a commercial kit. The sensitivity was 100% (95% CI: 85-100) and specificity 90.91% (95% CI: 62-99). Excellent agreement (Kappa index = 0.9) was obtained between ELISA assays. CONCLUSION: The careful choice of epitopes and the high epitope density, coupled with simple-step purification, pinpoints rMERUB as a promising alternative for rubella diagnosis, with potential for the development of a diagnostic kit.

Vancomycin-loaded N,N-dodecyl,methyl-polyethylenimine nanoparticles coated with hyaluronic acid to treat bacterial endophthalmitis: Development, characterization, and ocular biocompatibility.

Vancomycin-loaded N,N-dodecyl,methyl-polyethylenimine nanoparticles coated with hyaluronic acid (VCM-DMPEI nanoparticles/HA) were synthesized as an adjuvant for the treatment of bacterial endophthalmitis. The nanoparticles were formulated by experimental statistical design, thoroughly characterized, and evaluated in terms of bactericidal activity and both in vitro and in vivo ocular biocompatibility. The VCM-DMPEI nanoparticles/HA were 154 ± 3 nm in diameter with a 0.197 ± 0.020 polydispersity index; had a + 26.4 ± 3.3 mV zeta potential; exhibited a 93% VCM encapsulation efficiency; and released 58% of the encapsulated VCM over 96 h. VCM and DMPEI exhibited a synergistic bactericidal effect. The VCM-DMPEI nanoparticles/HA were neither toxic to ARPE-19 cells nor irritating to the chorioallantoic membrane. Moreover, the VCM-DMPEI nanoparticles/HA did not induce modifications in retinal functions, as determined by electroretinography, and in the morphology of the ocular tissues. In conclusion, the VCM-DMPEI nanoparticles/HA may be a useful therapeutic adjuvant to treat bacterial endophthalmitis.

Anti-CEA loaded maghemite nanoparticles as a theragnostic device for colorectal cancer.

Nanosized maghemite particles were synthesized, precoated (with dimercaptosuccinic acid) and surface-functionalized with anticarcinoembryonic antigen (anti-CEA) and successfully used to target cell lines expressing the CEA, characteristic of colorectal cancer (CRC) cells. The as-developed nanosized material device, consisting of surface decorated maghemite nanoparticles suspended as a biocompatible magnetic fluid (MF) sample, labeled MF-anti-CEA, was characterized and tested against two cell lines: a high-CEA expressing cell line (LS174T) and a low-CEA expressing cell line (HCT116). Whereas X-ray diffraction was used to assess the average core size of the as-synthesized maghemite particles (average 8.3 nm in diameter), dynamic light scattering and electrophoretic mobility measurements were used to obtain the average hydrodynamic diameter (550 nm) and the zeta-potential (-38 mV) of the as-prepared and maghemite-based nanosized device, respectively. Additionally, surface-enhanced Raman spectroscopy (SERS) was used to track the surface decoration of the nanosized maghemite particles from the very first precoating up to the attachment of the anti-CEA moiety. The Raman peak at 1655 cm(-1), absent in the free anti-CEA spectrum, is the signature of the anti-CEA binding onto the precoated magnetic nanoparticles. Whereas MTT assay was used to confirm the low cell toxicity of the MF-anti-CEA device, ELISA and Prussian blue iron staining tests performed with both cell lines (LS174T and HCT116) confirm that the as-prepared MF-anti- CEA is highly specific for CEA-expressing cells. Finally, transmission electron microscopy analyses show that the association with anti-CEA seems to increase the number of LS174T cells with internalized maghemite nanoparticles, whereas no such increase seems to occur in the HCT116 cell line. In conclusion, the MF-anti-CEA sample is a biocompatible device that can specifically target CEA, suggesting its potential use as a theragnostic tool for CEA-expressing tumors, micrometastasis, and cancer-circulating cells.