Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Electrochemical magneto-immunoassay for detection of zika virus antibody in human serum.

Zika virus (ZIKV) is a flavivirus transmitted by infected Aedes genus mosquitoes. An infected person may be asymptomatic or present symptoms such as fever, arthralgia, and in pregnancy it may lead to neurological disorders in the fetus, such as microcephaly. Based on the high dissemination potential of ZIVK and its similar antigen composition to other arboviruses, new approaches for selective virus detection are urgently needed. This work reports the development of an electrochemical immunoassay for detection of anti-ZIKV antibodies, using magnetic beads functionalized with recombinant protein derived from the non-structural protein 1 (ΔNS1-ZIKV) and anti-IgG antibodies labeled with horseradish peroxidase (HRP) enzyme. The magneto-immunoassay uses disposable microfluidic devices for detection of anti-ZIKV in serum samples. A linear response was obtained for a wide concentration range from 0.01 to 9.80 × 105 pg mL-1 (r2 = 0.982), with a limit of detection of 0.48 pg mL-1. The proposed immunoassay proved to be highly efficient for the detection of anti-ZIKV antibodies in serum, offering promising perspectives for the development of fast, simple, and affordable point-of-care diagnosis devices for ZIKV.

Electrochemical magneto-immunoassay for detection of zika virus antibody in human serum

Zika virus (ZIKV) is a flavivirus transmitted by infected Aedes genus mosquitoes. An infected person may be asymptomatic or present symptoms such as fever, arthralgia, and in pregnancy it may lead to neurological disorders in the fetus, such as microcephaly. Based on the high dissemination potential of ZIVK and its similar antigen composition to other arboviruses, new approaches for selective virus detection are urgently needed. This work reports the development of an electrochemical immunoassay for detection of anti-ZIKV antibodies, using magnetic beads functionalized with recombinant protein derived from the non-structural protein 1 (ΔNS1-ZIKV) and anti-IgG antibodies labeled with horseradish peroxidase (HRP) enzyme. The magneto-immunoassay uses disposable microfluidic devices for detection of anti-ZIKV in serum samples. A linear response was obtained for a wide concentration range from 0.01 to 9.80 × 105 pg mL−1 (r2 = 0.982), with a limit of detection of 0.48 pg mL−1. The proposed immunoassay proved to be highly efficient for the detection of anti-ZIKV antibodies in serum, offering promising perspectives for the development of fast, simple, and affordable point-of-care diagnosis devices for ZIKV.

Validation of Serological Methods for COVID-19 and Retrospective Screening of Health Employees and Visitors to the São Paulo University Hospital, Brazil.

Reliable serological tests for the detection of SARS-CoV-2 antibodies among infected or vaccinated individuals are important for epidemiological and clinical studies. Low-cost approaches easily adaptable to high throughput screenings, such as Enzyme-Linked Immunosorbent Assays (ELISA) or electrochemiluminescence immunoassay (ECLIA), can be readily validated using different SARS-CoV-2 antigens. A total of 1,119 serum samples collected between March and July of 2020 from health employees and visitors to the University Hospital at the University of São Paulo were screened with the Elecsys® Anti-SARS-CoV-2 immunoassay (Elecsys) (Roche Diagnostics) and three in-house ELISAs that are based on different antigens: the Nucleoprotein (N-ELISA), the Receptor Binding Domain (RBD-ELISA), and a portion of the S1 protein (ΔS1-ELISA). Virus neutralization test (CPE-VNT) was used as the gold standard to validate the serological assays. We observed high sensitivity and specificity values with the Elecsys (96.92% and 98.78%, respectively) and N-ELISA (93.94% and 94.40%, respectively), compared with RBD-ELISA (90.91% sensitivity and 88.80% specificity) and the ΔS1-ELISA (77.27% sensitivity and 76% specificity). The Elecsys® proved to be a reliable SARS-CoV-2 serological test. Similarly, the recombinant SARS-CoV-2 N protein displayed good performance in the ELISA tests. The availability of reliable diagnostic tests is critical for the precise determination of infection rates, particularly in countries with high SARS-CoV-2 infection rates, such as Brazil. Collectively, our results indicate that the development and validation of new serological tests based on recombinant proteins may provide new alternatives for the SARS-CoV-2 diagnostic market.

Histidine-based hydrogels via singlet-oxygen photooxidation.

The formation of hydrogels by photosensitized oxidation and crosslinking of histidine-derived polymers is demonstrated for the first time. The photooxidation of pendant His mediated by singlet oxygen was used to promote covalent coupling by its dimerization. As a proof-of-concept, two systems were studied: (i) chondroitin sulfate (CS) functionalized with His, and (ii) an elastin-like peptide (ELP) containing His produced by recombinant techniques. Both materials were crosslinked by irradiation at 425 nm in the presence of Zn-porphyrin derivatives yielding His-based hydrogels. The molecular structure and physicochemical properties of ELP-His and other 5 ELPs with photooxidizable amino acids were studied in silica by computer simulation. A correlation between the protein conformation and its elastic properties is discussed. CS-His hydrogels demonstrate larger storage moduli than ELPs with other amino acids. The obtained results show the potential use of photooxidation to create a new type of His-based hydrogels.

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 Replication by Hypertonic Saline Solution in Lung and Kidney Epithelial Cells.

An unprecedented global health crisis has been caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We performed experiments to test if a hypertonic saline solution was capable of inhibiting virus replication. Our data show that 1.2% NaCl inhibited virus replication by 90%, achieving 100% of inhibition at 1.5% in the nonhuman primate kidney cell line Vero, and 1.1% of NaCl was sufficient to inhibit the virus replication by 88% in human epithelial lung cell line Calu-3. Furthermore, our results indicate that the inhibition is due to an intracellular mechanism and not to the dissociation of the spike SARS-CoV-2 protein and its human receptor. NaCl depolarizes the plasma membrane causing a low energy state (high ADP/ATP concentration ratio) without impairing mitochondrial function, supposedly associated with the inhibition of the SARS-CoV-2 life cycle. Membrane depolarization and intracellular energy deprivation are possible mechanisms by which the hypertonic saline solution efficiently prevents virus replication in vitro assays.

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

Inhibition of severe acute respiratory syndrome coronavirus 2 replication by hypertonic saline solution in lung and kidney epithelial cells

An unprecedented global health crisis has been caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We performed experiments to test if a hypertonic saline solution was capable of inhibiting virus replication. Our data show that 1.2% NaCl inhibited virus replication by 90%, achieving 100% of inhibition at 1.5% in the nonhuman primate kidney cell line Vero, and 1.1% of NaCl was sufficient to inhibit the virus replication by 88% in human epithelial lung cell line Calu-3. Furthermore, our results indicate that the inhibition is due to an intracellular mechanism and not to the dissociation of the spike SARS-CoV-2 protein and its human receptor. NaCl depolarizes the plasma membrane causing a low energy state (high ADP/ATP concentration ratio) without impairing mitochondrial function, supposedly associated with the inhibition of the SARS-CoV-2 life cycle. Membrane depolarization and intracellular energy deprivation are possible mechanisms by which the hypertonic saline solution efficiently prevents virus replication in vitro assays.

CD4+ T Cells Cross-Reactive with Dengue and Zika Viruses Protect against Zika Virus Infection.

The underlying mechanisms by which prior immunity to dengue virus (DENV) affords cross-protection against the related flavivirus Zika virus (ZIKV) are poorly understood. Here, we examine the ability of DENV/ZIKV-cross-reactive CD4+ T cells to protect against versus exacerbate ZIKV infection by using a histocompatibility leukocyte antigen (HLA)-DRB1∗0101 transgenic, interferon α/ß receptor-deficient mouse model that supports robust DENV and ZIKV replication. By mapping the HLA-DRB1∗0101-restricted T cell response, we identify DENV/ZIKV-cross-reactive CD4+ T cell epitopes that stimulate interferon gamma (IFNγ) and/or tumor necrosis factor (TNF) production. Vaccination of naive HLA-DRB1∗0101 transgenic mice with these peptides induces a CD4+ T cell response sufficient to reduce tissue viral burden following ZIKV infection. Notably, this protective response requires IFNγ and/or TNF secretion but not anti-ZIKV immunoglobulin G (IgG) production. Thus, DENV/ZIKV-cross-reactive CD4+ T cells producing canonical Th1 cytokines can suppress ZIKV replication in an antibody-independent manner. These results may have important implications for increasing the efficacy and safety of DENV/ZIKV vaccines and for developing pan-flavivirus vaccines.

Expression of a soluble IL-10 receptor enhances the therapeutic effects of a papillomavirus-associated antitumor vaccine in a murine model.

The presence of IL-10, produced either by tumor cells or immunosuppressive cells, is frequently associated with a poor prognosis for cancer progression. It may also negatively impact anticancer treatments, such as immunotherapies, that otherwise would promote the activation of cytotoxic T cells capable of detecting and destroying malignant cells. In the present study, we evaluated a new adjuvant approach for anticancer immunotherapy using a plasmid vector encoding a soluble form of the IL-10 receptor (pIL-10R). pIL-10R was coadministered to mice with a DNA vaccine encoding the type 16 human papillomavirus (HPV-16) E7 oncoprotein genetically fused with glycoprotein D of herpes simplex virus (HSV) (pgDE7h). Immunization regimens based on the coadministration of pIL-10R and pgDE7h enhanced the antitumor immunity elicited in mice injected with TC-1 cells, which express HPV-16 oncoproteins. The administration of the DNA vaccines by in vivo electroporation further enhanced the anticancer effects of the vaccines, leading to the activation of tumor-infiltrating polyfunctional E7-specific cytotoxic CD8+ T cells and control of the expansion of immunosuppressive cells. In addition, the combination of immunotherapy and pIL-10R allowed the control of tumors in more advanced growth stages that otherwise would not be treatable by the pgDE7h vaccine. In conclusion, the proposed treatment involving the expression of IL-10R enhanced the antitumor protective immunity induced by pgDE7h administration and may contribute to the development of more efficient clinical interventions against HPV-induced tumors.

Nano-multilamellar lipid vesicles (NMVs) enhance protective antibody responses against Shiga toxin (Stx2a) produced by enterohemorrhagic Escherichia coli strains (EHEC).

Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems.

Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive System.

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Persistence and intra-host genetic evolution of zika virus infection in symptomatic adults: A special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus

Biosensor-based selective detection of Zika virus specific antibodies in infected individuals.

Zika virus (ZIKV) recently emerged as a global threat subsequent to its global spread because it induces microencephaly and other brain damages in infants born to infected mothers. Epidemiological monitoring of infection has been hampered by the absence of reliable serological tests capable to distinguish between ZIKV and other Flavivirus infections, in particular Dengue virus (DENV). As both viruses are transmitted by the same mosquito-species, their distributions largely overlap and reliable serological distinction between the viruses is essential. Here we develop a novel biosensor which is based on recombinant forms of ZIKV non-structural protein 1 (NS1) and the domain III of the envelope protein (EDIII). Using electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), we demonstrate that in addition to extremely sensitive detection of ZIKV-specific antibodies in serum and saliva, the biosensor promptly distinguished ZIKV and DENV-specific antibodies. Hence, this novel biosensor allows assessing ZIKV antibodies in blood and saliva and results are unaffected by presence of DENV virus-specific antibodies.

Persistence and intra-host genetic evolution of Zika virus infection in symptomatic adults: a special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Persistence and intra-host genetic evolution of Zika virus infection in symptomatic adults: a special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Induction of resident memory T cells enhances the efficacy of cancer vaccine.

Tissue-resident memory T cells (Trm) represent a new subset of long-lived memory T cells that remain in tissue and do not recirculate. Although they are considered as early immune effectors in infectious diseases, their role in cancer immunosurveillance remains unknown. In a preclinical model of head and neck cancer, we show that intranasal vaccination with a mucosal vector, the B subunit of Shiga toxin, induces local Trm and inhibits tumour growth. As Trm do not recirculate, we demonstrate their crucial role in the efficacy of cancer vaccine with parabiosis experiments. Blockade of TFGß decreases the induction of Trm after mucosal vaccine immunization, resulting in the lower efficacy of cancer vaccine. In order to extrapolate this role of Trm in humans, we show that the number of Trm correlates with a better overall survival in lung cancer in multivariate analysis. The induction of Trm may represent a new surrogate biomarker for the efficacy of cancer vaccine. This study also argues for the development of vaccine strategies designed to elicit them.

Diarrheagenic Escherichia coli

ABSTRACT Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

Diarrheagenic Escherichia coli

Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

Diarrheagenic Escherichia coli.

Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.