Optimization of Zika virus envelope protein production for ELISA and correlation of antibody titers with virus neutralization in Mexican patients from an arbovirus endemic region.

BACKGROUND: Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. METHODS: We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera. RESULTS: Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro. CONCLUSIONS: Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.

Prevalence of Human Papillomavirus in Women from the State of Michoacan, Mexico, Showed High Frequency of Unusual Virus Genotypes.

BACKGROUND: Human papillomaviruses (HPVs), the leading cause of cervical cancer, are distributed worldwide, with high prevalence in developing countries. OBJECTIVE: The objective of the study is to know the prevalence and genotypes of HPV in women from the state of Michoacán and the Women's Hospital in Morelia, Michoacán. MATERIALS AND METHODS: Cervical smear samples (159,288) were subjected to HPV detection by hybrid capture 2. A subsample of 484 patients from the Women's Hospital was studied by Papanicolaou test and linear array HPV genotyping, and when positive, patients were also examined by colposcopy and histopathology. RESULTS: The overall prevalence for HPV in Michoacán State was 7.74%; 7.11% in 2009, 6.46% in 2010, 9.58% in 2011, and 8.43% in 2012. The highest prevalence was found in the age groups < 25 and 25-34 years. The prevalence at the Women's Hospital was 8.51%. Cytological examination revealed normal cytology in 64.44% of samples, 26.66 % with low-grade and 8.88 % with high-grade squamous intraepithelial lesion (HSIL). However, by colposcopy, normal tissue appearance was found only in 26.66%; 51% were reclassified as low-grade squamous intraepithelial lesion, 17.77% as HSIL, and in 4.4% atrophy was observed. The most prevalent genotype in single infections was HPV59, followed by HPV51 and HPV45. Double infections occurred with the following genotypes: 52-53, 51-59, 61-67, 66-11, 16-62, 53-62, 59-CP6108, 45-66, and 45-51. Triple infections were identified as: 6-31-39, 51-59-62, 51-62-81, 54-55-59, 16-58-71, and 16-59-62. CONCLUSIONS: The prevalent genotype found among women from Michoacán, HPV59, was different to the rest of the country. The high prevalence of HPV59 could be due to cases imported to Michoacán by agricultural workers migrating to the USA or may be associated to ethnicity differences. Implications of this finding for immunization programs should be explored.

Temperature-dependent secretion of Zika virus envelope and non-structural protein 1 in mammalian cells for clinical applications.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus associated with congenital Zika syndrome and Guillain-Barré syndrome in adults. The recombinant ZIKV envelope (E) antigen can be useful for serodiagnosis of ZIKV infection and for monitoring immune responses during preclinical and clinical ZIKV vaccine development. In this study, we describe production of ZIKV E using the modified polyethyleneimine (PEI) transfection in HEK293 cells to improve cost-effective large-scale production. We show that the secretion of ZIKV E in HEK293 cells is dependent on cell culture incubation temperatures where incubation at a low temperature of 28 °C improved protein secretion of both, E-CD4 and E, whereas a substantial decrease in secretion was observed at 37 °C. The resulting E-CD4 produced at low temperature yielded similar binding profiles in ELISAs in comparison with a commercially available E protein using human seropositive sera to ZIKV. We also show that ZIKV NS1 and NS1 ß-ladder antigens produced in HEK293 cells, have similar binding profiles in ELISA which suggests that both NS1 or NS1 ß-ladder can be used for serodiagnosis of ZIKV. In conclusion, we propose a cost-effective production of the ZIKV E and NS1, suitable for both, clinical and research applications in endemic countries.

Investigation of a food-borne Salmonella Oranienburg outbreak in a Mexican prison.

INTRODUCTION: Gastroenteritis outbreaks in prisons represent a public health risk worldwide. Identifying and characterizing the etiological agents of gastroenteritis outbreaks in prisons is important for implementing effective prevention and infection control measures. We present the first studied case of a gastroenteritis outbreak in a Mexican prison. METHODOLOGY: Rectal swab samples were obtained from affected inmates. Standard microbiological techniques were used for isolating Salmonella enterica. Isolates were typed by PCR assays of DNA repetitive elements (ERIC, BOX, REP) and RAPD. Antibiotic resistance profiles were performed by the Kirby-Bauer method. RESULTS: S. enterica serotype Oranienburg was responsible for the outbreak affecting 150 inmates. All patients presented diarrhea, and 70% of them also presented vomiting, with no fatal cases. The origin of the outbreak was undetermined due to the difficulty of gathering epidemiological information, but was likely the result of consumption of shrimp broth or a cantaloupe melon beverage. REP, BOX, and ERIC analyses of 26 serotype Oranienburg strains resulted in Simpson discrimination index (D) values of 0, 0.5507, and 0.5661, respectively. The D values from DG93-RAPD analyses and from the combined ERIC-BOX-DG93 markers were 0.7753 and 0.6092, respectively. All strains showed multiresistance to antibiotics. CONCLUSIONS: This is the only studied case of a gastroenteritis outbreak in a Mexican prison, and of the first such outbreak caused by serotype Oranienburg. The combined ERIC, BOX, and RAPD markers adequately assessed the genotype diversity of analyzed strains. Penitentiary personnel or inmates involved in outbreaks might spread multiresistant strains outside of the facility.

Calidad sanitaria de las salsas crudas y cocidas en 50 restaurantes de la ciudad de Morelia / Health quality of raw and boiled sauces in 50 restaurants of Morelia city

Antecedentes: las enfermedades transmitidas por alimentos tienen gran importancia en el terreno epidemiológico, así como implicaciones económicas y laborales. Objetivo: determinar la calidad sanitaria de las salsas crudas y cocidas en restaurantes de la ciudad de Morelia, Michoacán. Material y métodos: se obtuvieron 216 muestras de salsas de 250 g cada una, 161 cocidas y 55 crudas, en 50 restaurantes de Morelia, las cuales se procesaron conforme a los métodos oficiales para detectar microorganismos indicadores de contaminación. Resultados: el 81 por ciento de las muestras de salsas cocidas y el 69 por ciento de las crudas se hallaron fuera de los valores establecidos en la normatividad sanitaria para bacterias mesófilas aerobias (BMA), organismos coliformes totales (OCT) y organismos coliformes fecales (OFC). Los resultados indican una elevada contaminación microbiana debida, tal vez, a deficiente lavado y desinfección antes de la elaboración de los productos referidos, además de que su manipulación propicia contaminación cruzada. Las elevadas cifras de bacterias mesófilas aerobias y organismos coliformes totales en las salsas cocidas indican una contaminación posproceso debida posiblemente a su manipulación y/o uso de equipos y utensilios contaminados. Conclusiones: se recomienda la verificación sanitaria y el muestreo continuo de los alimentos, además del fomento y asesoría correspondientes, con lo que se contribuirá a disminuir la frecuencia de las enfermedades transmitidas por alimentos.

Contaminación bacteriana de pescados y mariscos de venta callejera en el medio urbano / Bacterial contamination of street sold fish and sea-food

Antecedentes. Las infecciones gastrointestinales son una de las primeras causas de morbilidad en nuestro país, y su origen está ligado, entre otros factores, al consumo de alimentos contaminados. De entre ellos, los pescados y mariscos, por ser ricos en nutrientes, son un campo propicio para el desarrollo bacteriano. Sin embargo, no existen datos fidedignos acerca de sus condiciones microbiológicas en sitios de venta callejera en el medio urbano. Material y método. de 74 puestos callejeros de pescados y mariscos, se tomaron 132 muestras de pescado, así como de mariscos e ingredientes utilizados en su preparación y/o presentación, ya listos para su consumo. Se determinaron la existencia de coliformes fecales y bacterias mesofílicas aerobias, como marcadores de contaminación fecal, y de Salmonella sp. y Vibrio Cholerae, como agentes patógenos. Lo anterior se hizo mediante cultivo en medios específicos y pruebas bioquímicas y serológicas. Resultados. Se encontró una contaminación con coliformes fecales de 89 y 88 por ciento con bacterias mesofílicas aerobias. Se encontró sólo un caso positivo para Salmonella y dos para Vibrio Cholerae no O:I. Los alimentos más contaminados fueron el pulpo, los ostiones, los cocteles ya preparados, la cebolla y el cilantro. Conclusiones. Se confirma la grave contaminación fecal de estos alimentos y la necesidad imperiosa de establecer programas de educación para la salud entre los expendedores y los consumidores de estos productos