Analysis of influenza data generated by four epidemiological surveillance laboratories in Mexico, 2010-2016.

The disease caused by the influenza virus is a global public health problem due to its high rates of morbidity and mortality. Thus, analysis of the information generated by epidemiological surveillance systems has vital importance for health decision making. A retrospective analysis was performed using data generated by the four molecular diagnostic laboratories of the Mexican Social Security Institute between 2010 and 2016. Demographics, influenza positivity, seasonality, treatment choices and vaccination status analyses were performed for the vaccine according to its composition for each season. In all cases, both the different influenza subtypes and different age groups were considered separately. The circulation of A/H1N1pdm09 (48.7%), influenza A/H3N2 (21.1%), influenza B (12.6%), influenza A not subtyped (11%) and influenza A/H1N1 (6.6%) exhibited well-defined annual seasonality between November and March, and there were significant increases in the number of cases every 2 years. An inadequate use of oseltamivir was determined in 38% of cases, and the vaccination status in general varied between 12.1 and 18.5% depending on the season. Our results provide current information about influenza in Mexico and demonstrate the need to update both operational case definitions and medical practice guidelines to reduce the inappropriate use of antibiotics and antivirals.

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

Establishment and cryptic transmission of Zika virus in Brazil and the Americas.

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Complete Genome Sequence of Human Respiratory Syncytial Virus Isolated in Mexico.

Human respiratory syncytial virus (HRSV) is a member of the Paramyxoviridae family, which causes lower respiratory tract infections in neonates and children younger than 5 years. Here, we report the complete genome sequence of HRSV, isolated from a nasopharyngeal swab of a pregnant woman with cardiac complications.

Vaccination with Leishmania mexicana LPG induces PD-1 in CD8⁺ and PD-L2 in macrophages thereby suppressing the immune response: a model to assess vaccine efficacy.

Leishmania lipophosphoglycan (LPG) is a molecule that has been used as a vaccine candidate, with contradictory results. Since unsuccessful protection could be related to suppressed T cell responses, we analyzed the expression of inhibitory receptor PD-1 in CD8(+) and CD4(+) lymphocytes and it is ligand PD-L2 in macrophages of BALB/c mice immunized with various doses of Leishmania mexicana LPG and re-stimulated in vitro with different concentrations of LPG. Vaccination with LPG enhanced the expression of PD-1 in CD8(+) cells. Activation molecules CD137 were reduced in CD8(+) cells from vaccinated mice. In vitro re-stimulation enhanced PD-L2 expression in macrophages of healthy mice in a dose-dependent fashion. The expression of PD-1, PD-L2 and CD137 is modulated according to the amount of LPG used during immunization and in vitro re-stimulation. We analyzed the expression of these molecules in mice infected with 1×10(4) or 1×10(5)L. mexicana promastigotes and re-stimulated in vitro with LPG. Infection with 1×10(5) parasites increased the PD-1 expression in CD8(+) and diminished PD-L2 in macrophages. When these CD8(+) cells were re-stimulated in vitro with LPG, simulating a second exposure to parasite antigens, PD-1 expression increased significantly more, in a dose dependent fashion. We conclude that CD8(+) T lymphocytes and macrophages express inhibition molecules according to the concentrations of Leishmania LPG and to the parasite load. Vaccination with increased amounts of LPG or infections with higher parasite numbers induces enhanced expression of PD-1 and functional inactivation of CD8(+) cells, which can have critical consequences in leishmaniasis, since these cells are crucial for disease control. These results call for pre-vaccination evaluations of potential immunogens, specifically where CD8 cells are required, since inhibiting molecules can be induced after certain thresholds of antigen concentrations. We propose that the analysis of PD-1 and PD-L2 are useful tools to monitor the optimal dose for vaccination candidates.

TUNEL-positive cells in the surgical border of an amputation due to infected diabetic foot.

Diabetic infected foot is the outcome of progressive vascular and neurological damage caused by persistent chronic hyperglycemia. Due to acute hypoxia and infection, the tissues develop extensive necrosis and gangrene, which often require amputation. The decision regarding the level of amputation relies mainly on the personal experience of the surgeon who must identify the healthy tissue without necrosis. However, tissue cells under stress may succumb before clear evidence of necrosis is present. In this study, dying cells with DNA damage were identified in the necrotic lesions and surgical borders of amputations. Therefore, the main purpose of this study was to identify apoptosis in the surgical borders of amputations required to treat infected diabetic foot. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated bio-dUTP nick-end labeling (TUNEL) in the superficial and deep tissues of wounds, and in the surgical borders of 10 consecutive adult patients with diabetes mellitus type 2 (DM2) who underwent amputation due to infected diabetic foot. The severity of the disease was classified by the Acute Physiological and Chronic Health Evaluation II (APACHE II) score on admission, and laboratory data were collected and bacteriological cultures were obtained from the lesions. The ankle/arm blood pressure index was measured, the blood flow in the affected limb was evaluated by high-resolution ultrasonography and color Doppler and pulse oximetry were performed during surgery. A total of 5 males and 5 females, aged 45-84 years (58.8 ± 14.1), were included. The APACHE II score was 2-18 points (8 ± 5.7). A total of 9 patients developed sepsis and 2 succumbed. A total of 5 patients required above-ankle amputation, and 5 required toe disarticulation. The ankle/arm blood pressure index ranged from 0.23-0.85 (0.51 ± 0.23). Apoptotic cells were found in ulcers and abscesses, and in areas without necrosis. In the surgical borders of the amputations, apoptotic cells were found in skeletal muscle, blood vessels and peripheral nerves, particularly Schwann cells. Morphometric analysis revealed that the extent of apoptosis was 2-3 logarithms higher in the surgical borders of the infected diabetic foot compared to the venous ulcers, which were used as the reference. In conclusion, apoptosis was identified in regenerating tissues within diabetic foot wounds and in the surgical borders of amputations, where the surgeon considered the tissues to be undamaged. This information suggests that apoptosis may be present before visible signs of necrosis appear in the diabetic foot and may be caused by hypoxia, acidosis or proinflammatory cytokines. The extent of apoptosis in tissues proximal to necrotic areas may anticipate the development of diabetic foot and help the surgeon to make decisions regarding the need and extent of amputation.

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice.

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210-239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.

2-Methoxyestradiol (2-ME) reduces the airway inflammation and remodeling in an experimental mouse model.

Patients with asthma experience airway structural changes, termed airway remodeling, in response to persistent inflammation. 2-Methoxyestradiol (2-ME) is an anti-angiogenic agent and downregulates hypoxia-inducible factor 1 (HIF-1) and inhibits HIF-1alpha-induced transcriptional activation of vascular endothelial growth factor (VEGF) expression. We hypothesized that 2-ME may interfere with the development of the clinical manifestations of asthma. We used a chronic murine model of allergic airway inflammation with subepithelial fibrosis in BALB/c mice. Mice were sensitized with ovalbumin (OVA) that was administered intraperitoneally at days 0-5 and challenged intratracheally (IT) with OVA on days 12-22. The mice received 2-ME IT at days 24, 26 and 28 and sacrificed at day 32. The sensitized/challenged mice developed an extensive cell inflammatory response of the airways. 2-ME administration significantly reduced the cellular infiltrate in the perivascular and peribronchial lung tissues, reduced goblet mucous production, reduced airway fibrosis and thickness of smooth muscle and blood vessels, and reduced eosinophil infiltration. Mice treated with 2-ME had a significant decrease of HIF-1 and VEGF expression in the perivascular, peribronchial, and interstitium of lung tissues. Collagen IV expression was also significantly reduced in 2-ME treated mice compared to untreated mice. The 2-ME treatment was associated with a significant decrease of OVA-specific IgE antibodies. These findings provide the first indication that IT administration of 2-ME is effective in preventing and reversing antigen-induced airway remodeling in the OVA allergen inflammatory murine model. The potential role of 2-ME in patients is discussed.

CDIP-2, a synthetic peptide derived from chemokine (C-C motif) ligand 13 (CCL13), ameliorates allergic airway inflammation.

Airway inflammation is characterized by selective recruitment of mononuclear and granulocytic cells. This recruitment is mediated by the action of chemotactic cytokines, such as chemokines. A number of chemokines and their receptors have been identified and proposed as potential therapeutic agents in allergic airway inflammation. One of these chemokines is chemokine (C-C motif) ligand 13 (CCL13), a CC chemokine that has been associated with allergic inflammatory diseases such as asthma and allergic rhinitis. To investigate alternative therapeutic agents to alleviate allergic inflammatory diseases, a number of chemokine-derived synthetic peptides were designed and tested for their ability to modulate in vitro and in vivo chemokine-mediated functions. Our results show that one of these peptides, CDIP-2, displayed antagonist functions in in vitro chemotaxis assays using monocytic cell lines. In addition, we found that CDIP-2 significantly reduced peribronchial, perivascular infiltrate and mucus overproduction in an ovalbumin-induced allergic lung inflammation murine model. Thus, CDIP-2 may be considered as part of a novel group of anti-inflammatory agents based on chemokine-derived synthetic peptides.

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope.

Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.

Detection of Chlamydia trachomatis by immunofluorescence, papanicolaou and immunoperoxidase in women with leucorrhea

Se realizó la determinación de la incidencia de Chlamydia trachomatis por los métodos de Papanicolaou, inmunofluorescencia e inmunoperoxidasa y su asociación con otras bacterias en 245 mujeres con manifestaciones clínicas de leucorrea, que acudieron a los centros de salud "Gabriel Garzón Cosa" y "Manuel González Rivera" de la Ciudad de México. Se obtuvieron muestras de fondo de saco posterior a la vagina y del endocérvix a partir de los cuales se sembraron diferentes medios de cultivo para realizar el aislamiento e identificación microbiológica de otro tipo de microorganismos y los frotis correspondientes para realizar las técnicas de Gram, inmunofluorescencia directa (IFD) e inmunoperoxidasa, para investigar la presencia de alteraciones citológicas y Chlamydia trachomatis, detectándose este microorganismo en 8 casos (3.3 por ciento) por los tres métodos, de los cuales 4 se detectaron por IFD, 7 por el método citológixco y 6 por inmunoperoxidasa. La infección por Chlamydia se asoció desde el punto de vista citológico con displasia ya sea leve o moderada pero no se encontró una relación directa con un tipo particular de microorganismo como ya ha sido reportado por otros investigadores

Detection of Chlamydia trachomatis by immunofluorescence, papanicolaou and immunoperoxidase in women with leucorrhea.

Bacteriological, Papanicolaou, direct immunofluorescense (DIF) and immunoperoxidase studies were done in 245 samples from women with vaginal discharge and the frequency of Chlamydia trachomatis was investigated. The samples were obtained from two hospitals of the Public Health Services of Mexico City. Samples were taken from the endocervical and posterior fornix areas and streaked in different cultures media for the isolation and microbiological differentiation. Smears were done and stained by Gram, Papanicolaou, DIF and immunoperoxidase techniques and the presence of cytological alterations and Chlamydia trachomatis was investigated. The microorganism was detected in 8 patients by the three methods, only 4 of these were found by DIF, 7 by cytological and 6 by immunoperoxidase techniques. The infection caused by C. trachomatis cytologically was associated with mild or moderate dysplasia but not with some special microorganism.

[Analysis of 1185 strains of Shigella isolated in Mexico from 1982 to 1993]. / Análisis de 1185 cepas de Shigella aisladas en México de 1982 a 1993.

During the period from 1982 to 1993, 1185 Shigella strains from the National Network of Diarrhoeal Laboratories were sent to the Enteric Bacteriology Laboratory of INDRE. These strains from patients of various ages with diarrhoeal illness were serologically confirmed. The frequency was as follows: S. flexneri (61.35%), S. sonnei (26%), S. dysenteriae (6.4%) and S. boydii (6.2%). S. dysenteriae 1 is an epidemiologicaly important species because it has caused diarrhoeal outbreaks on the southern border of Mexico that later spread through Central America. It must be considered that the 20 isolates obtained in 1989 were from an intentional search focused on S. dysenteriae. Authors pretend to continue with epidemiological surveillance focused on Shigella and intensify the intentional search in order to identify possible human or environmental S. dysenteriae 1 reservoires.

[Seroepidemiology of cholera in Mexico]. / Seroepidemiología del cólera en México.

Antibodies against Vibrio cholerae were determined in 2352 serum samples obtained from patients with clinical diagnosis of cholera. Samples from their contacts and from healthy people living in the same communities were also analyzed. Vibriocidal antibodies with titers 1:160 or higher were observed in 25% of the samples. An increase of vibriocidal and antitoxin antibody titers were observed in 56 to 60% of the patients in which paired samples were available, one obtained in the acute phase of the disease and the other in the convalescence, confirming the diagnosis of cholera. Differences in the antibody titers were noticed when comparing the serotype according to the geographic area and the season of the year.

[Identification of Vibrio cholerae O1 by flow cytometry]. / Identificación de Vibrio cholerae O1 por citometría de flujo.

A total of 72 peptonated water samples suspected of carrying Vibrio cholerae were assessed by laser flow cytometry (LFC) and compared with positive culture. We used a direct fluorescence technique using polyclonal (PolAb) and monoclonal antibodies (MoAb) conjugated to fluorescein. The PolAb were able to detect 33 positive samples. A clear difference among the 20 positive samples was found with only three V. cholerae O1 false negatives when MoAb were used whereas all 13 V. cholerae Non O1 samples were detected. The correlation index comparing control autofluorescence with peptonated water samples show a R = 0.69, versus 0.96 with pure V. cholerae O1 strains. Our data suggest that the LFC technique is able to recognize V. cholerae O1 from a mixture of microorganisms with high sensitivity and specificity in a few hours.

[Principal serotypes of Salmonella identified in 10703 strains in Mexico from 1982 to 1993]. / Principales serotipos de Salmonella identificados en 10703 cepas en México entre 1982 y 1993.

From 1982 to 1993, 10703 Salmonella strains from The National Network of Diarroheal Laboratories of Mexico were sent to the Enteric Bacteriology Laboratory of INDRE. The strains were confirmed by serology and 119 different Salmonella serotypes were found. The most frequent serotypes were as follows: S. typhimurium, S, enteritidis, S. agona & S. typhi. The strains were classified according to the source of isolation as follows: 6671 strains (62.33%) from clinical samples, mainly of faecal origin; 2903 (27.1%) from food for human consumption; 425 from food for animal consumption, 665 (6.21%) from environment or fomites and 39 (0.36%) from animals. The most frequent serotype in clinical samples was S. typhimurium among 96 different serotypes. The main serotype from blood cultures was S. typhi although 27 other serotypes were found. Of thirteen serotypes related to diarrhoeal outbreaks the higher frequency of S. typhimurium was observed but S. typhi caused more outbreaks. A frequency of 119/> 2000 serotypes was observed, that means less than 5% of Salmonella known serotypes. A yearly variability on serotype predominance was observed as well as changes on source of isolation. This results suggest that epidemiological surveillance of salmonellosis should be continued and improved, looking for cases, asymptomatic carriers and contaminated food for human consumption.

[Evaluation of the ELISA method for cholera toxin determination in Vibrio cholerae cultures]. / Evaluación del método de ELISA para determinación de toxina colérica en cultivos de Vibrio cholerae.

ELISA test was evaluated in 503 cultures of Vibrio cholerae O1 y 303 Non-O1. The cultures were isolated from sewage from different states of México between june 1991 and october 1992. The sensitivity was 100% and specificity was 96%. Only 12 strains of V. cholerae Non-O1 were positive for CT toxin. When these cultures were confirmed by polymerase chain reaction (PCR) for cholera toxin, the results were negative. ELISA test is a good alternative to be used for toxin production in cultures of V. cholerae, it needs confirmation only with O1 negative and Non-O1 positive reactions.

[Preparation and evaluation of coagglutination reagents for the identification of Vibrio cholerae 01. Its trial during a cholera outbreak in Minatitlán, Veracruz]. / Preparación y evaluación del reactivo de coaglutinación para la identificación de Vibrio cholerae 01. Su ensayo durante un brote de cólera en Minatitlán, Veracruz.

This study was realized in Minatitlán, Veracruz during a cholera outbreak. 169 rectal swabs were taken from hilles and their contacts. They were transfer alkaline peptone water for enrichment to V. cholerae and incubated for 8 hrs to 37 degrees C, 70 were positive for V. cholerae in both techniques. The coagglutination was done with a reagent prepared at the Instituto de Diagnóstico y Referencia Epidemiológicos of México and the culture were also performed in the same Institute. We obtained 100% of sensitivity and specificity of co-agglutination in relation with culture. This results gave the possibility to use this kind or reagents for a rapid presumptive diagnosis of cholera.