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ASPECTOSCLÍNICOSYEPIDEMIOLÓGICOS

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STRATEGIES IN SEARCH OF RESPIRATORY SYMPTOMS FOR THE EARLY DETECTION OF CASES OF TUBERCULOSIS

R. E. Reyes Enríquez y E. Acosta Gutiérrez

Centro de Salud Urbano Sahuaro. Hermosillo, Son., México. centrosahuarotb@hotmail.com

Introduction With sadness and helplessness I found that after several doctor visits and many treatments, most patients with tuberculosis were diagnosed in the Secondary Care, causing with this, deterioration of the patient, its transmission to many people, and the dreaded drug resistance. The aim of this study was to demonstrate that TB exists and is a serious health problem that is increasing rapidly. This was achieved through raising awareness and training of staff and population at the Sahuaro Urban Health Centre* to identify and timely diagnose respiratory symptoms, to begin treatment in cases diagnosed to prevent deterioration of the patient and to break the chain of transmission. Methods Talks, delivery of promotional material to private and public institutions and to the general public were provided. There were news reports in the newspaper, radio and television and a large amount of work carried out with schools and doctors. House by house visits and sputum smear microscopy were performed to diagnose accurately and timely. Results In a year we implemented awareness strategies and training of the population, increasing the detection of respiratory symptoms from 39 to 814 and the diagnosis of positive cases by 140%. Because people know the symptoms, this currently generates timely diagnosis. Additionally, 1,700 people were trained in HIV / AIDS and 13,800 condoms were provided. The existence of TB in our community and a simple way to diagnose it and treat it for free was ignored. Most doctors neglect thinking about tuberculosis. Consequently, awareness must be permanent in schools. We saw the impact of its implementation in mental hospitals, diagnosing 2 cases. Currently, people who are trained and aware became health promoters and have come to "diagnose their own disease." Our greatest satisfaction is that users who became aware through talks, inquiries, visits, and brochures given house to house in previous months continue to attend the health unit to get a sputum smear microscopy. Conclusions To conduct this work, there is no need of large amounts of people, money or time. We only need vocation, attitude, self-confidence and desire to achieve objectives. Nevertheless, if these successful strategies do not have a continuity, which occurred, there is no early detection which in turn brings its subsequent increase in incidence and prevalence of the disease. Currently, the figures may not be alarming because the disease is still diagnosed in Secondary Care, and according to epidemiological studies of these patients, we can conclude that besides being diagnosed late, they are not given proper follow-up. Due to the results obtained before and after this project, it is clear to me that the most important part is the attitude of health workers, and the problem of public health represented by tuberculosis will only be solved by working.

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Mycobacteria in archaeological sites in Mexico

R. Alcalde-Vázquez1,2, N. B. Medina-Jaritz2, R. Olvera-Ramírez2 and J. A. González-Y-Merchand1 Instituto Politécnico Nacional. Escuela Nacional de Ciencias Biológicas. Department of Microbiology. Laboratory of Molecular Microbiology1 and Department of Botany. Laboratory of Plant Physiology2.

Prol. Carpio and Plan de Ayala. Santo Tomas. 11340. Miguel Hidalgo México, D.F. raulalcalde@hotmail.com

Introduction Mycobacteria are short, immobile aerobic bacilli and have a high G + C content in their DNA. They are classified into two groups: Mycobacterium tuberculosis complex (MTC) and the group of non-tuberculous mycobacteria (NTM) or environmental mycobacteria. Some of these mycobacteria may cause infections to humans. Others can colonize and form biofilms that eventually generate damage to monuments and historic buildings (biodeterioration). However, their study has primarily been aimed at clinical and veterinary area, so the search is limited to soil, water and mammals. In the present work we pretended to extend this search to regions of great economic and cultural interest such as monuments and archaeological ruins of Mexico, as a pioneering study in the description of the diversity of non-tuberculous mycobacteria within these habitats. Methods. Sampling was carried out in 17 archaeological sites with different physiographic features and different building materials: Atetelco and Malinalco in Estado de México, Guachimontones in Jalisco, Tulum and Cobá in Quintana Roo, Uxmal, Ek Balam and Chichén Itzá in Yucatán, Cacaxtla, Xochitécatl, Ocotelulco and Tizatlán in Tlaxcala, Mitla, Monte Albán and Zaachila in Oaxaca, Yohualichan in Puebla and La Antigua in Veracruz. This was a non-invasive sampling during the rainy season and under aseptic conditions. For mycobacteria isolation, a 1:10 dilution of the sample was performed and washed with a solution of hexadecylpyridinium chloride 0.75%, centrifuged at 5000 x g for 20 min and the pellet was cultured on Lowenstein-Jensen slants. A Ziehl-Neelsen stain was used to confirm the acid-fast characteristic of this genus; those acid-fast bacilli (AFB) were plated in Middlebrook 7H10 media in order to describe the macroscopic and microscopic features. For identification of the genus Mycobacterium the V2 region of rrs gene (16S rRNA) was amplified and the species identification was made by three molecular markers: the sequencing of V2 region of rrs gene, the PRA pattern (hsp65 gene) and the sequencing of rpoB gene. Results. A total of 90 samples from biofilms located on archaeological monuments were collected and processed. Thirty seven AFB isolates were obtained and described. All the 37 strains belonged to the genus Mycobacterium. Most strains have been isolated from the archaeological site of Guachimontones in Jalisco (20) and Atetelco in Estado de Mexico (11) the rest were isolated from Xochitécatl in Tlaxcala (1), Zaachila in Oaxaca (1), La Antigua in Veracruz (1), Malinalco in Estado de Mexico (2) and Ek Balam in Yucatan (1). Conclusions Microorganisms belonging to the genus Mycobacterium were identified in 40% of the archaeological sites studied. All had the inherent morphological characteristics of that genus and corresponded to NTM group.

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Acknowledgments.This research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT). We thank our colleagues from the Instituto Politécnico Nacional (IPN) who provided insight and expertise that greatly assisted the research. Keywords. Non-tuberculous mycobacteria (NTM), Biodeterioration, Archaeological sites.

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Meningeal Tuberculosis in the Children’s Hospital of the State of Sonora: Predictions of Lethality.

Olivera-Sandoval S. Cano-Rangel M.A.

Universidad Autónoma De Baja California. Escuela de Ciencias de la Salud, Valle de las Palmas. Tijuana, Baja California, México.

sara.olivera@uabc.edu.mx Introduction Meningeal tuberculosis is the most serious extra-pulmonary infection and is the cause of death in over half of infected patients.1 In Mexico little is known on the associated risk factors of death, nevertheless, in other countries these have been described as. Such for the diagnosis exist criteria as: Criteria of Lincoln 2, Glasgow scale, hiperproteinorraquia and tomographical: (Basal Meningitis and strocke). 3

Material and Methods Transversal, descriptive study. Dependent and independent variables interpreted with the NCSS 2007 program with square analysis were analyzed square test and t of student for qualitative and quantitative variables. A odss radio was also obtained. Results 56 patients were divided as follows: Group 1 deaths (10) and Group 2, alive (46). They were grouped in 4 common characteristics: socioepidemiological criteria, clinical criteria, laboratory criteria and cabinet criteria. The majority of the patients entered stage II and III. The result of stages to the entrance for mortality is at OR (0.011), IC 95% (0,00 - 0,11), p= 0.01. Proteins in cerebrospinal fluid (LCR) as a lethality factor, taking into account the protein value limit (202 proteins > mg/decilitro) with a OR (0.45), IC 95% (0,152 - 0,747), Value of statistically significant P= 0,003. The findings of inflammation in initial stages of the disease were aracnoiditis and hydrocephalus. The patients who did not received treatment with steroids have 11,3 times a greater risk of passing away with a P= 0. 34, OR 11,3 with 6 IC 95% (2.10-61.3). Sequels in motor abilities were found in 86% of all living patients. Conclusions: The clinical characteristics were associated to lethality in stages II and III of Lincoln. In the same way as international criteria the scale of Glasgow is established in the local population in three stages. Stages II and III are directly associate with lethality. Hiperproteinorraquia is the only reasonable biological factor that relates to a direct risk to lethality. A per-clinic diagnosis was established, treatment with steroids has been associated as a protective factor. Keywords: meningeal tuberculosis, extra-pulmonary tuberculosis, risk to lethality, criteria of Lincoln, Scale of Glasgow, hiperproteinorraquia, basal meningitis and heart attacks.

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Benzimidazole derivates as antimicobacterial agents W. Cruz-Chavez1, R. Ruiz-Nicolás1, K. Leyva-Paredes1, R. Jiménez-Juárez2, J. Luna-Herrera1, B.E.

García-Pérez1. 1Laboratorio de Inmunoquímica II, Escuela Nacional de Ciencias Biológicas, IPN, México D.F.

2Laboratorio 6 Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas, IPN, México D.F.

Background Tuberculosis is one of the most widespread infectious diseases worldwide caused by Mycobacterium tuberculosis. Although the first line drugs to treatment of the disease came to control it effectively, the disease continued to progress silently around worldwide, due mainly to the increase of the number of cases caused by resistant strains. For that, the investigation for new antimicobacterial drugs with biological activity towards the broad spectrum of species of Mycobacterium genus is an urgent need. The benzimidazole system, as a pharmacophore group is very known and confers biological activity to organic structures. The goal of this study was evaluate the antimicobacterial activity and toxicity of benzimidazol derivates substituted with acceptor groups. Methods The antimicobacterial activity in vitro was evaluated using the microtiter alamar blue assay (MABA). The intracellular effect was assessed in a macrophage based-assay. The effect on proliferation of one eukaryotic cell line (J774A.1 cell line) was evaluated with a colorimetric assay. Results The four benzimidazole derivates assessed show a MIC between 3.125 and 6.25 µg/ml. The results of intracellular activity demonstrated that all benzimidazole derivates inhibit the intracellular growth of M. tuberculosis between 24 and 48 h post-infection and maintained their effect until 96 h post-infection, with exception the 2-(2’-pirido)-5(6)-cloro benzimidazole which at 72 h showed a high intracellular replication of mycobacteria. The benzimidazole derivates did not exhibited cytotoxicity against the J774A.1 cell line. Conclusions These benzimidazole derivates may thus represent a new group of antimycobacterial agents with promising effects. Acknowledgments This project was funded by the “Fondo Sectorial de Investigación para la Educación" Conacyt 222001 and SIP 20151035. Keywords Mycobacterium tuberculosis, benzimidazole and antimicobacterial agents.

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Molecular and epidemiological characteristics of Mexican population with Tuberculosis living in Florida, USA

E. Fernández1, D. Munro1, A. García1, P. Hamsho2, J. May2, M.Lauzardo2., Zenteno-Cuevas R.1*

1. Instituto de Salud Pública, Universidad Veracruzana, Veracruz México. 2. South Eastern National Tuberculosis Center, Dept. of Medicine. University of Florida, Florida,

USA. *robzencue@gmail.com

Background Migration has an important influence on the spread of tuberculosis (TB) and promotes its repositioning as a threat to public health. Mexico and the United States of America (USA) have one of the most significant migratory flows in the world. However little is known about the influence that could have TB from Mexican population living in the USA. Considering the above, the aim of this study was to conduct a molecular-epidemiological characterization of Mexicans diagnosed with TB living in Florida, USA. Methods The clinical and socio-demographic data, and molecular fingerprint from MIRU-VNTR- 24 loci and spoligotyping of Mexicans with a diagnosis of TB living in Florida was recovered from the Genotyping Information Management System (GIMS). Lineages assignments and epidemiological characteristics were done according standard procedures Results From 2006-2014, 257 Mexicans with TB were diagnosed and included in GIMS-Florida database. Pulmonary TB was observed in 91% (234) of individuals. The age 25-44 years was found in 47% (121) of population. Male was observed in 73% (188), and M:F ratio was 1.7:1. Association to type 2 diabetes mellitus and HIV was observed in 12% (30) and 10% (25) respectively. The 18% (48) of individuals mentioned to have less to one year of being residing in USA, 38% (99) from 2-4 years and 37% (94) from 5-14 years. Drug- and multidrug-resistance was observed in 12% (32) and 1% (4) of strains. Resistance against S was observed in 7% (18), 6% (15) to I, 4% (10) to K, 2% (6) to R and 1% (4) to E. Finally, eleven lineages were found: 29% (52) strains were assigned as LAM, 20% (35) Haarlem, 19% (34) Orphans, 10% (17) EAI, 4% (8) Ghana, X, and Delhi-Cas, 3% (5) Beijing, 2% (4) Bovis and S and by last 1% (2) H37. Conclusions: Males and young population economically active were the most affected groups. Type 2 DM was found more frequently associated to TB, a low number of MDR-TB cases were observed. A high diversity of lineages were found; the two most frequent were LAM and Haarlem; otherwise Delhi-Case and Ghana were rarely described. The Spanish ethnicity, especially the Mexican, has one of the most important growing rates in the USA, for this reason it will be important develop a close surveillance of this population, in order to have a better understanding of the influence of this ethnic group in the development of TB in Florida and the rest of USA. Acknowledgements DM and AG are CONACyt fellowship No. of the Doctorado en Ciencias de la Salud. RZ has a grant from CONACyT- Problemas nacionales program No. 213712. Please consider as a Poster presentation.

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Development of one molecular epidemiology surveillance system of tuberculosis in Veracruz: preliminary results

R. Alamaraz-Velasco1, D. Munro1, A. García1, P. Hamsho2, M. Lauzardo2, R. Muñiz-Salazar3,

Zenteno-Cuevas R.1* 1. Instituto de Salud Pública, Universidad Veracruzana, Veracruz México.

2. South Eastern National Tuberculosis Center, Dept. of Medicine. University of Florida, Florida, USA.

3 Laboratorio de Epidemiología y Ecología y Molecular, Escuela de Ciencias de la Salud, Universidad Autónoma de Baja California, Baja California, México.

*robzencue@gmail.com

Background The molecular epidemiology surveillance systems on Tuberculosis are well established procedures in several national TB programs in countries such as USA, Japan, etc. These systems are considered a very useful tool for the understanding of the behavior and dynamic of the TB in those countries, nevertheless this system is not developed in Mexico. In addition, the state of Veracruz is one of the most important contributors of Tuberculosis in México, however there are no reports about the genotypic characteristics of M. tuberculosis. Considering all the above, the aim of this work was to develop a molecular epidemiology surveillance system on tuberculosis (TB), considering information of patients and the TB isolates circulating in Veracruz. Methods Respiratory samples of patients from the state of Veracruz with TB confirmed by AFB, were recollected from the period 2014 to 2015. Recovery of clinical isolate, DNA extraction and genotyping by MIRU-VNTR 24-loci was developed according to standard procedures. Additionally, sociodemographic information of patients was also obtained by the application of a short survey. All information was stored in an Excel file considering three formats: First, including all the epidemiological, sociodemographic and genotypic information; second, containing the information according to the format of the MIRU-VNTRplus system database; and third, considering the data in the format of the SPOLDB4 database. Results: 80 isolates were recovered. The average age was 42 ± 17 years, 65% were men, 30% had type 2 diabetes mellitus, and 6% were MDR-TB. Eight lineages were observed in 40% of isolates: LAM 11%, EAI with 9%, Haarlem 8%, H37 and S with 4%, Turkey 2%, Ghana and Canetti with 1%. The remaining isolates (60%) were classified as orphans. Twelve clusters were identified in the orphan strains mine while seven were observed in the strains with lineage. Some epidemiological characteristics such as age and drug resistance were associated with some clusters of specific lineages and orphans clusters respectively. Conclusions: Despite the information is preliminary, the procedure here developed could help in the construction of one molecular epidemiological surveillance system of TB. The inclusion of more strains from Veracruz and other states will be of great help in the identification of the lineages circulating in the state and the country, and in a second level in the strengthening of the surveillance system. The information derivate from this system could have important implications for the development of specific actions at the local, regional or national TB program.

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Acknowledgements: RAM, is a fellowship of the Maestria en Salud Pública, and DM and AG of the Doctorado en Ciencias de la Salud, Universidad Veracruzana. RZ was funded by a grant from CONACyT- Problemas nacionales program No. 213712.

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ASPECTOSINMUNOLÓGICOS

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Effect of silymarin in response to infection with Mycobacterium in macrophages

E.M.Rodríguez-Flores, M. Castañón-Arreola. Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México.

México D.F. Introduction Tuberculosis is an infectious disease that causes thousands of deaths worldwide. Treatment is not always effective. It lasts at least 6 months and induces liver damage. It has been proposed administration of a treatment with drugs with antioxidant activity, mainly flavonoids, to counteract the hepatotoxic effect. One of these is the flavonoid silymarin from seeds of Silybum marianum plant, which has been used to treat liver disorders , due it presents a hepatoprotective effect and has immunomodulatory activity. Induces an increase in INF-γ , IL-4 and IL-10 secretion, and inhibit nitric oxide production in macrophages. M. tuberculosis is a pathogen whose control depends on the bactericidal mechanisms of macrophage and the establishment of a Th1 response. It seems important to study the antioxidant and anti-inflammatory effect of silymarin on mycobacterium infection. In addition silymarin has a favorable activity in liver damage, it suggests that it may reduce the toxic effects of treatment. Methods The objective of this study was to determine the effect of treatment of macrophages with silymarin in controlling mycobacterium infection. In this study the production of cytokines expressed was measured in dose-response testing in macrophages treated with different concentrations of silymarin and silibilin by real time quantitative PCR and by flow cytometry. Results We found an increase in the expression of INF- γ and TNF-α to stimulate cells with a 50 µM concentration in both cases. These cytokines were assessed by flow cytometry, and we also determined the presence of NFk- B and IL-12. Similarly, the macrophages stimulated with silymarin and silibilin have an increased production of IFN- γ, TNF-α and IL-12, however a lower production of NF- kβ was found. These results are important because they are essential in the control of bacterial growth. In addition, macrophage infection with Mycobacterium bovis BCG were performed. These were treated with silymarin and silibilin and expression of INF-γ and TNF-α was determined. In both cases the expression of cytokines was twice in presence of silymarin and 1.5 times higher in presence of silibilina, when macrophages were infected with BCG (MOI 1: 5). Conclusions This results suggest it is possible that silymarin can regulate the immunopathology induced by mycobacterial infection. The findings are important, and it could be necessary to describe the role of silymarin in regulating the immune response against mycobacteria, its role in bacterial growth and potential therapeutic use.

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Acknowledgments This work was supported by the National Council of Science and Technology (CONACYT) No. 290908. Keywords. Tuberculosis, silymarin, immunomodulation

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Autophagy in Mycobacterium abscessus infection

N. S. Castrejón-Jiménez1, A. D. Hernández-Pérez2, J.C. Hernández-González3, J. Luna-Herrera1 and B.E. García Pérez1.

1 Laboratorio de Inmunoquímica II, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F.

2. Laboratorio de Microscopia Electrónica, Instituto Nacional de Rehabilitación, Av. México-Xochimilco No. 289, Col. El Arenal de Guadalupe, Tlalpan, México, D.F,

México

3 Laboratorio de Inmunología y Virología, Instituto de Ciencias Agropecuarias, Universidad Autónoma del Estado de Hidalgo

Background Autophagy is a cytoplasmic degradation process that has recently been linked to the regulation of innate immunity, the adaptive immunity and inflammation. Several studies have been shown that the induction of autophagy can eliminate microorganisms whereas other studies demonstrate that other microorganisms can replicate within autophagosomes. It has been demonstrated that autophagy contribute to control M. tuberculosis infection. Therefore, the aim of this work was evaluate the role of autophagy in the infection of human pneumocytes by M. abscessus (MAB). Methods The cell line A549 (type II pneumocytes) was used in this study. Infection kinetics were performed and were followed for 2h of infection and to 6, 24, 48 and 72 h post-infection. To demonstrate whether mycobacterial infection induces autophagy an antibody specific for the LC3-II (important marker of autophagy) protein was used. To evidence if the induction of autophagy in A549 cells promotes elimination of MAB, peptidoglycan was used as an agonist of TLR2 and moreover vitamin D also was used as an inductor of autophagy. To demonstrate whether the activation of the autophagy pathway induces mycobacterial elimination, autophagy was induced with peptidoglycan and vitamin D prior to mycobacterial infection and the CFU were determined. Autolysosomal fusion was evaluated with LAMP-1 and LC3-II. Results The results showed that MAB can invade and replicate in type II pneumocytes. Moreover, the bacillus induces expression of LC3-II at 6 and 24 h after infection, which increased when cells were stimulated with TLR2 agonist also when they were stimulated with Vitamin D. The autophagic vacuoles fused with lysosomes were more evident in cells stimulated with agonist of TLR2, however, did not decrease the bacterial burden. TEM assays showed the intracellular replication of bacilli in cytosol and mitophagy induction. Conclusions Autophagy induced by peptidoglycan and Vitamin D stimulation did not contributes to control MAB infection, although there is evidence of autolysosomal fusion. Our results suggest that MAB escapes into the cytosol which could contribute to its intracellular replication. Mitophagy could be a rescue response to maintain the viability cell.

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Acknowledgments: NSCJ would like to acknowledge CONACYT and BEIFI for their fellowships. BEGP and JLH received fellowships from COFAA, EDI and SNI. The authors acknowledge the financial support from "Fondo Sectorial de Investigación para la Educación" Conacyt 222001. Keywords: Autophagy, M. abscessus, Vitamin D, TLR2

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HGF induces protection in a model of Experimental Pulmonary Tuberculosis. Bello-Monroy O1,2, Enríquez-Cortina C1, Rosales-Cruz D. P. 1, Escobedo-Calvario O.

A. 1, Juárez-Hernández U2, Ramos-Robles B2, Miranda R. U. 1, Bucio L1, Souza V1, Mata-

Espinosa D2, Barrios-Payán J2, Marquina-Castillo B2, Gutiérrez-Ruiz M.C. 1, Hernández-Pando R2, Gómez-Quiroz L. E. 1

1Laboratory of Cellular Physiology. Department of Health Sciences. Universidad Autónoma Metropolitana Iztapalapa, México Distrito Federal.

2Department of Pathology. National Institute of Medical Sciences and Nutrition "Salvador Zubirán". México, Distrito Federal.

Introduction The increase in the number of cases of infections with multidrug resistant strains (MDR) of Mycobacterium tuberculosis is pointing out the necessity to find new therapeutics options. It has been proposed the elevation in the doses of drugs, such as rifampicin (RIF) or isoniazid (INH) could be useful for the elimination of the MDR bacteria, but organs can be compromised to damage. Hepatocytes growth factor (HGF) has been proved to induce protective effects in epithelial tissues. The aim of the present study was to address the effect of HGF in the treatment with high doses of RIF and INH in mice infected with a MDR strain of Mycobacterium tuberculosis and the impact in the bacterial load. Material and Methods In this preclinical approach, we used BALB/c mice that were infected with a MDR strain of Mycobacterium tuberculosis. After mice have established the pulmonary progressive tuberculosis animals were treated with high doses or RIF and INH, using both, intratracheal or intragavage administration, and co-treated or not with HGF. Then mice were sacrificed at 30, 60 and 90 days. We determined colony formation units, and reactive oxygen species (ROS) by DHE fluorescence in lungs, histology was assessed by routing H&E staining. Results Data show that HGF treatment alone decreases bacteria in lungs, presenting a differential role in presence or absence of Mycobacteria, in addition the content of ROS decreased when mice were not infected and, increased under the infection, suggesting the activation of immunocompetent cells, this data correlated with the reduction in bacterial load and in the percentage of pneumonia, contributing to the lung healing. Conclusion HGF could be considered as a good adjuvant in the treatment of tuberculosis due to the protective effect in the lungs and the possible immunomodulatory activities. Bibliography. 1) Clavijo-Cornejo, D., Enriquez-Cortina, C., Lopez-Reyes, A., Dominguez-Perez, M., Nuno, N., Dominguez-Meraz, M., Bucio, L., Souza, V., Factor, V.M., Thorgeirsson, S.S., et al. (2013). Biphasic regulation of the NADPH oxidase by HGF/c-Met signaling pathway in primary mouse hepatocytes. Biochimie 95, 1177-1184.

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2) Chang, K.C., and Leung, C.C. (2010). The best approach to reintroducing tuberculosis treatment after hepatotoxicity is still open to debate. Clin Infect Dis 51, 366-367; autor reply 367-368. 3) Enriquez-Cortina, C., Almonte-Becerril, M., Clavijo-Cornejo, D., Palestino-Dominguez, M., Bello-Monroy, O., Nuno, N., Lopez, A., Bucio, L., Souza, V., Hernandez-Pando, R., et al. (2013). Hepatocyte growth factor protects against isoniazid/rifampicin-induced oxidative liver damage. Toxicological sciences: an official journal of the Society of Toxicology 135, 26-36. 4) Hernandez-Pando, R., Orozcoe, H., Sampieri, A., Pavon, L., Velasquillo, C., Larriva-Sahd, J., Alcocer, J.M., and Madrid, M.V. (1996). Correlation between the kinetics of Th1, Th2 cells and pathology in a murine model of experimental pulmonary tuberculosis. Immunology 89, 26-33. 5) OMS (2014). Global Tuberculosis Report. Acknowledgments Conacyt 131707 Keywords. Hepatocyte Growth Factor, MDR Tuberculosis, Reactive Oxygen Species.

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Hyperglycemic conditions affect Mycobacterium tuberculosis antigen processing

G. Monroy-Mérida1, O. Medina-Contreras2, M.P. Sierra-Vargas3, K. Bobadilla-

Lozoya1 1Departamento de Investigación en Inmunología

2Laboratorio de Investigación en Inmunología y Proteómica, Hospital Infantil de México Federico Gómez

3Laboratorio de Investigación en Bioquímica y Medicina Ambiental,Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas”

Background It has been recognized that Diabetes Mellitus (DM) increases the risk for Tuberculosis (TB). The increased prevalence of Mycobacterium tuberculosis (Mtb) infection in patients with DM2 is associated with alterations in innate immune responses, including a decrease in phagocytosis, adhesion, and chemotaxis in macrophages, and increased expression of pro-inflammatory cytokines, such as TNF-α, IFN-γ, and IL-17A. However, most studies on DM2/TB have focused on the innate responses, whereas the adaptive responses are not well characterized. Since there is a low frequency of T cells, and reduced IFN-γ production, we propose there is an impairment in Mtb processing and formation of antigen–MHC-II complexes, which leads to a reduced antigen presentation to CD4+ T cells. In this study we assessed the processing and presentation of mycobacterial antigens by the phagolysosomal pathway under different glucose concentrations. Methods Human monocyte derived macrophages (MDMs) were cultured for 7 days in RPMI medium supplemented with 5.5, 11 and 30 mM of glucose. MDMs were infected with Mtb (H37Ra) at MOI of 10 and incubated 1 h at 37ºC, then washed with ice-cold RPMI-1640 to remove any extracellular bacteria. Pre-warmed medium was added and the cells were incubated for 0-180 min (chase time) at 37°C, and antigen processing was stopped by fixation with 1% paraformaldehyde. Detection of peptide–MHC-II complexes on the plasma membrane was detected by a classical antigen-presentation assay, in which MDMs were incubated with autologous Mtb-specific CD4+ T cells, and IFN-γ production was measured in culture supernatants by ELISA after 24 h. Results Here we show the Mtb processing and presentation kinetics in MDMs under in vitro normoglycemic (5.5 mM) and hyperglycemic (11, 30 mM) conditions. We observed a decrease in IFN-γ production by autologous Mtb-specific T cells after 30, 60 and 120 min post-infection at the highest glucose concentration (30 mM); however, under normoglycemic (5.5 mM) conditions IFN-γ production was the highest. Interestingly, we did not observe differences at 11mM when compared to normoglycemic condition Conclusions Hyperglycemia impairs Mtb processing and subsequent antigen presentation to CD4+ T cells.

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Keywords: Mycobacterium tuberculosis, hyperglycemia, antigen processing.

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In vitro persistence of M. avium, M. celatum and M. tuberculosis in human macrophages.

M.D. Soto-Ramírez1,2, A.C. Helguera-Repetto1, J.F. Cerna-Cortés2, S. Rivera-

Gutiérrez2, L. García-Morales, J.A. González-Y-Merchand2. 1Department of Immunobiochemistry. INPer. Mexico, D.F.

2Department of Microbiology. ENCB-IPN. México, D.F. jgonzal1212@yahoo.com.mx mdsotoram@outlook.com

Background: Nontuberculous mycobacteria (NTM) are free-living microorganisms that are able to colonize different environments. In recent years these bacteria have been identified as etiological agents for several diseases in immunocompetent and immunodeficient patients; nowdays they are considered as emergent pathogens. It has been reported that some slow-growing NTM such as M. avium and M. celatum, in the same way than M. tuberculosis, are capable to evade the immune response generated by their host macrophages. Therefore, the objective of this study was to evaluate the immune response of human macrophages to the infection with M. avium, M. celatum, using M. tuberculosis infection as a control. Methods:

• The infection model was standardized in macrophages derived from cell line THP-1, using a MOI of 1:1 with M. avium, M. celatum and M. tuberculosis H37Rv; cells were infected and the infection kinetic was determined at 0 to 120 h post-infection (hpi), cells were stained using the Kinyoun technique.

• The intracellular multiplication of each mycobacteria was determined by viable counting up to 96 hpi.

• Production of ROS and RNS (reactive oxygen and nitrogen species, respectively) were determined by NBT reduction and the Griess reagent system, respectively (up to 48 hpi).

• Cytotoxic effect on macrophages mycobacteria was performed with the "LDH Citotoxicity KitPLUS Detection" (Roche) kit.

• TNF-α, IL-8, IL-1β, IL-10 and TGF-β profiles were determined by the TiterZyme EAI, from Enzo Life Sciences.

Results: NTM did not caused damage to the cell monolayer integrity during the kinetic infection. Macrophages could control the intracellular growth of mycobacteria: in the case of M. avium, up to 72 hpi, and in the case of M. celatum, up to 96hpi. Both mycobacteria induced high production of ROS after 24hpi and RNS after 48 hpi. NTM generated the same cytotoxic effect on macrophages than the one produced by M. tuberculosis. Production of TNF-α and IL-1β induced by NTM was lower than the one induced by M. tuberculosis; M. celatum was the only mycobacteria that induced the production of IL-8 in this study. Finally, both NTM induced high levels of TGF-β in comparison with M. tuberculosis.

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Conclusions: Although our results suggest that M. avium and M. celatum are able to persist during macrophage infection in a similar fashion than M. tuberculosis, the levels of cytokines response differs between this last species and NTM. Acknowledgments: MDSR received a scholarship from CONACYT (No. 509250) and PIFI (No. A130462), México. Their projects was supported by CONACYT, México grant No. 156347 Keywords: Non-tuberculous-mycobacteria, human infection, persistence, M. avium, M. celatum.

Effect of EsxA in the miRNA expression profile of human macrophages M. G. Bonilla Muro, O. N. Hernández de la Cruz, E. M. Rodríguez-Flores, M.

Castañón-Arreola. Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México

Background Tuberculosis remains as a major worldwide health problem in XXI century, with a higher incidence in developing countries. The WHO proposed to eradicate disease by this year however though it was not possible to achieve this goal since no treatments or vaccine has been developed, and there's still a lot unknown on the biology of Mycobacterium tuberculosis. In order to survive within the host, M. tuberculosis has multiple proteins, which helps the mycobacteria to survive within the macrophages. One of the most remarkable is the 6-kDa EsxA. Due to the very important role of miRNA's in transcriptional regulation we decided to study the changes in expression profile of macrophage miRNA's in response to EsxA stimulation. Methods To define the optimal concentration and time for macrophage stimulation we evaluate the cytotoxicity of EsxA by neutral red assay, the nitric oxide production by DAF-FM assay and the expression of proinflamatory cytokines by RT-PCR. The evaluation of miRNA expression profile was done using TaqMan Low Density Assays (TLDA). Results The best concentration of EsxA was 5µg/ml and 24 hours of treatment. Upon these conditions we found the deregulation of 36 miRNA’s (16 up-regulated and 16 down regulated). The most significant was the mir-155 and is remarkable which has been observed in all analysis carried out with three different endogenous controls for the normalization of expression analysis. Conclusions EsxA induce the deregulation of miRNA's which possible targets in important pathways for the infection control and the immune response, like the MAPK signaling pathway, the endocytic pathway and the cytokines (like TGF-β, TNF-α, IL-12, etc.) and chemokine signaling pathways.

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Acknowledgments This work was supported by the National Council of Science and Technology (CONACYT) grant No. 160503. Keywords. miRNA, Tuberculosis, Macrophages

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Infection of macrophages with dormant Mycobacterium tuberculosis adapted to cholesterol environment

S. Yong-Mendoza1,2, N.M. Alonso-Aguilar1,2, S. Rivera-Gutiérrez2, J.F. Cerna-Cortés2,

J.A. González-Y-Merchand2, A.C. Helguera-Repetto1, 1Immunobiochemistry Department. INPer. Mexico, D.F.

2Microbiology Department. ENCB-IPN. México, D.F. ceciliahelguera@yahoo.com.mx : syongm0800@egresado.ipn.mx

Background: Tuberculosis is still one of the leading causes of mortality throughout the world and it is estimated that 2 billion people are latently infected, representing potential cases of reactivation and transmission of the disease. The mechanisms involved in reactivation and persistence of bacilli have not been completely clarified. It has been reported that cholesterol is essential for Mycobacterium tuberculosis (Mtb) persistence within the host, contributing to its survival; however, the role that this molecule plays during reactivation is unknown. In this study, we cultured Mtb in presence of cholesterol as carbon source until exponential-phase and NRP2-dormancy stage and we infected macrophages with each one of the Mtb cultures. Infection rate and macrophages integrity were analyzed, and bacterial intracellular multiplication and production of some interleukins were measured. Overall, we described a new model of in vitro infection using dormant Mtb; furthermore, this approach provided new information on the role of cholesterol during persistence and reactivation of Mtb. Methods:

• The infection model was standardized in the THP-1 macrophages infected with exponentially-growing or NRP2-dormant M. tuberculosis H37Rv, which were cultured in presence of cholesterol or dextrose as carbon sources. A MOI of 1 was used for all experiments.

• Infection rate was evaluated at different times: from 4h post-infection (hpi) to 96 hpi; cells were stained using the Kinyoun technique.

• Mtb intracellular multiplication was determined by colony forming units from 4 to 96 hpi.

• TNF-α, IL-8, IL-1β, IL-10 and TGF-β production were quantified at 6 and 24 hpi with the "Platinum ELISA" (Affymetrix, eBioscience) kit.

Results: THP-1 macrophages were infected at similar rates, independently of the metabolic phase of Mtb (active/NRP2 dormant phase) when bacilli were previously growing into glucose media, inducing the monolayer destruction since 24hpi. In contrast, when infection was carried out using Mtb adapted to cholesterol environment, NRP2 dormant bacilli persisted viable with a non-replicating profile within the cell during 96hpi; besides, the macrophage monolayer remained undamage. Finally, intracellular log-phase and dormant bacteria (cholesterol-adapted) induced lower production of pro/anti inflammatory cytokines, than bacilli adapted to glucose media. Conclusions:

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Overall, these results suggest that dormant Mtb cultured in hypoxic conditions and in presence of cholesterol, are prepared to survive and persist within macrophages without causing damage, inducing a discreet cell-cytokine response. Acknowledgments: Department of Immunobiochemistry of INPer and Molecular Microbiology Laboratory of Department of Microbiology of ENCB-IPN. Keywords: Tuberculosis, persistence, reactivation, macrophages infection, cholesterol, Mycobacterium tuberculosis.

Immunoinformatic approach to select Mycobacterium spp. antigen candidates towards the development of an oral vaccine

C. Angulo1*, P. Carlos1, S. Rosales-Mendoza2, M. Castro-Liera3

1Grupo de Inmunología & Vacunología. Centro de Investigaciones Biológicas del

Noroeste, SC. Instituto Politécnico Nacional 195, Playa Palo de Santa Rita Sur, La Paz, B.C.S. C.P. 23096, México.

2Laboratorio de biofarmacéuticos recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Av. Dr. Manuel Nava 6, SLP, C.P. 78210,

México 3Instituto Tecnológico de La Paz, Boulevard Forjadores 4720, 8 de Octubre Segunda

Sección, 23080 La Paz, BCS. eangulo@cibnor.mx

Introduction. Mycobacterial species cause several diseases in animals and humans. Modern reverse vaccinology is based on immunoinformatic tools to select appropriate antigens with vaccine potential. This concept currently involves not only the subcellular localization of the proteins but also new immunoinformatic tools to predict the process and presentation of relevant epitopes. Notably, the Mycobacterium spp, for which the genomes have been sequenced, share almost 98% of gene homology. Therefore, potential protein vaccine candidates in any specie of Mycobacterium could be useful to fight against several mycobacterial diseases. Here our model of study was Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis disease affecting ruminants worldwide and that has been associated to Crohn’s disease in human beings. Thus, the aim of this study was to identify potential candidate antigens and epitopes by immuno-informatic tools that could be later evaluated as vaccines, and to establish an immunoinformatic procedure to select vaccine candidates. Methods 110 protein sequences were selected from MAP K-10 genome database: 48 classified as putative enzymes involved in surface polysaccharide and lipopolysaccharide synthesis, as membrane associated and secreted proteins, 32 as conserved membrane proteins, and 30 as absent from other mycobacterial genomes. These 110 proteins were preliminary screened for Major Histocompatibility Complex (MHC) class II affinity and promiscuity using ProPred program. In addition, subcellular localization and host protein homology was analysed. Results

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From these analyses, 23 MAP proteins were selected for a more accurate inmunoinformatic analysis including T cell and B cell epitopes analysis and homology with mycobacterial proteins. Finally, eleven MAP proteins were identified as potential candidates for further immunogenic evaluation: six proteins (MAP0228c, MAP1239c, MAP2232, MAP3080, MAP3131 and MAP3890) were identified as presenting potential T cell epitopes, while 5 selected proteins (MAP0232c, MAP1240c, MAP1738, MAP2239 and MAP3641c) harboured a large number of epitopes predicted to induce both cell- and antibody-mediated immune responses. Moreover, immunogenicity of selected epitopes from the candidate protein MAP1239c were evaluated in IFN-γ release assay resulting in positive outcomes when leucocyte cells form naturally MAP-infected cattle were stimulated. Conclusion Eleven M. avium subsp. paratuberculosis proteins were identified by in silico analysis and need to be further evaluated for their vaccine potential in vivo. The potential of new selected antigens and their epitopes from MAP should be also analysed in the context of other mycobacterial diseases. Acknowledgment: SEP-CONACYT for financial support (Project No: 151818). Key words: Inmmunoinformatic; Reverse Vaccinology; Antigen selection; Vaccine candidates. Immunoendocrine interactions in pulmonary tuberculosis: Influence of infection

on the morphology of the male reproductive system

B. Ramos-Robles1, R.A. Valdéz1, U. Juárez-Hernández2,3, D. Mata-Espinosa3, J. Barrios-Payán3 , O. Bello- Monroy4, R.E. Hernández-Pando3, M. C. Romano-Pardo 1

1 Department of Physiology, Biophysics and Neuroscience of the Center for Research and Advanced Studies, Mexico.

2Department of Immunology, School of Medicine , National Polytechnic Institute, Mexico

3Department of Experimental Pathology of the National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico

4Laboratory of Cellular Physiology, Health Science Department of the Metropolitan Autonomous University, Iztapalapa, México

Background Tuberculosis (TB) is a bacterial infection caused by Mycobacterium tuberculosis (Mtb) that mainly affects the lungs but can spread to other organs as the male reproductive system. In the course of the disease remarkable changes occur in the expression of cytokines that could cause endocrine changes. So far, it is not known whether tuberculosis localized only in the lung can cause changes in the male reproductive system. Therefore, the aim of this study was to investigate whether pulmonary TB produces morphological alterations in the mice reproductive organs, spermatogenesis and serum testosterone concentrations. Methods BALB/c mice were infected intratracheally with Mtb strain H37Rv (250, 000 bacteria). Six animals from each time were sacrificed on days 1, 3, 7, 14, 21, 28, 60, 90 and 120 post-infection. CFUs counting was performed in lung, testis, prostate and seminal

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vesicle tissues to detect the presence of the mycobacteria. Spermatobioscopy was conducted to assess spermatozoid number and quality. The tissues were processed for histology and the epithelia from seminal vesicals and prostates were measured with the morphometry system Answering Qwin Leica. In addition, we have measured the serum androgen concentrations by radioimmunoanalysis (RIA) in control and infected mice in each time of sacrifice. Results Mycobacterium colonies only grew in lung tissue. Histopathological studies revealed no abnormalities in the testes, but a significant atrophy of the seminal vesicles and prostate epitheliums was found in the late stages of disease (p<0.05). Spermatobioscopy revealed a decreased number of spermatozoids in the later stages of the disease. Furthermore, serum androgens showed a trend to decrease in the infected mice compared to the healthy animals in the later TB stages. Conclusions The results indicate that the lung Mtb infection affects the reproductive organs and the sperm production despite being free of infection; these alterations are probably caused by the decreased production of androgens. The immunological alterations that occur in the course of pulmonary Mtb may play a role in the found alterations, by causing an imbalance in androgen production that may affect the function and morphology of their target organs. Acknowledgments: to Conacyt, Mexico, for M.C. Brenda Ramos Robles PhD fellowship. Keywords: Mycobacterium tuberculosis, H37Rv, male reproductive system, hormone, androgens.

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ASPECTOSMOLECULARES

Evaluation of Xpert MTB / RIF Assay for the diagnosis of Mycobacterium tuberculosis complex in pulmonary and extra pulmonary samples.

DA. Orozco-Jauregui*, N. Ayala-Chavira*, MF Flores-Torres*, Y. Vega-Laguna *, M Lopez-Rodríguez*

*State Laboratory of Public Health. Jalisco Health Ministry, Av. Zoquipan # 1000 Zap. Jalisco. Mex. ceeslab@hotmail.com

Introduction For an optimal management of patients with tuberculosis it is indispensable to obtain the microbiological identification of the etiological agent in the fewer amount of time possible and the new diagnostic technologies are alternatives acknowledged by the World Health Organization which mentions that the expansion of the rapid tests is allowing a timely diagnosis for a significantly higher number of patients, emphasising the use of the polymerase chain reaction and rifampicin resistant quick test for the

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early identification of the Mycobacterium tuberculosis complex, we believe important therefore, to present the experience obtained in the laboratory when using such methodology, undertaking a comparative analysis of the results to determine the sensibilities and specificities regarding the conventional techniques used such as staining, growing and classification.

Methods Comparative study carried out in the State Laboratory of Public Health of the Jalisco Health Ministry with the results of the analysis of 334 samples of patients with a clinical suspicion of pulmonary and extra pulmonary tuberculosis using a PCR rapid test in real time and rifampicin sensible (Xpert MTB/RIF)MR in relation to the results obtained with other conventional techniques such as a smear and Ziehl-Neelsen stain, Lowenstein Jensen growth in solid medium, Middlebrook 7 H9 Versa Trek (IL) liquid medium and classification by rapid test of immunochromatography (SD Tb Ag MPT 64) estimating the sensibilities, specificities and the predictive values of the tests using the growth as “gold standard” to compare the effectiveness of the tests. Results The use of the Xpert raised to 160 people with a positive result in regards to 143 achieved by growth, also detecting in 18.12% of patients, rifampicin resistance, with a sensibility of 100% and specificity of 99%, positive predictive value of 99% and negative predictive value of 100 %; the immunochromatography TB Ag MPT-64 test obtained a sensibility of 99 % and specificity of 100%, positive predictive value of 100% and negative predictive value of 92%; showing a concordance of 100% in the results regarding the Xpert with no evidence of a crossed reaction with MNTB achieving to identify MTB in 91.78% of the isolations obtained by growth and 8.21% were MNTB. Conclusions The identification of MTB by GeneXpert showed advantages over other used techniques obtaining positive results in less time as they were applied directly to the sample, sensibility and high specificity, simplicity of the technique and additional determination to rifampicin resistance; as disadvantages: does not detect micro bacteria different to complex M. tuberculosis and a high cost. On the other hand, the immunochromatography, presents excellent sensibility and specificity with the disadvantage of being applied only to the isolations achieved by growth. Acknowledgements For their help, to the management of the State Laboratory of Public Health of the state of Jalisco and to Distribuidora Química y Hospitalaria GAP SA de CV. Key words: Tuberculosis, pulmonary tuberculosis, extra pulmonary tuberculosis, laboratory quick tests, laboratory diagnosis.

Molecular and metabolic events in the production of antigen ESAT-6 from Mycobacterium tuberculosis using a thermo-inducible system in recombinant

Escherichia coli

S. Restrepo-Pineda, C.G. Bando-Campos, N.A. Valdez-Cruz, M.A. Trujillo-Roldán Unidad de Bioprocesos, Instituto de Investigaciones Biomédicas, UNAM. Tercer

circuito exterior, Edificio C, C.P. 04510, D.F., México

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sarestrepo90@gmail.com Introduction The early secreted antigenic target-6 (ESAT-6) of M. tuberculosis has been proposed to be used as part of a vaccine or diagnosis of Tuberculosis. One of the strategies to improve industrial production of recombinant proteins in E. coli is the expression system induced by temperature, because it is based on finely regulated promoters and the use of chemical inducers is avoided. However, the increase in temperature can causes decrease in cell growth and activation of bacterial heat shock response (HSR). These molecular events have been studied in separate contexts, but it converge in the activation of chaperones and proteases genes. In this study, the main objective was to analyze the kinetic behavior of a thermo-inducible strain of E. coli and to evaluate expression of recombinant ESAT-6 using different thermo-induction strategies. Methods First, transformation of E. coli was performed. Then, cells were grown in shake flasks (250 mL) and bioreactors (1 L) at different temperatures (30, 39 and 42 ° C) and recombinant protein production were tested using SDS-PAGE and Western Blot. Results and conclusions We found that growth of recombinant E. coli decreased in cultures at 39°C and 42°C compared to the cultures without induction (30°C), which can be attributed to the thermal stress and metabolic burden. In shake flasks, the accumulation of acetate was observed. While in bioreactors, the residual acetate began to be consumed at the end of the culture. Finally, under both induction temperatures (39 and 42°C) the recombinant protein ESAT-6 was trapped in inclusion bodies regardless of the culture system used. Acknowledgments CONACYT 178528, 214404 & 220795; PAPIIT-UNAM IN-210013 & IN-209113. Key words: ESAT-6, Thermo-inducible System, Heat Shock Response, Inclusion Bodies.

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Zmp1 gene detection in a hypervirulent strain from the Mycobacterium tuberculosis Beijing Family

U. Juárez-Hernández1,2; B. Ramos-Robles3; O. Bello-Monroy4; D. Mata-Espinosa2; S. Rivera-Gutiérrez5;M. A. Torres-Vega6 ; R. Hernández-Pando2 ;P. Figueroa-Arredondo1

Department of Immunology, School of Medicine , National Polytechnic Institute, Mexico.1

Department of Experimental Pathology of the National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico2

Department of Physiology, Biophysics and Neuroscience of the Center for Research and Advanced Studies, Mexico3

Laboratory of Cellular Physiology, Health Science Department of the Metropolitan Autonomous University, Iztapalapa, México4

Department of Microbiology, National School of Biochemical Sciences of the National Polytechnic Institute, México5

Department of Gastroentherology of the National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico6

Background It is estimated that one third of the world´s population is infected by Mycobacterium tuberculosis (Mtb), resulting in almost 2 million deaths every year. Mtb infection shows a variety of clinical and histopathological features. The most frequently Mtb genotype causing disease globally is Beijing and several lines of evidence suggest that members of the Beijing genotype might be hypervirulent in comparison to most of the other strain genotypes. Speaking about the host, assembly and activation of the inflammasome are essential processes of the innate immune response, and they involve the processing and maturation of IL-1β and IL-18 cytokines. Rv0198c gene (zmp1), plays a critical role in preventing caspase-1-dependent activation and secretion of IL-1β. Infection with Mtb triggers the inflammasome assembly, including the adaptor molecule ASC and caspase-1. Inflammasome activation results in IL-1β release, followed by the engagement of its receptor (IL-1R). Subsequent IL-1R signaling activation, promotes maturation of the phagosome, that ultimately leads to the fusion of lysosomes to activate the phagolysosome and therefore achieve bacterial degradation. Therefore, Mtb strains encoding the zmp1 gene are capable of preventing maturation of the phagosome, by inhibition of the inflammasome activation, increasing Mtb intracellular survival into the vacuole. From all the above, we hypothesize that the hypervirulent strain Beijing 31, should present the zmp1 gene and perhaps over express its product. In this work we would like to show first, confirmation of hypervirulence of Beijing 31 strain by testing its mortality rates in progressive tuberculosis murine model and presence of zmp1 gene in the bacteria. Methods Mortality was determined in a group of 18 mice after infection with Mycobacterium tuberculosis. Genera and specie was confirmed using the acid-alcohol resistance technique (Ziehl-Neelsen) and genetic characterization was carried out by the spoligotype methodology. For genetic identification of zmp1 gene, an in vitro culture of Mtb Beijing 31 was grown and direct PCR was performed. The PCR amplified fragment of Mtb was sequenced and analyzed in silico.

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Results After infection of mice, the Ziehl-Neelsen was positive to Mtb and the spoligotype pattern corresponded to the Beijing strain. In these experiments the mortality induced by the bacteria was 100%, 21 days after infection. Besides, the zpm1 gene was identified by PCR amplification from our Beijing 31 strain Conclusions The zpm1 gene presence strongly suggest that the Beijing 31 strain used in our experiments possess the capability to inhibit the phagolysosome assembling and the inflammasome activation; thus explaining the high mortality rates observed. Presence of the zmp1 enhances the bacterial possibility to survive inside macrophages, which therefore increase the bacterial virulence reflected as a high mortality rate, observed in earlier phases of the disease. Acknowledgments: CONACyT Keywords: Mycobacterium tuberculosis, Beijing strain, hypervirulent, zmp1 gene, inflammasome, phagolysosome.

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Mycobacterium abscessus infection and replication into lung epithelial cells (A549 cells):

role of structural and secretory mycobacterial components

J. C. Hernández-González1, N. S. Castrejón-Jiménez2, V. Vanzzini-Zago3, B. E. García-Pérez2, J. Luna-Herrera2.

1) Área Académica de Medicina Veterinaria y Zootecnia, Instituto de Ciencias Agropecuarias-UAEH. Tulancingo de Bravo, Hgo., México.

2) Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala s/n, 11340 Mexico City,

D.F., México. 3) Centro Nacional para Prevención de la Ceguera. Mexico City, D.F., México.

Background Previously, our research group described that a clinical isolate of Mycobacterium abscessus (MABs) was able to infect lung epithelial cells (human type II pneumocytes, A549 cell line), since the first hour of infection, the mycobacteria stimulated the formation of abundant cytoplasm endocytic vacuoles and long membrane protrusions suggesting interiorization by macropinocytosis. Infected cells also produced nitric oxide (NO) in response to the infection. The aim of this study was to establish if the entry of MABs into the A549 cells was due to macropinocytosis, and to analyze the role of secretory or structural components from the mycobacteria in the internalization of this pathogen. Methods Intracellular multiplication of Mycobacterium abscessus. Confluent monolayers of A549 cells prepared in 24 well plates were infected with MAB in suspension at a multiplication of infection (MOI) of 10:1, then they were incubated for 2, 6, 24 y 48 h. At each time point four wells were lysed to determine the number of viable bacteria. Cells lysates were plating onto Middlebrook 7H11 agar plates to determine colony-forming units (CFUs). Fluid phase uptake. Confluent monolayers of A549 cells were prepared in the same conditions as it was mentioned before. The cells were infected with MAB suspension, treated with MAB culture supernatant to determine the role of possible secretory components or with NaN3– treated MAB or heat-killed MAB. In all cases Dextran-FITC 70 kDa was added to quantify the fluid phase uptake by fluorometry after 30, 60, 90 y 120 min. Effect of biochemical inhibitors on fluid phase. The effect of biochemical inhibitors was analyzed by plate fluorometry as mentioned before. The cells A549 were infected with MABs or treated with its supernatant, along with amiloride (an specific inhibitor of micropinocytosis), and wortmannine (an PI3K inhibitor). Results MABs showed a high infectious and replication ability in the A549 cell line, recovering up to 3.9 ± 15 x 106 UFC/ml after 72 h post-infection. The fluid phase uptake was high and statistically significant since 30 min until 2 h after infection; in contrast, fluid phase uptake was lower with bacterial supernatant-treatment or NaN3-inactivated bacteria the fluid phase, the lower uptake was triggered with heat killed bacteria. The biochemical inhibitors, amiloride and wortmannine inhibited fluid phase uptake induced by viable bacteria and its supernatant.

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Conclusions The clinical isolate of MAB used in this study showed high invasive and replicative abilities in the A549 non-phagocytic cells. Bacterial supernatant induce fluid phase uptake suggesting the participation of secretory factors in MAB internatization. Bacteria inactivated with NaN3, promote phase fluid uptake but not as high as that observed with viable cells. These outcomes suggest that secretory and structural components of MAB participate in bacterial internalization. Acknowledgments. SIP20150914 y CONACYT CB-180401. Keywords. M. abscessus, Pneumocytes type II, macropinocytosis.

Assessment of the antimycobaterial activity of the venom of Conus brunneus and Conus nux

E.E. León-Acosta 1, A.P. Gutiérrez-Ordoñez 1, J. Bernáldez-Sarabia1 and A.F. Licea-

Navarro 1. 1 Applied and Experimental Biology Division, Biomedical Innovation Department,

Center for Scientific Research and Higher Education of Ensenada (CICESE), B. C., Mexico.

eleon@cicese.edu.mx Introduction Tuberculosis (Tb) is an infectious disease caused by the bacillus Mycobacterium tuberculosis. Is a significant public health concern since is an important cause of death worldwide due to bacterial resistance to the available anti-tuberculosis agents. This problem has led to a continuous search for new drugs for the treatment of this disease. In this search it had been found a wide range of active compounds present in the venom of several species of animals, including cone snails. The venom of each specie contains a variety of highly structured peptides known as conotoxins, each have a different biological activity, such as inhibition of ion channels and membrane receptors on different cell types, making them potential molecules for development of new treatments and therapy for different diseases, including tuberculosis Methods The organisms were collected in the beach "El Bajo" in Loreto B.C., Mexico. Venom purification was performed by high resolution liquid chromatography in reverse phase (RP-HPLC). The assessing of cellular metabolic activity using M. tuberculosis strain H37Rv, was performed with colorimetric assays in 96-well microplate using the reagent MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), as a positive control isoniazid was used. Fractions of each snail venom were evaluated in duplicate, at an initial concentration of 200 ug/ml with a bacillary suspension of 1.5 x 107 cells/ml. All the assays were performed in a Biosafety Level 3 Laboratory. Results For Conus brunneus venom, 12 fractions were tested in the assay for cell viability; the fraction called CB10 obtained a greater percentage of inhibition of the mycobacteria. For Conus nux venom, 13 fractions of were tested in the cell viability assay, in the CN3, CN4 and CN7 fractions, inhibitory activity was found

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Conclusions. We report the evaluation of the venom of two species of snails with anti-tuberculosis potential, which showed some promising results for M. tuberculosis inhibition, this results leads to obtain sub fractions and determine its activity and perform a characterization of this conotoxins. Acknowledgments. Center for Scientific Research and Higher Education of Ensenada (CICESE), Leslie Otero and Pavel Lugo. Keywords. Mycobacterium tuberculosis, tuberculosis, Conus brunneus, Conus nux.

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Isolation and characterization of toxin from marine snails of the genus Conus with activity in Mycobacterium tuberculosis

J. Bernáldez-Sarabia1 and A. Licea-Navarro1

1 Biomedical Innovation Department, Scientific Research and Higher Education Center from Ensenada (CICESE), B. C. México.

jbernald@cicese.edu.mx, alicea@cicese.edu.mx

Introduction Tuberculosis disease is considered the leading cause of death from a single infectious agent (Mycobacterium tuberculosis). It is estimated that one-third of the world population carries the tuberculosis bacillus (WHO Report 2006). In 2011, Baja California reported the largest number of deaths in the country for pulmonary TB. The permanence of this mycobacteria through history can be linked to current chemotherapy, which has been proven to be a very complex prophylaxis with 4 drugs administration for a period of 6 to 8 months. Therefore, this work focused on the search for marine molecules with potential for future application in tuberculosis therapy. Methods We performed biochemical and biological characterization of the venom of an endemic marine snail, employing HPLC technique for purification of our conotoxin of interest. The evaluation of the in vitro antimycobacterial activity of several compounds of vemon was performed against Mycobacterium tuberculosis H37Rv strain using MTT and blue alamar assays. The full-length sequence of conotoxin was cloned and sequenced using 3’ RACE and 5’ RACE. Results One molecule of protein origin was identified, which was able to inhibit the in vitro growth of bacilli. The results showed that conotoxin inhibits to H37Rv strain with a MIC of 4.08 µg/ml. Primary structure of peptide shows 4 disulfide bonds, 4 modified prolines and C-terminal amidated. Conclusions This placed to our conotoxin as a candidate for future studies focused on inclusion of this peptide to the conventional antibiotic therapy to assess their synergistic activity and behavior on MDR strains assays. Acknowledgments Dra. Elizabeth Ponce Rivas, Dr. Manuel Aguilar Ramírez, Dr. Rogelio Hernández Pando, Dr. Tomás Rentería Evangelista, Dra. Iris Estrada García, Dr. Rafael Laniado Laborin, Samuel Navarro Alvarez. Keywords: Tuberculosis, conotoxins, anti-tuberculosis drugs.

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Molecular identification of isoniazid resistance in Mycobacterium Tuberculosis complex isolated

A. Santiago-Pineda1 and L. L. Martinez-Martínez1**

1Lab. Molecular Biology, Center of Research, Faculty of Medicine UNAM-UABJO, Oaxaca, Oax., Mexico

Introduction Tuberculosis (TB) is caused by members of the M. tuberculosis complex (CMtb). It is considered one of the leading causes of death by infectious diseases, accounting for 2 to 3 million deaths per year worldwide. The most common manifestation is pulmonary TB and its treatment is based on the association of a number of drugs which have different mechanisms to prevent the resistance occurrence. The isoniazid (INH) and rifampin(RIF) are reference drugs in short-course chemotherapy for Mtb and the increase of resistant strains to these drugs have been intensified in later years. We identify in this work the INH-resistance of clinical isolates of CMtb by determining the mutation characteristic. Methods 1.- Clinical specimens (pulmonary and extrapulmonary) from patients suspected of TB and likely drug-resistance were obtained. 2. The sputum samples were decontaminated by adding N-acetyl-L-cysteine and NaOH.3.- For DNA total extraction of every sample, extraction kit (DNA Purification Kit) was used following the manufacturer's directions. 4. To the tube multiplex PCR-reaction was added dNTPs, MgCl2, primers (MTUBr, MTUBf, Katg0F, R315mut, MabAF, inhARmut), Buffer, DNA polymerase GoTaqFlexi 5. For the katG gene PCR were added the same reagents from the previous step but the primers were KatG904 and KatG1523. 6. Finally, 10 µL of each of the amplified gene KatG samples will be subjected to digestion with satI enzyme. Results Forty two of 104 samples amplified a 1020 bp fragment from gyrB gene by multiplex PCR, 5 amplified two bands, one of 1020 bp and other of 296 bp (mutation AGC - ACC), one sample amplified two bands of de1020 bp and other of 146 bp (mutation inhAc-15T). The 56 remaining samples did not amplify any fragment. The 42 samples amplified a 620 bp fragment corresponding to the gene katG which will be digest with the sati enzyme to check other mutations in katG gene, It could be present[AGC---AAC (Ser-Asp), AGC---ATC(Ser-Ile),AGC---ACA(Ser-Thr)]. Conclusions Multiplex PCR results showed that 10.41% (5) of isolates have a mutation in katG315 AGC-ACC (Ser315Thr), 2% (1) has a mutation in theinhAC-15T gene of the regulatory region from mabA-inhA operon. Acknowledgments. The Integral of strengthening institutional program (PIFI) for financial support. Keywords. Tuberculosis, Mycobacteirum tuberculosis, resistance, isoniazid.

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"Drug-resistance and its relationship with Mycobacterium tuberculosis genotypes of clinical isolates from the State of Oaxaca"

Valencia-Carmona OD1,2, Palma-Nicolás JP3, Martínez Martínez LL2, Nakamura-

López Y1* 1 Consejo Estatal para la Prevención y Control del Sida-Centro Ambulatorio para la

Prevención y Atención del Sida e ITS (COESIDA-CAPASITS) Oaxaca. 2 Posgrado en Biomedicina Experimental, Facultad de Medicina y Cirugía, Universidad

Autónoma Benito Juarez de Oaxaca. 3 Departamento de Microbiologia y Parasitología, Facultad de Medicina, Universidad

Autónoma de Nuevo Leon. yuko@unam.mx

Background Tuberculosis (TB) is considered by WHO as an emergency public health worldwide and one of the reemerging diseases of highest mortality since 1993. It is an infectious disease caused by the Mycobacterium tuberculosis complex, which consists of eight species; the most important is M.tuberculosis. In 2013, 9 million people developed TB and 1.5 million died from this infection. Mexico ranks third in number of cases of tuberculosis in the Americas, the State of Oaxaca has incidence and mortality rates above the national average, and there is no information about the circulating genotypes. Moreover, there is a global trend towards drug resistance, which makes more complicated the disease control. Detection of patterns of drug resistance and genotyping of strains are powerful tools that help to the disease management. Objective Correlate the genotypes and patterns of drug resistance of clinical isolates of M. tuberculosis obtained during the year 2014 in the State of Oaxaca, México. Methods We obtained 25 clinical isolates of M. tuberculosis. Patterns of drug resistance to first line drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SM), were determined by the proportion method, and they were genotyped by MIRU-VNTR 24 loci using the Cavalli-Sforza Dc algorithm. Results The drug-suceptibility testing showed that 8% of isolates were RMP mono-resistance, 12% STR mono-resistance and 4% multi-drug resistant TB. We obtained 24% of strains close to the Haarlem lineage, 20% to EAI, 16% to LAM, 12% to Ghana and 12% could not be classified in any lineage. The MDR-TB isolate is close to Haarlem lineage. The lineage more frequently associated with drug-resistant to at least one drug was Haarlem (33.3%), followed by LAM (25%). Conclusions These preliminary results show a high diversity of genotypes and drug-resistant patterns. Apparently, the strains belonging to Haalem lineage have more probability to be drug resistant to at least one drug of first line. To enrich this project, 15 more isolates will be study, and all strains will be analyzed by spoligotyping.

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Acknowledgments. This study is supported by the Fondo Mixto Consejo Nacional de Ciencia y Tecnología (CONACYT) and Oaxaca State Government, grand “FOMIX 193298”. Keywords. Tuberculosis, genotyping, drug-resistant.

MICOBACTERIASNOTUBERCULOSAS(NTM)

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Occurrence of nontuberculous mycobacteria from ready-to-eat sprouts

A. L. Cortés Cueto, N. León Montes, L. P. Salas Rangel, A. C. Helguera Repetto, S. Rivera Gutiérrez, E. Fernández Rendón, R. L. García Reyes, J. F. Cerna Cortés y J.

A. González y Merchand Laboratorio de Microbiología Molecular, Depto. de Microbiología, ENCB-IPN. Prol. de

Carpio y Plan de Ayala S/N. C.P.11340. México. D.F. cocaoc82@gmail.com

Keywords: Nontuberculous mycobacteria, sprouts, microbiological quality. Background. Nontuberculous mycobacteria (NTM) are opportunistic pathogens found in the environment that may cause life-threatening infections in humans. The incidence of NTM disease is increasing worldwide, in both immunocompetent and immunocompromised subjects1. NTM have been isolated from various kinds of food, and many studies support the hypothesis that food, especially raw or partially cooked products, plays a role as a source of NTM for humans2. The aims of this study were to evaluate the microbiological quality of ready-to-eat (RTE)-sprouts collected in Mexico City and to analyze the occurrence and identity of NTM in these samples. Methods. One hundred samples of RTE-sprouts were collected from different boroughs of Mexico City: 50 from different supermarkets (SPM) and 50 from street-vendor stalls (SVS). The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC) and fecal coliforms (FC) in all sprouts were analyzed following the methods approved by the FDA's Bacteriological Analytical Manual. Samples were decontaminated with 0.1% cetylpyridinium chloride and inoculated onto Middlebrook 7H10 agar supplemented with different antibiotics. The identification of acid-fast bacilli was carried out by the Ziehl-Neelsen (Z-N) stain. A PCR assay was used for the identification of the genus Mycobacterium3. Subsequently, a second PCR was performed to identify if the mycobacterial isolate belonged to Mycobacterium tuberculosis complex or to the group of the NTM3. NTM species identification was performed using three molecular markers (rrs, rpoB and hsp65 genes)1. Results. AMB and TC were present in 100% of samples with limits ranging from 1.2 x106 ─ 6.2x108 CFU/g and 6.1 ─ >1100 MPN/g for AMB and TC, respectively. All samples

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exceeded the permissible limit of AMB established by the International Commission on Microbiological Specifications for Foods (ICMSF)4. Twenty-four percent of the samples contained FC outside the permissible limits established by the ICMSF. Mycobacteria were present in 12 samples; five isolates were obtained from sprouts purchased from SPM and seven from SVS. All isolates belonged to the genus Mycobacterium; none of them belonged to the MTC. Sequence analysis of the genes rrs, rpoB and hsp65 revealed five isolates of M. porcinum, two of M. abscessus, two of M. gordonae, two of M. mucogenicum and one of M. avium. Conclusions. RTE-sprouts had an unsatisfactory microbiological quality and the 12% of them harbored NTM. Therefore, sprouts consumption could be considered a health risk for consumers particularly if their immune response is diminished. References 1. Cerna-Cortes, J.F., et al.2009. J. Food Prot. 72:182-184. 2. Yoder, S. et al. 1999. Appl. Environ. Microbiol. 65: 2650-2653 3. Cobos-Marin, L. et al. 2003. Epidemiol. Infect. 130:485-490 4. ICMSF. 2011. Microorganisms in Foods 8, 147-153.

Assessment of protein fractions culture supernatant of Mycobacterium bovis in interferon gamma release assay by ELIspot

Ortega ARA2, Díaz OF1*, Jaramillo ML1, Martínez MJJ2, Pérez GR3, Martínez EH4,

Lascuraín LR5, Santillán FMA1, Hernández AL1 1CENID-Microbiología Animal INIFAP

2FMVZ-UNAM 3FES-Cuautitlán UNAM

4Profesionista independiente 5FM-UNAM

diaz.fernando@inifap.gob.mx Introduction Interferon gamma (IFN-γ) is one of the most critical effector molecules to the establishment of a cell-mediated immune response required for control of Mycobacterium bovis infection, so its evaluation have been investigated as a biomarker of disease. The EliSpot assay allows for the identification of a wide range of cell populations based upon their cell function and their production of specific immune molecules. In tuberculosis diagnosis, this assay has been used as a research tool to detect cell populations that are releasing IFN-γ following exposure to mycobacterial antigens. Furthermore, in recent years the research in the field of bovine tuberculosis has been focused on identifying specific antigens capable of inducing the production of IFN-γ. Thus, the aim of this work was to evaluate the ability of inducing IFN-γ fractions of the protein extract from the culture filtrate of M. bovis separated by isoelectric focusing in ELIspot assays.

Methods M. bovis strain AN5 was grown in Dorset-Henley liquid medium at 37 °C for 6 wk; subsequently, cultures were filtered obtaining free-bacteria medium. Filtered

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constituent proteins were precipitated with ammonium sulfate, then preparative liquid-phase isoelectric focusing was used for the separation of culture filtrate protein (CFP) of M. bovis. This procedure resolved CFP into 20 fractions with a pI range of 2.59 to 12.9. These fractions were subjected to immunological analysis by ELIspot; for which, were separated PBMC from 24 cows tuberculin reactors and 22 non-reactors. The cells culture were also stimulated with bovine PPD and avian PPD, ESAT-6 and CFP-10 antigens, as well as with pokeweed mitogen and the no-antigen control. The quantification of reaction points was performed using an automatic counter ImmunoSpot 3.2. The levels of IFN-γ produced with different antigens were also evaluated by ELISA. A Kruskal-Wallis test was performed to analyze the results obtained between groups and the Mann Whitney test was used to compare the different antigens

Results The analysis results indicate statistical difference among bovine PPD, the CFPE, ESAT-6 and the fractions 1, 2, 3, 4, 9, 11, 14, 15, 16 and 19. However, greater specificity was determined to bovine PPD itself, the ESAT-6 protein fractions 1, 2, 3, 4, 17 and 19 for the group of reactors cows. By electrophoretic analysis of the fractions it was determined that the MPB70 and MP83 proteins are located in fractions 3 and 4, which correspond to specific proteins of M. bovis. Highlight study the reactivity of cows reactors to antigens fractions 5, 6 and 7, in which heat shock proteins and enzymatic activity proteins are located, but due show high levels of homology to other mycobacterial its diagnostic usefulness is questionable.

Conclusions According to results the fractions 1, 2, 3, 4 and 17 showed greater specificity in assays release of IFN-γ assessed by ELISA and ELIspot, by which are suitable for the purpose searched of disease diagnosis.

Acknowledgments: INIFAP 13453132021 Keywords: Diagnosis, tuberculosis, bovine, ELIspot

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Differential recognition of M. Tuberculosis antigens by leprosy and Tuberculosis sera

Patricia Arce Paredes, Sergio Islas Trujillo, Oscar Rojas-Espinosa.

Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala, Col. Santo Tomás, 11340 México, D.F.,

México Background. From preliminary experiments, we have seen that the sera from patients with leprosy and tuberculosis react distinctly with antigens extracted from M. tuberculosis (MTB). Although there is strong cross-reactivity between leprosy and tuberculosis sera with the antigens extracted from MTB, there are also some antigens that are distinctly recognized by these sera. In this investigation we have extended the number of leprosy and tuberculosis sera to analyze their reactivity with secreted proteins from MTB. Materials and methods. We worked with the secretion proteins of MTB grown in PBY medium and used them for Western blot analysis. Strips of nitrocellulose membrane with the transferred proteins were tested against 10 leprosy (lepromatous) sera and 10 tuberculosis (pulmonary) sera. Sera were taken by qualified professionals from Dr. Gea Gonzalez hospital (leprosy) and the General Hospital (tuberculosis), under informed consent of the patients. In a complementary study we tested the sera from mice having different infection times with MTB, and from infected-mice treated with antifimic drugs. Western blot of their sera and histopathologic analysis of their lungs were then performed. Results.

1. We found that all of the tuberculous patients and none of the healthy controls tested distinctly reacted with a 14.5 kDa protein.

2. This 14.5 kDa protein was not recognized by the lepromatous sera. 3. Lepromatous sera very strongly recognized a group of proteins of around 33

kDa; this reactivity disappeared with anti-leprosy treatment. 4. This 33 kDa complex was but weakly recognized by the tuberculosis sera. 5. Mice infected with MTB developed a progressive reactivity with the 14.5 kDa

and this reactivity vanished with the antifimic treatment.

Conclusions. 1. Protein 14.5 kDa of MTB may be an antigen useful for the diagnosis of

extrapulmonary tuberculosis were clinical signs or symptoms of the disease are not clearly evident.

2. Complex 33 kDa might be useful for the confirmatory diagnosis or leprosy or for case detection.

3. Identification of 14.5 kDa and complex 33 kDa need to be accomplished. 4. Many studies have still to be done on this project.

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Research carried out with the financial help of COFAA (IPN), EDI (IPN), and SNI (CONACyT).

Molecular Identification of Tuberculous and Non-Tuberculous Mycobacteria from Pulmonary and Extrapulmonary Infections Using Real Time PCR

F. Nani-Rodríguez1, C. Lara-Ochoa2, M. A. Pavón3, J. A, Yáñez4, Y. Martínez-

Laguna5, M. L. Cedillo2. 1Facultad de Ciencias Químicas, BUAP. Puebla, México.

2Centro de Detección Biomolecular, V.I.E.P., B.U.A.P., Puebla, México. 3Centro Médico Nal. Manuel Ávila Camacho, Hosp. de Especialidades San José.

IMSS. Puebla, México. 4Laboratorio de Investigación en Microbiología Oral, Facultad de Estomatología.

BUAP. Puebla, México. 5Vicerrectoría de Investigación y Estudios de Posgrado, B.U.A.P., Puebla, México.

Introduction Tuberculosis (TB) is a reemerging disease considered one of the main world-health problems. In developing countries most of the diagnosis of TB is based on the Ziehl-Neelsen stain or bacterial culture. Smear microscopy is insensitive and microbiological methods may take several weeks to yield identification. By contrast, the use of molecular techniques such as real-time PCR allows specific identification in a shorter time and with greater sensitivity, which represents an ideal strategy that health services should be implement to help identify TB cases. The purpose of this study was to identify tuberculous (MTBC) and nontuberculous (NTM) Mycobacteria from pulmonary and extrapulmonary clinical samples using quantitative real-time PCR (qPCR). Methods Four hundred and eighteen biological samples (sputum, bronchoalveolar lavages, pleural fluid, cerebrospinal fluid, urine, peritoneal fluid, stools and blood) from patients with clinical signs of TB were included in the study. DNA was extracted from samples using the High pure PCR template preparation kit (Roche). Quantity and purity of DNA was determined by nanospectrophotometry. DNA was analyzed by qPCR using the LightMix® TIB MOLBIOL kit. This kit amplifies a fragment of the gene that codifies for 16S RNAr with two binding sites of specific probes for Mycobacterium tuberculosis complex (MTBC) and NTM. MTBC is detected in the 640/back 530 channel while NTM is detected in the 705/530 channel. PCR products were identified using the dissociation curve. The sensitivity of this kit was measured in ten biological samples of blood collected from healthy volunteers spiked with serial dilutions of 1, 10, 100, 1000 genomes of the strain BCG 172-1 and tested in duplicate in the qPCR assays.

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Results Three hundred and ninety samples (93.3%) were negative and twenty-eight samples (6.6%) were positive for Mycobacterium spp. Twenty-three samples were positive for MTBC and five for NTM. Ten samples from pulmonary sites (7 sputum, 2 bronchoalveolar lavages and 2 pleural fluid) and 18 from extrapulmonary sites (7 cerebrospinal fluid, 5 urine, 1 peritoneal fluid, 1 stool and 2 blood) were positive for Mycobacterium. This approach allowed us to detect as few as 10 to 100 organisms in 7 and 3 samples of blood seeded with mycobacteria. Conclusion Most of the MTBC and NTM were detected mainly from extrapulmonary sites. Our data suggest that it is important to identify for MTBC and NTM in patients with suspected TB due to the severe damage that the infection may produce if it is not diagnosed and treated promptly. The use of qPCR assay in the diagnostic of the TB allowed the specific detection in the short time of mycobacteria directly in biological samples. Acknowledgments: We are grateful to the Vicerrectoría de Investigación y Estudios de Posgrado (BUAP) for support. Keywords: Tuberculous Mycobacteria, Nontuberculous Mycobacteria, Real Time PCR.

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Follow up study immune response in calves by vaccination with Mycobacterium bovis BCG-Phipps strain in field conditions

E. Quevillon-Cardinal2, L. Jaramillo-Meza1*, F. Díaz-Otero1, R. Lascurain-Ledesma3,

J.A. Gutiérrez-Pabello4 1CENID-Microbiología Animal INIFAP

2Universidad de Montreal 3FM-UNAM

4FMVZ-UNAM jaramillo.laura@inifap.gob.mx

Introduction The persistence of bovine tuberculosis (bTB) poses a major economic problem and a global human health risk. The control of the disease is based on a test and slaughter strategy, a costly method for the developing world. Immunization of cattle with bacillus Calmette-Guérin (BCG), especially neonates, induces protection against Mycobacterium bovis and has been proposed as a strategy for bTB control. However, most of the experiments are generally conducted under controlled conditions and few studies have evaluated the immune responses and the protection induced by BCG in a more natural cattle-to-cattle transmission setting, which could influence the results found in controlled experiments. Therefore, the aim of this study was to provide information about immune responses induced in neonatal calves by vaccination with BCG-Phipps in field conditions. Methods Twenty Holstein-Friesian calves less than one month of age, belonging to a herd with bTB prevalence of 40% were used; ten calves were inoculated subcutaneously with 1x106 CFU of BCG Phipps, while the other ten accounted the control group. Calves remained within the herd throughout the study phase, and sampled weekly during the first month post vaccination, and monthly thereafter for a total period of seven months to assess levels of IFN-γ after in vitro stimulation of whole blood cultures with bovine PPD. The percentage of T cell subpopulations and degree of cellular activation by CD25 expression was determined by flow cytometry in stimulated cultures. For the detection of possible infections in the groups, the purified antigens ESAT-6 and CFP-10 were used for lymphocyte stimulation in vitro, considering that such antigens allow differentiation of infected from vaccinated animals. Results The data obtained were analyzed using Student's t-test employing the program JMP 5.0, a probability of p<0.05 was considered significant. In the vaccinated group showed a progressive increase in the levels of IFN-γ during the first month after vaccination, being significant in relation to the control group at 21 (p= 0.02) and 30 days (p=0.02). The percentages of CD8+ T cells and the activation levels of CD4+ and CD8+ T cells were observed, 3 to 4 weeks post-vaccination (p=0.001). Furthermore, any calf vaccinated turned out reactor to the intradermal comparative cervical tuberculin (ICCT) test. Conclusions Therefore, it can be concluded that BCG Phipps strain stimulated peripheral blood T

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cells activity and induced a cell-mediated immune response (IFN-γ production and increase in CD8+, CD4+/CD25+ and CD8+/CD25+ T cells). In addition, the vaccination did not interfere with the ICCT test as reported, which represents an important implication, as the vaccine could be applied in bTB control campaigns. However, further studies are required to evaluate the protective efficacy of this BCG strain over a long study period, especially in a natural transmission setting. Acknowledgments: INIFAP 13453132021 Keywords: BCG, tuberculosis, calves

Identification of non-tuberculous mycobacteria (NTM) in cervical lymphadenopathy biopsies from HIV-infected patients

B. P. Ruiz-Sánchez1, A. Hernández Solis2, M. González Villa3, M. I. Wong-Baeza1, H.

González González2, R. Cicero-Sabido2

1. Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México

D.F., México 2.Unidad de Neumología, Hospital General de México “Dr. Eduardo Liceaga”, y Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F.,

México 3. Instituto de Diagnóstico y Referencia Epidemiológica, Secretaria de Salud, México

D.F., México

Background Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. TB is predominantly a lung disease, but cervical lymphadenopathy has increased with the co-infection with HIV/AIDS. Cervical lymphadenopathy can be caused by M. tuberculosis or by non-tuberculous mycobacteria (NTM); the identification of the NTM is important to determine the most effective treatment and to predict the patient’s outcome. The objective of this study was to identify the most common NTM in the cervical lymphadenopathy lesions of HIV-infected patients. Methods: * Prospective and consecutive study of cervical lymphadenopathy in HIV-infected patients with clinical suspicion of NTM infection, from January 2010 to January 2013. * Patients with HIV infection, diagnosed by ELISA and WB, weight loss, decreased appetite and cervical lymphadenopathy. * The lymph node biopsy was used for histopathology (looking for granulomas) and bacteriology (Ziehl-Neelsen stain, cultures) studies, and for PCR amplification of IS6110 and Hsp65, with the following primers: IS6110F (5’-GGA-TCC-GCC-AGC-CCA-GGA-TCC-TGC-G-3’), IS6110R (5’-AGG-TGC-GGA-CCA-CCA-GCA-CCT-AAC-C-3’), Tb11 (5’-ACC-AAC-GAT-GGT-GTG-TCC-AT-3’) and Tb12 (5’-CTT-GTC-GAA-CCG-CAT-ACC-CT-3’). After the PCR, the Hsp65 amplicon was restricted with BstEII and HaeIII (Roche Diagnostics, Mannheim, Germany), the fragments were analyzed by electrophoresis on a 3% Nusieve 3:1 agarose gel (Lonza, Basel, Switzerland), and the results were interpreted according to the algorithms of Devallois, Brunello and Chimara. The patients received highly active antiretroviral therapy (HAART) and directly observed treatment, short-course (DOTS) therapy.

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Results: Forty patients with confirmed M. tuberculosis infection and cervical lymphadenopathy were included in this study; 20 of these patients were HIV-positive, with counts of CD4+ T cells below 100 cells/mm3. Unilateral cervical lymphadenopathy was present in 15 HIV-positive patients (75%) and 18 HIV-negative patients; the affection was predominantly in the right cervical lymph nodes, and the histopathology studies revealed the presence of caseificating granulomas in the lymph nodes. A positive Ziehl-Neelsen stain in the lymph node was observed in 7 (35%) of the HIV-positive and 10 (50%) of the HIV-negative patients, but mycobacteria were recovered by culture from the 40 lymph node biopsies. In 19 of the HIV-negative patients, the mycobacteria recovered from the lymph nodes were M. tuberculosis, and in the remaining HIV-negative patient (5%) the mycobacteria was M. fortuitum. In the HIV-positive patients, 17 (85%) mycobacteria recovered from the lymph nodes were members of the Mycobacterium tuberculosis complex (MTBC), 2 (10%) were M. intracellulare and one (5%) was M. gordonae. One year after treatment, only 4 of these HIV-positive patients survived, while 19 HIV-negative patients were still alive, and the other abandoned the treatment. Conclusion In Mexico, cervical lymphadenopathy has increased in TB patients with the co-infection with HIV/AIDS. Here we find that HIV-positive patients have increased susceptibility to infection with NTM. Acknowledgments: We appreciate the assistance of Arturo Reding (Dirección de Investigación, Hospital General de México “Dr. Eduardo Liceaga”) and Marco Gudiño (Unidad de Investigación Clínica, Facultad de Medicina, UNAM) for technical support and manuscript preparation. Keywords: Lymphadenopathy, non-tuberculous mycobacteria, HIV

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Evaluation of a Dot-ELISA for diagnosis of bovine tuberculosis in field trials

L. Jaramillo-Meza 1*, F. Díaz-Otero 1, BAV. Gil-Centeno 2

1CENID-Microbiología Animal INIFAP, 2Profesionista Independiente jaramillo.laura@inifap.gob.mx

Introduction Bovine tuberculosis (bTB) mainly caused by Mycobacterium bovis is an infection that predominantly induces cell-mediated immunity (CMI), therefore the main diagnostic techniques used in eradication programs are based on its evaluation, as the disease progresses a shift from Th1 to Th2 is associated with a decrease of CMI and the development of humoral immunity. However the onset and magnitude of response to individual antigens varied among the animals, situation that can be mitigated by multiantigen assay with well characterized antigens, such as MPB70 and MPB83 being among the most relevant in M. bovis. On the other hand, ESAT-6 and CFP-10 are antigens expressed in M. bovis but absent from environmental mycobacteria or BCG which have been proposed as relevant to differentiate infected from vaccinated animals DIVA candidates. In this work was evaluated a Dot-ELISA for diagnosis of bovine tuberculosis in field trials. Methods The assay was established using protein fractions separated by preparative liquid-phase isoelectric focusing obtained from culture filtrate protein (CFP) of M. bovis, which contain the antigen 85 (Ag85) complex, MPB70, MPB83, ESAT6 and CFP-10. This assay was applied in combination with intradermal tests, IFN-γ assay and ELISA in a 115 tuberculin reactors and 55 non-reactors cows Holstein-Fresian belonging to dairy farm with high prevalence of bTB. The agreement between the positive or negative results obtained with the reference tests was determined by the value from the kappa test. Additionally, the statistically significant association between different assays was confirmed by the chi-square test. Results The 98% of reactor cows to intradermal test were positives to IFN-γ assay, of these 85% were also positives to ELISA and 80% to the Dot-ELISA. For non-reactor cows, the 95% were negatives to IFN-γ assay, 97 % were negative to ELISA and 98% to Dot-ELISA. In the concordance analysis all κ values found to be significant (p <0.01). Conclusions There was strong correlation between tests employed to evaluate the CMI and between those that using to measure serological response. However, it requires further studies especially in cows that have been confirmed to be tuberculous by isolation of the mycobacteria to fully determine the diagnostic validity of the test. Regardless, serological assays utilizing defined antigens as ESAT6 for early bTB detection and MPB70 for detection of the advanced stages, can be used with the intradermal tests to determine the status of disease and reduce the frequency of misdiagnosis of animals in the herds. The multiantigen assay has therefore potential as a diagnostic tool, taking into account the evolution of this infection, the individual variation of immune response and herd history.

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Acknowledgments: INIFAP 13453132021 Keywords: Diagnosis, tuberculosis, bovine

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Biological characterization of the venom of Conus californicus on Mycobacterium bovis.

A. P. Gutiérrez Ordoñez 1, J. Bernaldez Sarabia1 and A. F. Licea Navarro1.

1 Applied and Experimental Biology Division, Biomedical Innovation Department, Center for Scientific Research and Higher Education of Ensenada (CICESE), B. C., Mexico.

anapaolagutierrez@gmail.com

In Mexico and other developing countries it have not been eradicated bovine tuberculosis, which causes public health problems and huge economic losses. This becomes a major issue when there is not an effective treatment for the infected cattle. Recent studies at CICESE have demonstrated that crude (raw) extracts venom of Conus californicus have activity on Mycobacterium tuberculosis. Hypothesis. Mycobacterium bovis growth can be inhibited by one or more toxins of Conus californicus venom. Objective Contribute to the characterization of Conus californicus venom as growth inhibitor of Mycobacterium bovis. Methods The organisms were collected at km 58 Tijuana-Ensenada, BC Mexico. The dissection and extraction of the venom were performed. The extracted venom was purified by HPLC. Simultaneously the isolation and culture of Mycobacterium bovis were performed, where different strains cultures were used. The evaluation of the inhibitory activity of the fractions and subfractions venom of C. californicus, were evaluated by cell viability assays and minimum inhibitory concentration (MIC) test on 3 different strains of M. bovis. Results. In the assay with fractions of whole venom the inhibitory activity was found in fractions 7, 8, 10, 11 and 12 on one strain of M. bovis. In the assays with sub fractions inhibitory activity was found in the subfraction 8.1 from fraction 8 on one strain of M. bovis and in all the subfractions from fractions 10, 11 and 12 inhibitory activity was found, with minimum inhibitory concentration (MIC) between 50 µg/ml and 1.5 µg/ml with effect to 1-3 strains. Conclusions. So far there have been no reports that have been found extracts from marine organisms with inhibitory activity against M. bovis. The results obtained in this study are encouraging, it is suggest to continue in deepen the study of the activity of sub fractions on a larger number of strains and determine its use as part of a therapy against bovine tuberculosis. Acknowledgement. Dr. Milian Feliciano and his team, Dr. Rosa Maria Bermudez, Dr. Ana Maria Escofet, Dr. Ana Denisse Re, Dr. Tomas Rentería and the Municipal Trail of Ensenada. Keywords. Conus californicus, Mycobacerium bovis, bovine tuberculosis.

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Effect of resuscitation promoting factor C (rpfC) in the growth and latency gene expression in Mycobacterium bovis BCG.

L. V. Reyes Gonzalez, O. N. Hernández de la Cruz, M. Castañón Arreola.

Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México Background Mycobacterium tuberculosis is the major etiological agent of human tuberculosis, in which the persistence and reactivation of the bacillus play a key role in the pathogenesis of the disease. The WHO estimates that a third of the world's population is infected with this microorganism without showing signs or symptoms of the disease, because the bacilli are dormant. The search of the factors that allow the bacteria reactivate and leave dormancy led to the identification of resuscitation promoting factors (Rpf's). In the genome of M. tuberculosis there are 5 genes that encode these factors (RpfA-E), however, its role in the reactivation of the bacillus has not been well determined. The aim of this study was to determine the effect of overexpression of RpfC in the growth and the expression of genes associated with latency in M. bovis BCG. Methods The rpfC gene was amplified from DNA of M. tuberculosis H37Rv and cloned into the expression vector pMV261 (pMV261-rpfC). The constructed plasmid was used to transform a M. bovis BCG strain by electroporation. In the transformed strains were evaluated the differences in culture growth. The expressions of a panel of genes associated with latency were assessed by RT-PCR under normal and hypoxia culture conditions. Results The bacilli transformed with the PMV261-rpfC show higher growth than the bacilli transformed with the empty plasmid. The RT-PCR results show that this strain have an increased expression of the pfkB, sigB, sigE, fdxA, nark2, rpfC and rpoA genes compared with the expression observed in the control group. Conclusions The resuscitation promoting factor RpfC induce the growth of M. bovis BCG and the expression of genes associated with latency, which could promotes its reactivation when it is dormant. Acknowledgments This work was supported by the Instituto de Ciencia y Tecnología del Distrito Federal, grant No. PIFUTP09-269. Keywords. Mycobacterium bovis, RpfC, growth, latency

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VNTR-typing of Mycobacterium bovis isolates from cattle in Mexico.

A. Nava-Vargas1; O.E. Pizano-Martínez2; E. Rodríguez-Hernández3; G.J. Cantó-Alarcón4; Y. Rubio-Venegas1; R.I. Guerrero-Solorio4; F. Milián-Suazo4.

1Doctorado en Ciencias Biológicas, UAQ, Qro. Mx. 2CUCS, Departamento de Clínicas Médicas, Universidad de Guadalajara, Jal. Mx.

3CENID-FyMA-INIFAP, Qro. Mx. 4FCN-UAQ, Qro. Mx. alexnavavargas@hotmail.com

Background Bovine tuberculosis (BTb) is a disease caused by Mycobacterium bovis (M. bovis), which affects cattle, other animal species and men. BTb generates severe economic losses and represents a risk to public health. In addition, it is a non-tariff barrier for free trade of cattle, mainly with the US, which is why Mexico has a national campaign aimed to BTb eradication. VNTR is a molecular biology tool useful in the diagnosis and genotyping of M. bovis. It is a PCR based tool that identifies 6-24 loci to establish genetic similarities in bacterial isolates, allowing the identification of M. bovis, establishing dissemination routes and sources of infection. The aim of this study was to compare genetic patterns among isolates of M. bovis from cattle from different regions of Mexico to determine their geographical distribution. Methods PCR was performed with 12 sets of primers and the resulting reaction mixures were examined by gel electrophoresis (2% agarose). The gel was stained with ethidium bromide for the size determination, which indicates the number of copies of each loci to generate a specific VNTR. Results The results show the grouping of 467 isolates of M. bovis from different states of Mexico into 13 VNTR-types. Combining VNTR's patterns with spoligotype patterns, 17 genetically related groups were generated. The dendrogram reveals that there is a tendency to grouping by region with at least two states and state. There are, however, groups which include states from different regions. A general discrimination for VNTR's of 0.99 was obtained. Diversity results for each of the loci ranged from 0.239-0.857. The loci that presented the highest discriminatory power was 2461 (D = 0.857) followed by 0577 (D = 0.804), the 2686 (0.239) had the lowest discriminatory power. Conclusions In this work we found that using both spoligotyping and MIRU-VNTR increases the discriminatory power for the characterization of isolates. Typing with 12 loci is a good option for genotyping isolates of M. bovis in Mexico. Acknowledgments: This project was funded by FORDECYT 2012 Number 193512. Keywords: VNTR’s, Bovine tuberculosis, Mycobacterium bovis, Genotyping.

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Lípid antigen of Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis for serological diagnosis of bovine tuberculosis and Johneʹs

disease.

E. Ponce-Barraza1, G. López-Valencia1, A. De la Mora-Valle1, D. Miranda-Guzmán1, G.E. Medina-Basulto1, T.B. Renteria-Evangelista1, S. Eda2, S. Hori-Oshima1*

1Research Institute of Veterinary Sciences, Autonomous University of Baja California, Mexicali, Baja California, Mexico.

2Institute of Agriculture, The University of Tennessee, Knoxville, Tennessee, USA. shori@uabc.edu.mx

Background: Bovine tuberculosis (TB) and Johne’s disease (JD) are infectious diseases in ruminant caused by Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis (Map), respectively. Both have significant economic impact on the livestock industry due to direct and indirect economic loss caused in affected herds. There are also potential concerns relating to the safety of dairy products derived from the infected animals. Diagnostic test based on the cell-mediated immune response such as the official skin test and interferon-γ can fail up to 20% in detecting infected animals. The studies on protein antigens of M.bovis for serological test are limited and humoral immune responses of infected animals are not consistent. Recently the lipid surface antigen of Map has been reported as highly specific and sensitive in diagnosis of bovine JD. The aim of this study was to evaluate the usefulness of lipid antigen of M.bovis and Map for the serological diagnosis of TB and ovine JD in Baja California. Methods: 50 cattle with TB compatible granulomatous lesion were sampled at an abattoir TIF of Mexicali Baja California. Lymphoid tissues were collected for histopathological analysis by Hematoxylin-Eosin and Ziehl-Neelsen staining. Sera were stored at -20°C until its use for ELISA test. Positive sera for ovine JD were collected from 43 sheep in a commercial flock in Mexicali Valley. All sheep were confirmed as JD positive by fecal PCR. To prepare lipid antigen of both pathogens, 80% ethanol at a bacterial concentration of 80 mg/ml were used to extract antigens according to Eda et al. (2006). Then the antigen-immobilized ELISA microplates were prepared, and used to detect specific antibodies in sera from cattle and sheep. 30 sera provided by the National Institute of Animal Health in Japan and 28 sheep sera from a flock of IICV-UABC were used as negative control for TB and JD, respectively. Results: Of 50 cattle with compatible TB lesions 38 were resulted positive in histopathological staining. ELISA results were obtained in the OD value of 450nm and positive groups could be classified applying 2 SD of the average negative group. However, the serology test of ovine JD was not successfully optimized due to the large amount of non-specific binding in JD negative sera. Conclusions: Lipid surface antigen of M. bovis could be a future candidate to be used in diagnosis of animal TB using economic and rapid platforms of serological tests, such as “Lab on chip”. To standardize ELISA test for diagnosis of ovine JD, further studies with animals

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with detailed description of disease status are required. Acknowlegdments: The first author is supported by CONACyT student scholarship. This research was partially supported by grant from PRODUCE-BC 2012. Keywords: Bovine tuberculosis, Mycobacteirum bovis, Map, lipid antigen, serology

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CONCLUSIONES

1.Falta mucha infraestructura para realizar el diagnóstico oportuno de la TB.

2.Es critico implementar nuevas tecnologías de biología molecular, como NGS que permitan tener una mejor resolución tanto para acelerar el diagnóstico y migrar la TBDR, así también puede utilizarse en el análisis epidemiológico

3.Es importante tener un cambio en el programa nacional de la TB, empezando desde la Norma Oficial Mexicana para el control de la tuberculosis.

4.Se logró una interacción muy fuerte entre clínicos e investigadores, lo cual, por lo general es muy difícil que se dé.

5.Se logró una actualización a los asistentes en los métodos actuales de diagnóstico

6.Es necesario continuar la Vinculación multidisciplinaria de la RemiTB

7.Es importante Implementar nuevas tecnologías como la de los Smartphone para incrementar la tasa de adherencia a la tuberculosis

8.Se debe de Fomentar la asistencia de estudiantes de pregrado y posgrado de medicina, enfermería, biología, química, lo cual permitirá incrementar el conocimiento de la enfermedad

9.Es importante hacer un seguimiento más estrecho en la vigilancia de la TB causada por M. bovis.

10.Es necesario desarrollar mas estudios de biología molecular con e;l fin de comprender la dispersión y transmisión de linajes considerados altamente virulentos.