Leonor Enciso-Moreno

Genetic diversity of the Mycobacterium tuberculosis Complex in San Luis Potosí, México

BackgroundAlthough epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce.MethodsComplete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined.ResultsPulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities.ConclusionsIn San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage.

Type I Interferon Gene Response Is Increased in Early and Established Rheumatoid Arthritis and Correlates with Autoantibody Production

Background Rheumatoid arthritis (RA) is an inflammatory debilitating disease that affects the joints in the early and productive phases of an individual’s life. Several cytokines have been linked to the disease pathogenesis and are known to contribute to the inflammatory state characteristic of RA. The participation of type I interferon (IFN) in the pathogenesis of the disease has been already described as well as the identity of the genes that are regulated by this molecule, which are collectively known as the type I IFN signature. These genes have several functions associated with apoptosis, transcriptional regulation, protein degradation, Th2 cell induction, B cell proliferation, etc. This article evaluated the expression of several genes of the IFN signature in different stages of disease and their correlation with the levels of anticitrullinated protein antibodies (ACPA) anticarbamylated protein (Anti-CarP) antibodies. Methods Samples from individuals with early and established RA, high-risk individuals (ACPA+ and ACPA−), and healthy controls were recruited at “Unidad de Artritis y Rheumatismo” (Rheumatism and Arthritis Unit) in Guadalajara Jalisco Mexico. Determinations of ACPA were made with Eurodiagnostica ACPA plus kit. Anti-CarP determinations were made according to previously described protocols. RNA was isolated, and purity and integrity were determined according to RNA integrity number >6. Gene expression analysis was made by RT-qPCR using specific primers for mRNAs of the type I IFN signature. Relative gene expression was calculated according to Livak and Schmitgen. Results Significant differences in gene expression were identified when comparing the different groups for MXA and MXB (P < 0.05), also when comparing established RA and ACPA− in both IFIT 1 and G15. An increased expression of ISG15 was identified (P < 0.05), and a clear tendency toward increase was identified for HERC5. EPSTRI1, IFI6, and IFI35 were found to be elevated in the chronic/established RA and early RA (P < 0.05). Significant correlations were identified for the IFN signature genes with the levels of ACPA and anti-CarP (P < 0.05). Conclusion Our data confirm previous observations in the role of IFN signature and the pathogenesis of RA. Also, we provide evidence of an association between several genes of the IFN signature (that regulate Th2 cells and B cell proliferation) with the levels of anti-CarP antibodies and ACPA.

Associated Risk Factors for Latent Tuberculosis Infection in Subjects with Diabetes.

BACKGROUND AND AIMS Type 2 diabetes mellitus (DM2) confers a higher risk for active tuberculosis (TB). However, information on associated risk factors for latent tuberculosis infection (LTBI) inpatients with DM2 is limited. We conducted a cross-sectional study to elucidate the prevalence of LTBI and its associated factors on Mexican adults with DM2 receiving medical care at the Mexican Social Security Institute (IMSS). METHODS Six hundred patients with DM2 without a prior history of TB from outpatient diabetes clinics were enrolled in the study. The tuberculin-skin-test (TST) was performed. The presence of LTBI was defined by a TST value of ≥ 5 mm. A standardized interview and physical examination were conducted to obtain clinical, demographic, and LTBI risk factor information; all subjects were laboratory tested to determine the presence of exclusion criteria. Microscopic examination of sputum samples and chest x-rays was performed to identify potential active TB. Subjects with any finding suggesting active TB or malignancy were excluded. A logistic regression model was used to identify variables associated with LTBI. RESULTS LTBI prevalence among patients with DM2 was 51.3%. Risk factors for LTBI were living with a relative with TB, having been in prison, having hemoglobin values >14 g/dL, and glycosylated hemoglobin (HbA1c) values of > 7%. Blood pressure, economic income, or anthropometric measurements were not associated risk factors. CONCLUSIONS Over one half of patients with DM harbor LTBI. Exposure to certain environmental conditions and poorly controlled DM2 (HbA1c > 7.0%) were risk factors for having LTBI in persons with DM2.

Transcriptional profiles discriminate patients with pulmonary tuberculosis from non-tuberculous individuals depending on the presence of non-insulin diabetes mellitus

Our objective was to identify transcriptional biomarkers in peripheral blood mononuclear cells (PBMC) that discriminate individuals with latent tuberculosis infection (LTBI) from those with pulmonary tuberculosis (PTB) in subjects with non-insulin-dependent diabetes mellitus (NIDDM) and in individuals without NIDDM. Using gene expression microarrays we identified differentially expressed genes from lungs of mice infected with Mycobacterium tuberculosis (Mtb) or a mutant (ΔsigH) representing a non-inflammatory model. Genes expressed in blood, with inflammatory related functions were evaluated in humans by RT-qPCR. NCF1 and ORM transcripts have the better discriminatory capacity to identify PTB subjects from LTBI and non-infected controls (NICs) independently of the presence of NIDDM. The sequential evaluation of the mRNA levels of NCF1 and ORM as multiple diagnostic tests showed 95% Sensitivity (Se) and 80% Specificity (Sp). In addition, FPR2 promises to be a good biomarker for the PTB detection in subjects with NIDDM (Se=100%; Sp=90%).

Description of the population structure and genetic diversity of tuberculosis in Estado de México, a low prevalence setting from Mexico

In order to identify the genetic characteristics of the strains of mycobacteria circulating in the Estado de México, one of the states with the lowest prevalence of tuberculosis in Mexico, spoligotyping and 12‐loci MIRU‐VNTR typing were used to genotype tuberculosis clinical isolates. The average age of the 183 patients analyzed was 50 (± 17) years, drug resistance was noted in 57 (31%) and multidrug resistance in 22 (12%) individuals. The results from the isolates recovered showed that 80% were located in four major Euro‐American lineages: Haarlem (17%), LAM (15%), T (20%) and X (29%). Other lineages found in lower proportions were: EAI, S, Beijing, West African, Turkey, Vole and Bovis. Eighteen isolates were orphans. Only 57 isolates were grouped in nine clusters and the SIT119 (X1) showed the highest number of members (23). The LAM lineage showed an increased risk for development of drug resistance (RR=4, IC: 95%: 1.05–14.2, p = 0.03). Despite the important prevalence of four major lineages found and the diversity of strains circulating in the population, we found the presence of one of the largest populations of isolates clustered to the X lineage in a setting from a Latin American country.

Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current enotyping procedures: a population-based study in northeast Mexico

The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman’s rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.

Concordance between IFNγ gene +874 A/T polymorphism and interferon-γ expression in a TB-endemic indigenous setting

INTRODUCTION: Interferon-γ (IFN-γ) plays a crucial role in resistance to mycobacterial diseases; accordingly, variants of the gene encoding this cytokine may be associated with elevated risk of contracting pulmonary tuberculosis (TB). METHODS: Blood samples were collected from 135 Warao indigenous individuals with newly diagnosed sputum culture-positive TB. Of these, 24 were diagnosed with active tuberculosis (ATB). The study comprised 111 participants, who were grouped as follows: 1) 14 tuberculin skin test (TST)-positive Warao indigenous individuals and 4 that were QuantiFERON-TB?Gold In-Tube (QFT-IT) test-positive, collectively comprising the latent TB infection group (LTBI), n = 18), and 2) healthy controls who were QFT-IT- and TST-negative, comprising the control group (CTRL, n = 93). Detection of the IFN γ gene (IFNG) +874A/T polymorphism was performed via PCR and quantification of IFNG expression via qPCR. RESULTS: Relative to indigenous and white Americans, ATB and CTRL groups had a higher frequency of the IFNG SNP (+874A): 23 (95.8%) and 108 (97.3%), respectively. Indigenous Warao individuals homozygous for the IFNG (+874) A allele exhibited 3.59-fold increased risk of developing TB (95% confidence interval, 2.60-4.96, p =0.0001). A decreased frequency of the AT genotype was observed in individuals with pulmonary TB (4.16%) and controls (0.90%). The frequency of the TT genotype was decreased among controls (1.80%); none of the patients with TB were found to have this genotype. The differences in IFNG expression between the groups, under unstimulated and stimulated conditions, were not statistically significant. CONCLUSIONS: Preliminary results demonstrate concordance between IFNG +874 A/A genotype and low expression of IFNG.

THU0018 Identification of a Specific Transcriptional Profile Associated to Early Rheumatoid Arthritis (RA)

Background The etiopathogenic mechanisms involved in the development of RA are complex and poorly understood. Microarray analysis, as part of the system biology strategies, may be useful to assess molecular mechanisms participating in the initiation of clinically apparent RA. Objectives To assess the transcriptional signatures that may be associated with the transition from preclinical to clinically evident RA. Methods In a cross-sectional design, we study 3 groups: a) Anti-CCP positive (ELISA), healthy first-degree relatives of patients with RA (FDR-RA); b) Anti-CCP negative, healthy FDR-RA subjects; c) Patients with early RA (eRA) (<1 year)(ACR/EULAR criteria). RNA labeled samples were obtained from each studied subject and used to assess the transcriptional profiles of each study groups, and hybridized to the Agilent 4x44k microarray chip. Data analyzes were performed using the Gene Spring software to identify the gene expression profiles of each group. Gene ontology and molecular pathways analysis were done as well. Results 34 subjects were included (ACCP- (n=12), ACCP+ (n=12), eRA (n=10). A specific transcriptional profile including 876 up-regulated genes and 7531 down-regulated genes in the group of FDR-RA ACCP+ were identified (Figure 1) (some of these genes are known to participate in innate immune response, IFN type 1 response, IFN-γ response, cellular response to chemical and insulin stimulus, etc.). In the eRA group, only 551 genes were up-regulated and 4402 down-regulated genes (the up-regulated genes that participate in the inflammatory and immune response, regulation of metabolic process, cellular response to chemical stimulus, etc.). These two groups were compared with the ACCP negative FDR-RA subjects. Using these strategies of gene ontology (GO), we found 19 genes associated to immune response and inflammation that are up regulated exclusively in eRA patients; it could represent an inflammation signature in eRA patients, and could be used as biomarkers of disease. After GO identification, we analyzed the induced pathways in ACCP+ and eRA. We found pathways with genes that regulate inflammatory genes in ACCP+, and genes that maintain inflammation and proliferation of immune cells in eRA. The identified cascades were shared between RA and ACCP+ subjects, but the up-regulated genes in each cascade were different for each group. Conclusions Characteristic transcriptional profiles related to the preclinical autoimmune stage of RA and RA of recent onset were identified. A unique set of nineteen up-regulated genes in eRA can be considered as biomarkers for the early diagnosis of RA. These transcriptional signature associated to eRA suggest a phenomenon of immune tolerance loss in the early stage of the disease. Disclosure of Interest None declared

AB0008 Type I Interferon Gene Response is Associated with Early Rheumatoid Arthritis (RA)

Background Rheumatoid arthritis (RA) is often accompanied by severe inflammation joint destruction and circulating autoantibodies. Recently it as been described that inflammatory responses associated with type I interferon are associated with the disease, however, there is no information regarding the role of the response of these associated gene signatures with other stages of the disease such as early RA or individuals at risk such as those with anticitrullinated antibodies and direct and family history of RA. Objectives To evaluate the relative gene expression of the genes IFI44L, IFI6, IFIT1, IFIT2, IFI35, ISG15, MXB, MXA, Ly6E, RSAD2, HERC5, EPSTRI1 in blood samples isolated from patients with RA and their family members with preclinical stages of the disease. Methods Blood samples from Mexican patients with early RA (eRA; n=10) and patients with chronic RA with more than 2 years with the disease (cRA; n=20) were collected. All cRA patients had received treatment with DMARDs. First-degree relatives of patients with RA were stratified in positive (ACCP+; n=20) or negative (ACCP-; n=20) to ACCP. Subjects without family history of autoimmune diseases were included in a healthy control group (HC; n=20). Serum levels of ACCP were evaluated using second generation ELISA. RNA was isolated and integrity was verified in a Bioanalyzer (Life Technologies), only samples with RIN scores>6 were used for cDNA synthesis by a superscript II reverse transcription system (Invitrogen), according to manufacturers instructions.qPCR analysis of the samples was carried out in a Lightcycler 480 (Roche diagnostics) and normalized using HPRT. Statistical analysis for group comparison was done using the Kruskal-Wallis test for non-parametric data, with p<0.05 considered significant. A spearman correlation was also performed. Results No differences were found for clinically relevant variables (HC, ACCP-, ACCP+, eRA and cRA). Differences between the chronic arthritis group and the healthy controls were identified for IFIT1, IFIT2, IFI35, ISG15, Ly6E, HERC5, RSAD2, EPSTRI1 and MXA (p<0.05) confirming previous reports in our population. However, differences among the healthy controls or the ACPA- individuals and the eRA cases were also identified for the genes IFI 5, MXA and MXB (P<0.05). A significant correlation was found for several genes in the signalling cascade such as IFIT1, EPSTRI1, IFI35, LY6E, HERC5 and MXA (P<0.05) suggesting that once that the cascade has been initiated, al genes belonging to the cascade are expressed to regulate the type I interferon gene response. Conclusions The gene expression signature of the type IFN response is overexpressed in the majority of the genes analysed, however, several of these genes are also overexpressed in the early RA subjects, suggesting that the expression of these genes might be used as an early detection system for patients with early disease symptoms. The use of the markers to further advance the classification of undifferentiated arthralgia and early RA needs to be further explored. Disclosure of Interest None declared