Case Report: Exposure to Relapsing Fever Group Borreliae in Patients with Undifferentiated Febrile Illness in Mexico.

Relapsing fever (RF) borreliosis is a neglected disease in Mexico. A retrospective serological survey using diagnostic antigens GlpQ and BipA from Borrelia turicatae was performed to evaluate human exposure to RF borreliae. Seventy serum samples were used from a cohort of patients with undifferentiated febrile illness in Mexico. Four samples were positive to GlpQ and three to BipA. Results indicate that RF borreliae continue to circulate in regions of Mexico and pose a risk to human health.

The Flp type IV pilus operon of Mycobacterium tuberculosis is expressed upon interaction with macrophages and alveolar epithelial cells.

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.

Multiregional Sequencing Analysis Reveals Extensive Genetic Heterogeneity in Gastric Tumors from Latinos.

Gastric cancer is a leading cause of cancer mortality and health disparities in Latinos. We evaluated gastric intratumoral heterogeneity using multiregional sequencing of >700 cancer genes in 115 tumor biopsies from 32 patients, 29 who were Latinos. Analyses focused on comparisons with The Cancer Genome Atlas (TCGA) and on mutation clonality, druggability, and signatures. We found that only approximately 30% of all mutations were clonal and that only 61% of the known TCGA gastric cancer drivers harbored clonal mutations. Multiple clonal mutations were found in new candidate gastric cancer drivers such as EYS, FAT4, PCDHA1, RAD50, EXO1, RECQL4, and FSIP2. The genomically stable (GS) molecular subtype, which has the worse prognosis, was identified in 48% of our Latino patients, a fraction that was >2.3-fold higher than in TCGA Asian and White patients. Only a third of all tumors harbored clonal pathogenic mutations in druggable genes, with most (93%) GS tumors lacking actionable clonal mutations. Mutation signature analyses revealed that, in microsatellite-stable (MSS) tumors, DNA repair mutations were common for both tumor initiation and progression, while tobacco, POLE, and inflammation signatures likely initiate carcinogenesis. MSS tumor progression was likely driven by aging- and aflatoxin-associated mutations, as these latter changes were usually nonclonal. In microsatellite-unstable tumors, nonclonal tobacco-associated mutations were common. Our study, therefore, contributed to advancing gastric cancer molecular diagnostics and suggests clonal status is important to understanding gastric tumorigenesis. Our findings of a higher frequency of a poor prognosis associated molecular subtype in Latinos and a possible new aflatoxin gastric cancer etiology also advance cancer disparities research. Significance: Our study contributes to advancing our knowledge of gastric carcinogenesis, diagnostics, and cancer health disparities.

Nested Polymerase Chain Reaction and Cutaneous Tuberculosis.

The role of Mycobacterium tuberculosis in the etiology and pathogenesis of cutaneous tuberculosis is controversial because of the difficulties associated with demonstrating the presence of these mycobacteria in tuberculid cutaneous lesions by routinely available microbiological and histological techniques. In this study, we aimed to demonstrate the presence of M. tuberculosis in cutaneous tuberculosis. Multiple polymerase chain reaction (PCR) followed by nested PCR was used to amplify genomic fragments from 3 different mycobacteria species. DNA was isolated from 30 paraffin-embedded skin biopsies. Samples were selected randomly from patients with a clinical and histopathological diagnosis of the most frequent groups of cutaneous tuberculosis in Mexico as follows: 5 cases of scrofuloderma tuberculosis; 2 cases of lupus vulgaris tuberculosis; and 5 cases of tuberculosis verrucosa cutis. The other cases denominated tuberculids in some countries such as Mexico and included the following: 7 cases of rosacea-like tuberculosis; one case of papulonecrotic tuberculosis; and 10 cases of erythema induratum of Bazin. Four normal skin biopsies were included as controls. M. tuberculosis DNA was amplified successfully by nested PCR in 80% of the samples (24 of the 30 samples) assayed. Mycobacterial DNA was not detected in the normal skin biopsies used as controls. Detection of M. tuberculosis DNA in 80% of cutaneous tuberculosis analyzed implicates this mycobacterium in the pathogenesis of multiple clinical forms of cutaneous tuberculosis.

The sigma factor SigD of Mycobacterium tuberculosis putatively enhances gene expression of the septum site determining protein under stressful environments.

This work examined the expression of the septum site determining gene (ssd) of Mycobacterium tuberculosis CDC1551 and its ∆sigD mutant under different growing conditions. The results showed an up-regulation of ssd during stationary phase and starvation conditions, but not during in vitro dormancy, suggesting a putative role for SigD in the control of ssd expression mainly under lack-of-nutrients environments. Furthermore, we elucidated a putative link between ssd expression and cell elongation of bacilli at stationary phase. In addition, a -35 sigD consensus sequence was found for the ssd promoter region, reinforcing the putative regulation of ssd by SigD, and in turn, supporting this protein role during the adaptation of M. tuberculosis to some stressful environments.

Identification of Mycobacterium tuberculosis in the cerebrospinal fluid of patients with meningitis using nested PCR.

Tuberculous meningitis (TBM) is the most severe form of tuberculosis. It is caused by Mycobacterium tuberculosis (M. tuberculosis; MT) and it is very difficult to diagnose. The symptoms are similar to other infectious neurological diseases, such as neurocysticercosis, neuroborreliosis, or herpes viral infection. The aim of this study was to identify tuberculosis (TB) in cases of meningitis with clinical and laboratory evidence suggestive of TBM, and to confirm our findings with molecular tests for TB infection. We recruited patients with neurological symptoms who were examined at the neurology services of Hospitals of Instituto Mexicano del Seguro Social (IMSS) in Mexico City. A total of 144 consecutive patients with suggestive infectious meningitis were initially included; 94 cases of meningitis with clinical and laboratory evidence suggestive of TBM were included, but only 50 of these cases fulfilled the criteria for probable TBM. As the controls, we included 50 cases of meningitis with clinical and laboratory evidence suggestive of non-TBM. Cerebrospinal fluid (CSF) was collected from all 100 patients (cases and controls) and tested for TB by multiplex and nested PCR analyses. Nested PCR detected 0.1 fg of M. tuberculosis DNA. TB infection was confirmed with molecular tests in 49 patients from the 50 cases suggestive of TBM and in 1 of the 50 non-TBM cases. The analysis exhibited a sensitivity of 98.0%, a specificity of 92.0%, a positive predictive value of 88.0% and a negative predictive value of 98.0%. The use CSF for the analyses proved to be effective for the rapid diagnosis of TBM using a developed system of multiplex and nested PCR analyses in patients presenting neurological symptoms.

Antibiotic resistance of gram-negative bacilli isolated from pediatric patients with nosocomial bloodstream infections in a Mexican tertiary care hospital.

BACKGROUND: Gram-negative bacilli are the most common bacteria causing nosocomial bloodstream infections (NBSIs) in Latin American countries. METHODS: The antibiotic resistance profiles of Gram-negative bacilli isolated from blood cultures in pediatric patients with NBSIs over a 3-year period in a tertiary care pediatric hospital in Mexico City were determined using the VITEK-2 system. Sixteen antibiotics were tested to ascertain the resistance rate and the minimum inhibitory concentration using the Clinical Laboratory Standards Institute (CLSI) broth micro-dilution method as a reference. RESULTS: A total of 931 isolates were recovered from 847 clinically significant episodes of NBSI. Of these, 477 (51.2%) were caused by Gram-negative bacilli. The most common Gram-negative bacilli found were Klebsiella pneumoniae (30.4%), Escherichia coli (18.9%), Enterobacter cloacae (15.1%), Pseudomonas aeruginosa (9.9%), and Acinetobacter baumannii (4.6%). More than 45 and 60% of the K. pneumoniae and E. coli isolates, respectively, were resistant to cephalosporins, and 64% of the E. coli isolates were resistant to fluoroquinolones. A. baumannii exhibited low rates of resistance to antibiotics tested. In the E. cloacae and P. aeruginosa isolates, no rates of resistance higher than 38% were observed. CONCLUSIONS: In this study, we found that the proportion of NBSIs due to antibiotic-resistant organisms is increasing in a tertiary care pediatric hospital of Mexico.

Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response.

We examined the ability of porins from Salmonella enterica serovar typhi to induce a long-term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long-lasting anti-S. typhi porin sera did not cross-react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal-binding activity to the wild-type S. typhi strain, whereas porin-specific antibody titres measured by enzyme-linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long-lasting antibody response. OmpC and OmpF induced long-lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody-mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant.

Induction of cellular immune response and anti-Salmonella enterica serovar typhi bactericidal antibodies in healthy volunteers by immunization with a vaccine candidate against typhoid fever.

Typhoid fever remains a serious public health problem. We have developed a vaccine from Salmonella enterica serovar typhi (S. typhi) outer-membrane proteins (OMPs) known as porins. A single subcutaneous dose of 10 microg of porins induced a five-fold (P = 0.05) seroconversion index consisting of IgM and IgG at 7 and 15 days after vaccination as well as the production of IgG1 and IgG2 isotypes. The porins-based vaccine induced a two-fold increase (P = 0.05) in bactericidal titres in volunteers, whom also developed a T-cell response characterized by the production of interferon-gamma (INF-gamma). Side effects after vaccination were mild and transient. The data showed that our S. typhi porins-based candidate vaccine is safe and immunogenic in healthy humans.