Differential expression of biomarkers in saliva related to SARS-CoV-2 infection in patients with mild, moderate and severe COVID-19.

BACKGROUND: Severe COVID-19 is a disease characterized by profound dysregulation of the innate immune system. There is a need to identify highly reliable prognostic biomarkers that can be rapidly assessed in body fluids for early identification of patients at higher risk for hospitalization and/or death. This study aimed to assess whether differential gene expression of immune response molecules and cellular enzymes, detected in saliva samples of COVID-19 patients, occurs according to disease severity staging. METHODS: In this cross-sectional study, subjects with a COVID-19 diagnosis were classified as having mild, moderate, or severe disease based on clinical features. Transcripts of genes encoding 6 biomarkers, IL-1ß, IL-6, IL-10, C-reactive protein, IDO1 and ACE2, were measured by RT‒qPCR in saliva samples of patients and COVID-19-free individuals. RESULTS: The gene expression levels of all 6 biomarkers in saliva were significantly increased in severe disease patients compared to mild/moderate disease patients and healthy controls. A significant strong inverse relationship between oxemia and the level of expression of the 6 biomarkers (Spearman's correlation coefficient between -0.692 and -0.757; p < 0.001) was found. CONCLUSIONS: Biomarker gene expression determined in saliva samples still needs to be validated as a potentially valuable predictor of severe clinical outcomes early at the onset of COVID-19 symptoms.

Interim safety and immunogenicity results from an NDV-based COVID-19 vaccine phase I trial in Mexico.

There is still a need for safe, efficient, and low-cost coronavirus disease 2019 (COVID-19) vaccines that can stop transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we evaluated a vaccine candidate based on a live recombinant Newcastle disease virus (NDV) that expresses a stable version of the spike protein in infected cells as well as on the surface of the viral particle (AVX/COVID-12-HEXAPRO, also known as NDV-HXP-S). This vaccine candidate can be grown in embryonated eggs at a low cost, similar to influenza virus vaccines, and it can also be administered intranasally, potentially to induce mucosal immunity. We evaluated this vaccine candidate in prime-boost regimens via intramuscular, intranasal, or intranasal followed by intramuscular routes in an open-label non-randomized non-placebo-controlled phase I clinical trial in Mexico in 91 volunteers. The primary objective of the trial was to assess vaccine safety, and the secondary objective was to determine the immunogenicity of the different vaccine regimens. In the interim analysis reported here, the vaccine was found to be safe, and the higher doses tested were found to be immunogenic when given intramuscularly or intranasally followed by intramuscular administration, providing the basis for further clinical development of the vaccine candidate. The study is registered under ClinicalTrials.gov identifier NCT04871737.

Pyridostigmine reduces mortality of patients with severe SARS-CoV-2 infection: A phase 2/3 randomized controlled trial.

BACKGROUND: Respiratory failure in severe coronavirus disease 2019 (COVID-19) is associated with a severe inflammatory response. Acetylcholine (ACh) reduces systemic inflammation in experimental bacterial and viral infections. Pyridostigmine increases the half-life of endogenous ACh, potentially reducing systemic inflammation. We aimed to determine if pyridostigmine decreases a composite outcome of invasive mechanical ventilation (IMV) and death in adult patients with severe COVID-19. METHODS: We performed a double-blinded, placebo-controlled, phase 2/3 randomized controlled trial of oral pyridostigmine (60 mg/day) or placebo as add-on therapy in adult patients admitted due to confirmed severe COVID-19 not requiring IMV at enrollment. The primary outcome was a composite of IMV or death by day 28. Secondary outcomes included reduction of inflammatory markers and circulating cytokines, and 90-day mortality. Adverse events (AEs) related to study treatment were documented and described. RESULTS: We recruited 188 participants (94 per group); 112 (59.6%) were men; the median (IQR) age was 52 (44-64) years. The study was terminated early due to a significant reduction in the primary outcome in the treatment arm and increased difficulty with recruitment. The primary outcome occurred in 22 (23.4%) participants in the placebo group vs. 11 (11.7%) in the pyridostigmine group (hazard ratio, 0.47, 95% confidence interval 0.24-0.9; P = 0.03). This effect was driven by a reduction in mortality (19 vs. 8 deaths, respectively). CONCLUSION: Our data indicate that adding pyridostigmine to standard care reduces mortality among patients hospitalized for severe COVID-19.

Effective Treatment of Patients Experiencing Primary, Acute HIV Infection Decreases Exhausted/Activated CD4+ T Cells and CD8+ T Memory Stem Cells.

Several studies have identified main changes in T- and B-lymphocyte subsets during chronic HIV infection, but few data exist on how these subsets behave during the initial phase of HIV infection. We enrolled 22 HIV-infected patients during the acute stage of infection before the initiation of antiretroviral therapy (ART). Patients had blood samples drawn previous to ART initiation (T0), and at 2 (T1) and 12 (T2) months after ART initiation. We quantified cellular HIV-DNA content in sorted naïve and effector memory CD4 T cells and identified the main subsets of T- and B-lymphocytes using an 18-parameter flow cytometry panel. We identified correlations between the patients' clinical and immunological data using PCA. Effective HIV treatment reduces integrated HIV DNA in effector memory T cells after 12 months (T2) of ART. The main changes in CD4+ T cells occurred at T2, with a reduction of activated memory, cytolytic and activated/exhausted stem cell memory T (TSCM) cells. Changes were present among CD8+ T cells since T1, with a reduction of several activated subsets, including activated/exhausted TSCM. At T2 a reduction of plasmablasts and exhausted B cells was also observed. A negative correlation was found between the total CD4+ T-cell count and IgM-negative plasmablasts. In patients initiating ART immediately following acute/early HIV infection, the fine analysis of T- and B-cell subsets has allowed us to identify and follow main modifications due to effective treatment, and to identify significant changes in CD4+ and CD8+ T memory stem cells.

Safety and immunogenicity of a live recombinant Newcastle disease virus-based COVID-19 vaccine (Patria) administered via the intramuscular or intranasal route: Interim results of a non-randomized open label phase I trial in Mexico.

There is still a need for safe, efficient and low-cost coronavirus disease 2019 (COVID-19) vaccines that can stop transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we evaluated a vaccine candidate based on a live recombinant Newcastle disease virus (NDV) that expresses a stable version of the spike protein in infected cells as well as on the surface of the viral particle (AVX/COVID-12-HEXAPRO, also known as NDV-HXP-S). This vaccine candidate can be grown in embryonated eggs at low cost similar to influenza virus vaccines and it can also be administered intranasally, potentially to induce mucosal immunity. We evaluated this vaccine candidate in prime-boost regimens via intramuscular, intranasal, or intranasal followed by intramuscular routes in an open label non-randomized non-placebo-controlled phase I clinical trial in Mexico in 91 volunteers. The primary objective of the trial was to assess vaccine safety and the secondary objective was to determine the immunogenicity of the different vaccine regimens. In the interim analysis reported here, the vaccine was found to be safe and the higher doses tested were found to be immunogenic when given intramuscularly or intranasally followed by intramuscular administration, providing the basis for further clinical development of the vaccine candidate. The study is registered under ClinicalTrials.gov identifier NCT04871737. Funding was provided by Avimex and CONACYT.

Increased Rate of Protease Inhibitor-resistance Associated Mutations in Human Immunodeficiency Viruses Infecting Mexicans who Had Been Living Abroad.

BACKGROUND: Migrants face multiple barriers to accessing health services and antiretroviral therapy (ART). We tested the hypothesis that HIV-infected ART-experienced Mexicans with a history of residence in the U.S. have a higher rate of viral drug-resistance associated mutations (RAMs) versus those without such a history. METHODS: Viral genotypic resistance tests obtained from 336 HIV-infected Mexican patients throughout the country were analysed for the presence of viral-RAMs and its rate was compared between migrants and non-migrants. Adjustment for potential confounders was done though a multivariate analysis. RESULTS: Eighty-four Mexicans who had lived for at least 3 months in the U.S. were more likely to have three or more protease inhibitor (PI)-major RAMs (aOR = 2.47; 95% CI = 1.06-5.76; p < 0.05) than in 252 individuals without this background, independently of the time spent on ART. CONCLUSIONS: A migration background is associated with a higher likelihood of the emergence of HIV variants with decreased susceptibility to several PI.

Integrative and Conjugative Elements of Helicobacter pylori Are Hypothetical Virulence Factors Associated With Gastric Cancer.

Helicobacter pylori is a bacteria with high genome plasticity that has been associated with diverse gastric pathologies. The genetic diversity of this bacteria has limited the characterization of virulence factors associated with gastric cancer (GC). To identify potentially helpful disease biomarkers, we compared 38 complete genomes and 108 draft genomes of H. pylori isolated worldwide from patients with diverse gastric pathologies and 53 draft genomes of H. pylori isolated from Mexican patients with GC, intestinal metaplasia, gastritis, peptic ulcer, and dyspepsia. H. pylori strains isolated from GC were 3-11 times more likely to harbor any of seven genes encoded within an integrative and conjugative element (ICE) than H. pylori isolated from subjects with other gastric pathologies. We tested the cytopathic effects on AGS cells of selected H. pylori strains with known cytotoxin-associated gene pathogenicity island (cag-PAI) and ICE status (H. pylori strains 29CaP, 29CaCe, 62A9, 7C, 8822, and 26695) and the histopathological damage of H. pylori 29CaP and 62A9 in a mouse model. H. pylori 29CaP, which harbors a complete ICEHptfs3 but lacks cag-PAI, elicited distinctive morphology changes and higher histopathological scores compared with other H. pylori strains carrying cag-PAI and hybrid ICE with incomplete TFSS. The presence of intact segments of ICE regions might be a risk factor to develop GC that needs to be addressed in future studies.

A parallel-group, multicenter randomized, double-blinded, placebo-controlled, phase 2/3, clinical trial to test the efficacy of pyridostigmine bromide at low doses to reduce mortality or invasive mechanical ventilation in adults with severe SARS-CoV-2 infection: the Pyridostigmine In Severe COvid-19 (PISCO) trial protocol.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), may lead to severe systemic inflammatory response, pulmonary damage, and even acute respiratory distress syndrome (ARDS). This in turn may result in respiratory failure and in death. Experimentally, acetylcholine (ACh) modulates the acute inflammatory response, a neuro-immune mechanism known as the inflammatory reflex. Recent clinical evidence suggest that electrical and chemical stimulation of the inflammatory reflex may reduce the burden of inflammation in chronic inflammatory diseases. Pyridostigmine (PDG), an ACh-esterase inhibitor (i-ACh-e), increases the half-life of endogenous ACh, therefore mimicking the inflammatory reflex. This clinical trial is aimed at evaluating if add-on of PDG leads to a decrease of invasive mechanical ventilation and death among patients with severe COVID-19. METHODS: A parallel-group, multicenter, randomized, double-blinded, placebo-controlled, phase 2/3 clinical trial to test the efficacy of pyridostigmine bromide 60 mg/day P.O. to reduce the need for invasive mechanical ventilation and mortality in hospitalized patients with severe COVID-19. DISCUSSION: This study will provide preliminary evidence of whether or not -by decreasing systemic inflammation- add-on PDG can improve clinical outcomes in patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov NCT04343963 (registered on April 14, 2020).

The Decline in HIV-1 Drug Resistance in Heavily Antiretroviral-Experienced Patients Is Associated with Optimized Prescriptions in a Treatment Roll-Out Program in Mexico.

A decrease in the rate of acquired antiretroviral (ARV) drug resistance (ADR) over time has been documented in high-income settings, but data on the determinants of this phenomenon are lacking. We tested the hypothesis that in heavily ARV-experienced patients in the Mexican ARV therapy (ART) roll-out program, the drop in ADR would be associated with changes in ARV drug usage. Genotypic resistance tests obtained from 974 HIV-infected patients with virological failure and at least 2 previously failed ARV regimens from throughout the country were analyzed for the presence of nucleos(t)ide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitor (PI) resistance-associated mutations (RAMs). Patients were divided into two groups according to their first ART start date: 488 patients initiated ART before mid-2003 (group 1) and 486 after mid-2003 (group 2). The rate of RAMs, median resistance score of several sentinel ARVs, and composition of ART drugs in patient's entire treatment history were compared between both groups. Patients in group 2 were less likely to have >3 thymidine analogue-associated mutations (TAMs) and >3 PI-mRAMs [adjusted odds ratio (aOR) = 0.37; 95% confidence interval (95% CI) = 0.25-0.54; p < .001 and aOR = 0.53; 95% CI = 0.36-0.77; p = .001, respectively] and had a significantly lower resistance score for zidovudine, tenofovir, ritonavir-boosted (r)-lopinavir, r-atazanavir, and r-darunavir than group 1 patients. A significantly lower proportion of patients in group 2 used monotherapy, bitherapy, thymidine analogue-containing regimens, nonboosted PI-containing regimens, and low resistance barrier PI-containing regimens. In Mexican ARV-experienced patients, the occurrence of TAM and PI-mRAM has significantly declined over time. This can be explained by treatment optimization in the national ART roll-out program in recent years.

Real Time Monitoring of Children, and Adults with Mental Disabilities Using a Low-Cost Non-Invasive Electronic Device.

There are a growing number of small children-as well as adults-with mental disabilities (including elderly citizens with Alzheimer's disease or other forms of age-related dementia) that are getting lost in rural and urban areas for various reasons. Establishing their location within the first 72 h is crucial because lost people are exposed to all kinds of adverse conditions and in the case of the elderly, this is further aggravated if prescribed medication is needed. Herein we describe a non-invasive, low-cost electronic device that operates constantly, keeping track of time, the geographical location and the identification of the subject using it. The prototype was made using commercial low-cost electronic components. This electronic device shows high connectivity in open and closed areas and identifies the geographical location of a lost subject. We freely provide the software and technical diagrams of the prototypes.

Electronegativity and intrinsic disorder of preeclampsia-related proteins.

Preeclampsia, hemorrhage, and infection are the leading causes of maternal death in underdeveloped countries. Since several proteins associated with preeclampsia are known, we conducted a computational study which evaluated the commonness and potential functionality of intrinsic disorder of these proteins and also made an attempt to characterize their origin. The origin of the preeclampsia-related proteins was assessed with a supervised technique, a Polarity Index Method (PIM), which evaluates the electronegativity of proteins based solely on their sequence. The commonness of intrinsic disorder was evaluated using several disorder predictors from the PONDR family, the charge-hydropathy plot (CH-plot) and cumulative distribution function (CDF) analyses, and using the MobiDB web-based tool, whereas potential functionality of intrinsic disorder was studied with the D2P2 resource and ANCHOR predictor of disorder-based binding sites, and the STRING tool was used to build the interactivity networks of the preeclampsia-related proteins. Peculiarities of the PIM-derived polar profile of the group of preeclampsia-related proteins were then compared with profiles of a group of lipoproteins, antimicrobial peptides, angiogenesis-related proteins, and the intrinsically disordered proteins. Our results showed a high graphical correlation between preeclampsia proteins, lipoproteins, and the angiogenesis proteins. We also showed that many preeclampsia-related proteins contain numerous functional disordered regions. Therefore, these bioinformatics results led us to assume that the preeclampsia proteins are highly associated with the lipoproteins group, and that some preeclampsia-related proteins contain significant amounts of functional disorders.

A multi-drug resistant Salmonella Typhimurium ST213 human-invasive strain (33676) containing the bla CMY-2 gene on an IncF plasmid is attenuated for virulence in BALB/c mice.

BACKGROUND: Classical strains of Salmonella enterica serovar Typhimurium (Typhimurium) predominantly cause a self-limiting diarrheal illness in humans and a systemic disease in mice. In this study, we report the characterization of a strain isolated from a blood-culture taken from a 15-year old woman suffering from invasive severe salmonellosis, refractory to conventional therapy with extended-spectrum cephalosporin (ESC). RESULTS: The strain, named 33676, was characterized as multidrug-resistant Salmonella serogroup A by biochemical, antimicrobial and serological tests. Multilocus sequence typing (MLST) and XbaI macrorestrictions (PFGE) showed that strain 33676 belonged to the Typhimurium ST213 genotype, previously described for other Mexican Typhimurium strains. PCR analyses revealed the presence of IncA/C, IncFIIA and ColE1-like plasmids and the absence of the Salmonella virulence plasmid (pSTV). Conjugation assays showed that the ESC-resistance gene bla CMY-2 was carried on the conjugative IncF plasmid, instead of the IncA/C plasmid, as found in previously studied ST213 strains. Although the IncA/C plasmid conferred most of the observed antimicrobial resistances it was not self-conjugative; it was rather able to conjugate by co-integrating with the IncF plasmid. Strain 33676 was fully attenuated for virulence in BALB/c mice infections. Both type-three secretion system (T3SS), encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), were functional in the 33676 strain and, interestingly, this strain produced the H2 FljB flagellin instead of the H1 FliC flagellin commonly expressed by S. enterica strains. CONCLUSIONS: Strain 33676 showed two main features that differentiate it from the originally described ST213 strains: 1) the bla CMY-2 gene was not carried on the IncA/C plasmid, but on a conjugative IncF plasmid, which may open a new route of dissemination for this ESC-resistance gene, and 2) it expresses the H2 FljB flagella, in contrast with the other ST213 and most Typhimurium reference strains. To our knowledge this is the first report of an IncF bla CMY-2-carrying plasmid in Salmonella.

Complete Genome Sequence of a Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Harboring a Multidrug Resistance IncA/C Plasmid and a blaCMY-2-Carrying IncF Plasmid.

Salmonella enterica subsp. enterica serovar Typhimurium strain 33676 was isolated in Mexico City, Mexico, from a patient with a systemic infection, and its complete genome sequence was determined using PacBio single-molecule real-time technology. Strain 33676 harbors an IncF plasmid carrying the extended-spectrum cephalosporin gene blaCMY-2 and a multidrug resistance IncA/C plasmid.

Antiretroviral purchasing and prescription practices in Mexico: constraints, challenges and opportunities.

OBJECTIVE: This study examines the antiretroviral (ARV) market characteristics for drugs procured and prescribed to Mexico's Social Protection System in Health beneficiaries between 2008 and 2013, and compares them with international data. MATERIALS AND METHODS: Procurement information from the National Center for the Prevention and the Control of HIV/AIDS was analyzed to estimate volumes and prices of key ARV. Annual costs were compared with data from the World Health Organization's Global Price Reporting Mechanism for similar countries. Finally, regimens reported in the ARV Drug Management, Logistics and Surveillance System database were reviewed to identify prescription trends and model ARV expenditures until 2018. RESULTS: Results show that the first-line ARV market is concentrated among a small number of patented treatments, in which prescription is clinically adequate, but which prices are higher than those paid by similar countries. The current set of legal and structural options available to policy makers to bring prices down is extremely limited. CONCLUSIONS: Different negotiation policies were not successful to decrease ARV high prices in the public health market. The closed list approach had a good impact on prescription quality but was ineffective in reducing prices. The Coordinating Commission for Negotiating the Price of Medicines and other Health Supplies also failed to obtain adequate prices. To maximize purchase efficiency, policy makers should focus on finding long-term legal and political safeguards to counter the high prices imposed by pharmaceutical companies.

Antiretroviral purchasing and prescription practices in Mexico: constraints, challenges and opportunities / Compras y prescripciones de medicamentos antirretrovirales en México: restricciones, retos y oportunidades

Objective. This study examines the antiretroviral (ARV) market characteristics for drugs procured and prescribed to Mexico's Social Protection System in Health beneficiaries between 2008 and 2013, and compares them with international data. Materials and methods. Procurement information from the National Center for the Prevention and the Control of HIV/AIDS was analyzed to estimate volumes and prices of key ARV. Annual costs were compared with data from the World Health Organization's Global Price Reporting Mechanism for similar countries. Finally, regimens reported in the ARV Drug Management, Logistics and Surveillance System database were reviewed to identify prescription trends and model ARV expenditures until 2018. Results. Results show that the first-line ARV market is concentrated among a small number of patented treatments, in which prescription is clinically adequate, but which prices are higher than those paid by similar countries. The current set of legal and structural options available to policy makers to bring prices down is extremely limited. Conclusions. Different negotiation policies were not successful to decrease ARV high prices in the public health market. The closed list approach had a good impact on prescription quality but was ineffective in reducing prices. The Coordinating Commission for Negotiating the Price of Medicines and other Health Supplies also failed to obtain adequate prices. To maximize purchase efficiency, policy makers should focus on finding long-term legal and political safeguards to counter the high prices imposed by pharmaceutical companies.

Objetivo. Este estudio analiza el mercado de los medicamentos antiretrovirales (ARV) adquiridos y prescritos a los beneficiarios del Seguro Popular entre 2008 y 2013, en México, comparándolo con información internacional. Material y métodos. Se analiza información sobre la compra de medicamentos por parte del Centro para la Prevención y el Control del VIH y el Sida (Censida) para estimar precios y volúmenes de compra de los principales ARV. Los costos anuales de tratamiento estimados fueron comparados con información del Global Price Reporting Mechanism (GPRM) de la Organización Mundial de la Salud, para países similares. Finalmente se revisaron los esquemas reportados en el Sistema de Administración, Logística y Vigilancia de ARV para identificar tendencias y proyectar el gasto en ARV hasta 2018. Resultados. El mercado mexicano de ARV está concentrado en pocos esquemas de primera línea y, aunque la prescripción es clínicamente adecuada, los precios son más altos que en otros países similares. El conjunto actual de opciones legales y estructurales disponibles para los formuladores de políticas para reducir los precios es muy limitado. Conclusiones. Las políticas de negociación han sido poco exitosas para disminuir los precios de los ARV en México. La Coordinating Commission for Negotiating the Price of Medicines and other Health Supplies y la integración de las guías de tratamiento han tenido impacto significativo en la calidad de la prescripción, pero moderado en la reducción de precios. Por ello es necesario buscar garantías jurídicas y políticas a largo plazo para hacer frente a los altos precios de los ARV.

The successful application of a national peer advisory committee for physicians who provide salvage regimens to heavily antiretroviral-experienced patients in mexican human immunodeficiency virus clinics.

BACKGROUND: Designing optimal antiretroviral (ARV) salvage regimens for multiclass drug-resistant, human immunodeficiency virus (HIV)-infected patients demands specific clinical skills. Our aim was to assess the virologic and immunologic effects of the treatment recommendations drafted by a peer advisory board to physicians caring for heavily ARV-experienced patients. METHODS: We conducted a nationwide, HIV clinic-based, cohort study in Mexico. Adults infected with HIV were assessed for a median of 33 months (interquartile range [IQR] = 22-43 months). These patients had experienced the virologic failure of at least 2 prior ARV regimens and had detectable viremia while currently being treated; their physicians had received therapeutic advice, by a panel of experts, regarding the ARV salvage regimen. The primary endpoint was the incidence of loss of virologic response (plasma HIV-RNA levels of <200 copies per mL, followed by levels above this threshold) during the follow-up assessment using an observed-failure competing risks regression analysis. RESULTS: A total of 611 patients were observed (median ARV therapy exposure = 10.5 years; median prior regimens = 4). The probabilities of virologic failure were 11.9%, 14.4%, 16.9%, and 19.4% at the 12-, 24-, 36-, and 48-month follow-up assessments, respectively. Of the 531 patients who achieved a confirmed plasma HIV-RNA level below 200 copies per mL, the median increase in blood CD4(+) T-cell count was 162 cells per mL (IQR = 45-304 cells per mL). CONCLUSIONS: In routine practice, a high rate of patients with extensive ARV experience, who received an optimized salvage regimen recommended by a peer advisory committee, achieved a long-term sustained virologic response and immune reconstitution.

Burden and transmission of zoonotic foodborne disease in a rural community in Mexico.

BACKGROUND: The foodborne transmission and human health impact of Salmonella and Campylobacter infections have rarely been evaluated at the population level in highly endemic settings. METHODS: A prospective 15-month cohort study of 127 infants and 119 elderly people was combined with animal and food surveillance to determine the incidence and severity of Salmonella and Campylobacter gastroenteritis in a comparatively prosperous rural community in Mexico. RESULTS: Salmonella and Campylobacter were isolated in up to 75% and 57%, respectively, of raw retail meat and in up to 4.5% of ready-to-eat foods. Rates of acute gastroenteritis of any etiology in infants and elderly people were, respectively, 2.1 and 0.7 episodes per person per year. The annual incidence density rate of Salmonella gastroenteritis was 17.8 per 100 infants and 7.9 per 100 elderly people; the rate of Campylobacter gastroenteritis was 11.7 per 100 infants and 0 per 100 elderly people. Pulsed-field gel electrophoresis analysis yielded multiple clusters of human, meat, and/or animal Salmonella and Campylobacter isolates with indistinguishable patterns. On average, gastroenteritis episodes with these pathogens lasted 3 days in infants and 2 days in elderly people. Medical attention was sought in 44% of diarrheal episodes in infants and in 26% of diarrheal episodes in elderly people; none required hospitalization. Infants with multidrug-resistant Salmonella gastroenteritis had a higher frequency of bloody stools and medical visits (50% vs 11%; odds ratio, 8.5; P = .04) than those with more susceptible strains. CONCLUSIONS: In this relatively advantaged Mexican rural community, the human health impact of a food chain heavily contaminated with Salmonella and Campylobacter was of low magnitude.

Nociceptin/orphanin FQ suppresses adaptive immune responses in vivo and at picomolar levels in vitro.

Nociceptin/orphanin FQ (N/OFQ), added in vitro to murine spleen cells in the picomolar range, suppressed antibody formation to sheep red blood cells in a primary and a secondary plaque-forming cell assay. The activity of the peptide was maximal at 10(-12) M, with an asymmetric U-shaped dose-response curve that extended activity to 10(-14) M. Suppression was not blocked by pretreatment with naloxone. Specificity of the suppressive response was shown using affinity-purified rabbit antibodies against two N/OFQ peptides and with a pharmacological antagonist. Antisera against both peptides were active, in a dose-related manner, in neutralizing N/OFQ-mediated immunosuppression, when the peptide was used at concentrations from 10(-12.3) to 10(-11.6) M. In addition, nociceptin given in vivo by osmotic pump for 48 h suppressed the capacity of spleen cells placed ex vivo to make an anti-sheep red blood cell response. These studies show that nociceptin directly inhibits an adaptive immune response, i.e., antibody formation, both in vitro and in vivo.

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains.

BACKGROUND: Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. RESULTS: We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. CONCLUSION: Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population.

Phenotypic differences between BCG vaccines at the proteome level.

To contribute to Mycobacterium bovis BCG characterization, two substrains were analyzed using two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS), based on their protective efficacy in a pulmonary-tuberculosis mouse model. Cell-fraction proteins of BCG Denmark and Phipps substrains were separated into approximately 500 spots in 2D-PAGE. The proteomes were similar in protein number, and isoelectric point (pI) and molecular mass (MM) distribution. Statistical analysis, resulted in 72 spots with no change, and 168 and 90 unique for BCG Phipps or Denmark, respectively. Two hundred and fourteen spots showed changes in intensity of >1-fold, 138 of Denmark, and 76 of Phipps. Seventeen spots were selected for MS-based identification (13 from Phipps and 4 from Denmark), including unique, as well as proteins with changes in intensity. The proteins identified participate in virulence, detoxification, adaptation, lipid metabolism, information pathways, cell wall and cell processes, intermediary metabolism and respiration, or still hypotheticals. Our findings contribute to phenotype characterization of BCG substrains and provide new elements to consider for the design of diagnostic tools, drug targets and a new vaccine against tuberculosis based upon protein expression through quantitative statistical analysis.