Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens.

We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.

Ecological patterns of blood-feeding by kissing-bugs (Hemiptera: Reduviidae: Triatominae)

Host use by vectors is important in understanding the transmission of zoonotic diseases, which can affect humans, wildlife and domestic animals. Here, a synthesis of host exploitation patterns by kissing-bugs, vectors of Chagas disease, is presented. For this synthesis, an extensive literature review restricted to feeding sources analysed by precipitin tests was conducted. Modern tools from community ecology and multivariate statistics were used to determine patterns of segregation in host use. Rather than innate preferences for host species, host use by kissing-bugs is influenced by the habitats they colonise. One of the major limitations of studies on kissing-bug foraging has been the exclusive focus on the dominant vector species. We propose that expanding foraging studies to consider the community of vectors will substantially increase the understanding of Chagas disease transmission ecology. Our results indicate that host accessibility is a major factor that shapes the blood-foraging patterns of kissing-bugs. Therefore, from an applied perspective, measures that are directed at disrupting the contact between humans and kissing-bugs, such as housing improvement, are among the most desirable strategies for Chagas disease control.

Dipolo impreso para aplicaciones Wi-fi y caracterización de la ganancia por medio de la celda TEM

Este trabajo recoge el estudio, diseño, fabricación y análisis de dos dipolos de media onda para aplicaciones Wi-fi, con objeto de caracterizar sus respectivas ganancias mediante el uso de dos sistemas de medida distintos, el primero basado en el análisis del balance de enlace entre antenas mediante un analizador de redes y el segundo mediante una celda TEM. Para obtener un correcto funcionamiento de los dipolos, resulta de vital importancia conseguir un buen ajuste de las dimensiones de los mismos durante el desarrollo práctico del proyecto, consiguiendo una máxima transferencia de potencia y un ancho de banda suficientemente amplio para asegurar que las antenas presenten una buena adaptación en la banda de los 2.4 GHz – 2.5 GHz.

Ultra high throughput sequencing in human DNA variation detection: a comparative study on the NDUFA3-PRPF31 region.

BACKGROUND: Ultra high throughput sequencing (UHTS) technologies find an important application in targeted resequencing of candidate genes or of genomic intervals from genetic association studies. Despite the extraordinary power of these new methods, they are still rarely used in routine analysis of human genomic variants, in part because of the absence of specific standard procedures. The aim of this work is to provide human molecular geneticists with a tool to evaluate the best UHTS methodology for efficiently detecting DNA changes, from common SNPs to rare mutations. METHODOLOGY/PRINCIPAL FINDINGS: We tested the three most widespread UHTS platforms (Roche/454 GS FLX Titanium, Illumina/Solexa Genome Analyzer II and Applied Biosystems/SOLiD System 3) on a well-studied region of the human genome containing many polymorphisms and a very rare heterozygous mutation located within an intronic repetitive DNA element. We identify the qualities and the limitations of each platform and describe some peculiarities of UHTS in resequencing projects. CONCLUSIONS/SIGNIFICANCE: When appropriate filtering and mapping procedures are applied UHTS technology can be safely and efficiently used as a tool for targeted human DNA variations detection. Unless particular and platform-dependent characteristics are needed for specific projects, the most relevant parameter to consider in mainstream human genome resequencing procedures is the cost per sequenced base-pair associated to each machine.

Revalidation and redescription of Triatoma brasiliensis macromelasoma Galvão, 1956 and an identification key for the Triatoma brasiliensis complex (Hemiptera: Reduviidae: Triatominae)

Triatoma brasiliensis macromelasoma is revalidated based on the results of previous multidisciplinary studies on the Triatoma brasiliensis complex, consisting of crossing experiments and morphological, biological, ecological and molecular analyses. These taxonomic tools showed the closest relationship between T. b. macromelasoma and Triatoma brasiliensis brasiliensis. T. b. macromelasoma is redescribed based on specimens collected in the type locality and specimens from a F1 colony. The complex now comprises T. b. brasiliensis, T. b. macromelasoma, Triatoma melanica, Triatoma juazeirensis and Triatoma sherlocki. An identification key for all members of the complex is presented. This detailed comparative study of the morphological features of T. b. macromelasoma and the remaining members of the complex corroborates results from multidisciplinary analyses, suggesting that the subspecific status is applicable. This subspecies can be distinguished by the following combination of features: a pronotum with 1+1 narrow brownish-yellow stripes on the submedian carinae, not attaining its apex, hemelytra with membrane cells darkened on the central portion and legs with an incomplete brownish-yellow ring on the apical half of the femora. Because the T. brasiliensis complex is of distinct epidemiological importance throughout its geographic distribution, a precise identification of its five members is important for monitoring and controlling actions against Chagas disease transmission.

Monitoring switch-type sensors and powering autonomous sensors via inductive coupling: application to removable seats in vehicles

Aquesta tesi explora la possibilitat de fer servir enllaços inductius per a una aplicació de l’automòbil on el cablejat entre la centraleta (ECU) i els sensors o detectors és difícil o impossible. S’han proposat dos mètodes: 1) el monitoratge de sensors commutats (dos possibles estats) via acoblament inductiu i 2) la transmissió mitjançant el mateix principi físic de la potència necessària per alimentar els sensors autònoms remots. La detecció d'ocupació i del cinturó de seguretat per a seients desmuntables pot ser implementada amb sistemes sense fils passius basats en circuits ressonants de tipus LC on l'estat dels sensors determina el valor del condensador i, per tant, la freqüència de ressonància. Els canvis en la freqüència són detectats per una bobina situada en el terra del vehicle. S’ha conseguit provar el sistema en un marge entre 0.5 cm i 3 cm. Els experiments s’han dut a terme fent servir un analitzador d’impedàncies connectat a una bobina primària i sensors comercials connectats a un circuit remot. La segona proposta consisteix en transmetre remotament la potència des d’una bobina situada en el terra del vehicle cap a un dispositiu autònom situat en el seient. Aquest dispositiu monitorarà l'estat dels detectors (d'ocupació i de cinturó) i transmetrà les dades mitjançant un transceptor comercial de radiofreqüència o pel mateix enllaç inductiu. S’han avaluat les bobines necessàries per a una freqüència de treball inferior a 150 kHz i s’ha estudiat quin és el regulador de tensió més apropiat per tal d’aconseguir una eficiència global màxima. Quatre tipus de reguladors de tensió s’han analitzat i comparat des del punt de vista de l’eficiència de potència. Els reguladors de tensió de tipus lineal shunt proporcionen una eficiència de potència millor que les altres alternatives, els lineals sèrie i els commutats buck o boost. Les eficiències aconseguides han estat al voltant del 40%, 25% i 10% per les bobines a distàncies 1cm, 1.5cm, i 2cm. Les proves experimentals han mostrat que els sensors autònoms han estat correctament alimentats fins a distàncies de 2.5cm.

The peri-urban interface and house infestation with Triatoma infestans in the Argentine Chaco: an underreported process?

Peri-urban infestations with triatomine bugs, their sources and their dynamics have rarely been investigated. Here, we corroborated the reported occurrence of Triatoma infestans in a peri-urban area and in neighbouring rural houses in Pampa del Indio, in the Argentine Chaco, and identified its putative sources using spatial analysis and demographic questionnaires. Peri-urban householders reported that 10% of their premises had triatomines, whereas T. infestans was collected by timed manual searches or community-based surveillance in only nine (3%) houses. Trypanosoma cruzi-infected T. infestans and Triatoma sordida were collected indoors only in peri-urban houses and were infected with TcV and TcI, respectively. The triatomines fed on chickens, cats and humans. Peri-urban infestations were most frequent in a squatter settlement and particularly within the recently built mud houses of rural immigrants, with large-sized households, more dogs and cats and more crowding. Several of the observed infestations were most likely associated with passive bug transport from other sources and with active bug dispersal from neighbouring foci. Thus, the households in the squatter settlement were at a greater risk of bug invasion and colonisation. In sum, the incipient process of domestic colonisation and transmission, along with persistent rural-to-urban migratory flows and unplanned urbanisation, indicate the need for active vector surveillance and control actions at the peri-urban interface of the Gran Chaco.

Activated protein C consumption and coagulation parameters in severe sepsis and septic shock.

Introduction Activated protein C (APC) deC ciency is prevalent in severe sepsis and septic shock patients. The aim of the study was to relate the anticoagulation activity evaluated by APC with other coagulation parameters adjusted to 28-day mortality. Methods A cohort study of 150 critically ill adults. Age, sex, sources of infection and coagulation markers within 24< hours from severe sepsis or septic shock onset, deC ned according to Surviving Sepsis Campaign (SSC) criteria, were studied. We analyzed APC activity using a hemostasis laboratory analyzer (BCS® XP; Siemens). A descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the UCI. The median age of the study sample was 64 (interquartile range (IQR): 22.30.001). See Figure 1. Conclusion Low levels of PC are associated with poor outcome and severity in severe sepsis, and it is well correlated with antithrombin III and INR.

Where do these bugs come from? Phenotypic structure ofTriatoma infestans populations after control interventions in the Argentine Chaco

House re-invasion by native triatomines after insecticide-based control campaigns represents a major threat for Chagas disease vector control. We conducted a longitudinal intervention study in a rural section (Area III, 407 houses) of Pampa del Indio, northeastern Argentina, and used wing geometric morphometry to compare pre-spray and post-spray (re-infestant bugs) Triatoma infestanspopulations. The community-wide spraying with pyrethroids reduced the prevalence of house infestation by T. infestans from 31.9% to < 1% during a four-year follow-up, unlike our previous studies in the neighbouring Area I. Two groups of bug collection sites differing in wing shape variables before interventions (including 221 adults from 11 domiciles) were used as a reference for assigning 44 post-spray adults. Wing shape variables from post-spray, high-density bug colonies and pre-spray groups were significantly different, suggesting that re-infestant insects had an external origin. Insects from one house differed strongly in wing shape variables from all other specimens. A further comparison between insects from both areas supported the existence of independent re-infestation processes within the same district. These results point to local heterogeneities in house re-infestation dynamics and emphasise the need to expand the geographic coverage of vector surveillance and control operations to the affected region.

Levels of immune cells in transcendental meditation practitioners.

CONTEXT Relationships between mind and body have gradually become accepted. Yogic practices cause modulation of the immune system. Transcendental meditation (TM) is a specific form of mantra meditation. We reported previously different plasma levels of catecholamines and pituitary hormones in TM practitioners comparing with a control group, and patterns of the daytime secretion of these hormones different from those normally described. AIMS The aim of the following study is to evaluate the immune system in these meditation practitioners, by determining leukocytes and lymphocytes subsets. METHODS TM group consisted of 19 subjects who regularly practice either TM or the more advanced Sidhi-TM technique. A control group consisted of 16 healthy subjects who had not previously used any relaxation technique. Total leukocytes, granulocytes, lymphocytes and monocytes were counted by an automated quantitative hematology analyzer, whereas lymphocytes subsets were determined by flow cytometry. Samples were taken from each subject at 0900 h after an overnight fast. RESULTS The results indicated that the TM group had higher values than the control group in CD3+CD4-CD8+ lymphocytes (P < 0.05), B lymphocytes (P < 0.01) and natural killer cells (P < 0.01), whereas CD3+CD4+CD8- lymphocytes showed low levels in meditation practitioners (P < 0.001). No significant differences were observed in total leukocytes, granulocytes, monocytes, total lymphocytes or CD3+ lymphocytes comparing both groups. CONCLUSIONS The technique of meditation studied seems to have a significant effect on immune cells, manifesting in the different circulating levels of lymphocyte subsets analyzed. The significant effect of TM on the neuroendocrine axis and its relationship with the immune system may partly explain our results.

Risk factors associated with Trypanosoma cruziexposure in domestic dogs from a rural community in Panama

Chagas disease, caused by Trypanosoma cruzi infection, is a zoonosis of humans, wild and domestic mammals, including dogs. In Panama, the main T. cruzi vector is Rhodnius pallescens, a triatomine bug whose main natural habitat is the royal palm, Attalea butyracea. In this paper, we present results from three T. cruzi serological tests (immunochromatographic dipstick, indirect immunofluorescence and ELISA) performed in 51 dogs from 24 houses in Trinidad de Las Minas, western Panama. We found that nine dogs were seropositive (17.6% prevalence). Dogs were 1.6 times more likely to become T. cruziseropositive with each year of age and 11.6 times if royal palms where present in the peridomiciliary area of the dog’s household or its two nearest neighbours. Mouse-baited-adhesive traps were employed to evaluate 12 peridomestic royal palms. All palms were found infested with R. pallescens with an average of 25.50 triatomines captured per palm. Of 35 adult bugs analysed, 88.6% showed protozoa flagellates in their intestinal contents. In addition, dogs were five times more likely to be infected by the presence of an additional domestic animal species in the dog’s peridomiciliary environment. Our results suggest that interventions focused on royal palms might reduce the exposure to T. cruzi infection.

Evaluation of two HbA1c point-of-care analyzers.

BACKGROUND Measurement of HbA1c is the most important parameter to assess glycemic control in diabetic patients. Different point-of-care devices for HbA1c are available. The aim of this study was to evaluate two point-of-care testing (POCT) analyzers (DCA Vantage from Siemens and Afinion from Axis-Shield). We studied the bias and precision as well as interference from carbamylated hemoglobin. METHODS Bias of the POCT analyzers was obtained by measuring 53 blood samples from diabetic patients with a wide range of HbA1c, 4%-14% (20-130 mmol/mol), and comparing the results with those obtained by the laboratory method: HPLC HA 8160 Menarini. Precision was performed by 20 successive determinations of two samples with low 4.2% (22 mmol/mol) and high 9.5% (80 mmol/mol) HbA1c values. The possible interference from carbamylated hemoglobin was studied using 25 samples from patients with chronic renal failure. RESULTS The means of the differences between measurements performed by each POCT analyzer and the laboratory method (95% confidence interval) were: 0.28% (p<0.005) (0.10-0.44) for DCA and 0.27% (p<0.001) (0.19-0.35) for Afinion. Correlation coefficients were: r=0.973 for DCA, and r=0.991 for Afinion. The mean bias observed by using samples from chronic renal failure patients were 0.2 (range -0.4, 0.4) for DCA and 0.2 (-0.2, 0.5) for Afinion. Imprecision results were: CV=3.1% (high HbA1c) and 2.97% (low HbA1c) for DCA, CV=1.95% (high HbA1c) and 2.66% (low HbA1c) for Afinion. CONCLUSIONS Both POCT analyzers for HbA1c show good correlation with the laboratory method and acceptable precision.

Assessment of melanocytic skin lesions with a high-definition laser Doppler imaging system.

BACKGROUND: Early detection is a major goal in the management of malignant melanoma. Besides clinical assessment many noninvasive technologies such as dermoscopy, digital dermoscopy and in vivo laser scanner microscopy are used as additional methods. Herein we tested a system to assess lesional perfusion as a tool for early melanoma detection.¦METHODS: Laser Doppler flow (FluxExplorer) and mole analyser (MA) score (FotoFinder) were applied to histologically verified melanocytic nevi (33) and malignant melanomas (12).¦RESULTS: Mean perfusion and MA scores were significantly increased in melanoma compared to nevi. However, applying an empirically determined threshold of 16% perfusion increase only 42% of the melanomas fulfilled the criterion of malignancy, whereas with the mole analyzer score 82% of the melanomas fulfilled the criterion of malignancy.¦CONCLUSION: Laser Doppler imaging is a highly sensitive technology to assess skin and skin tumor perfusion in vivo. Although mean perfusion is higher in melanomas compared to nevi the high numbers of false negative results hamper the use of this technology for early melanoma detection.

Prediction of fat-free mass using bioelectrical impedance analysis in young adults from five populations of African origin.

Background/objectives:Bioelectrical impedance analysis (BIA) is used in population and clinical studies as a technique for estimating body composition. Because of significant under-representation in existing literature, we sought to develop and validate predictive equation(s) for BIA for studies in populations of African origin.Subjects/methods:Among five cohorts of the Modeling the Epidemiologic Transition Study, height, weight, waist circumference and body composition, using isotope dilution, were measured in 362 adults, ages 25-45 with mean body mass indexes ranging from 24 to 32. BIA measures of resistance and reactance were measured using tetrapolar placement of electrodes and the same model of analyzer across sites (BIA 101Q, RJL Systems). Multiple linear regression analysis was used to develop equations for predicting fat-free mass (FFM), as measured by isotope dilution; covariates included sex, age, waist, reactance and height(2)/resistance, along with dummy variables for each site. Developed equations were then tested in a validation sample; FFM predicted by previously published equations were tested in the total sample.Results:A site-combined equation and site-specific equations were developed. The mean differences between FFM (reference) and FFM predicted by the study-derived equations were between 0.4 and 0.6âeuro0/00kg (that is, 1% difference between the actual and predicted FFM), and the measured and predicted values were highly correlated. The site-combined equation performed slightly better than the site-specific equations and the previously published equations.Conclusions:Relatively small differences exist between BIA equations to estimate FFM, whether study-derived or published equations, although the site-combined equation performed slightly better than others. The study-derived equations provide an important tool for research in these understudied populations.