A survey strategy for human respiratory syncytial virus detection among haematopoietic stem cell transplant patients: epidemiological and methodological analysis

Human respiratory syncytial virus (HRSV) causes severe infections among children and immunocompromised patients. We compared HRSV infections among Haematopoietic Stem Cell Transplant program (HSCT) patients and children using direct immunofluorescence (DFA), point-of-care RSV Bio Easy® and a polymerase chain reaction (PCR) assay. Overall, 102 samples from HSCT patients and 128 from children obtained positivity rate of 18.6% and 14.1% respectively. PCR sensitivity was highest mainly on samples collected after five days of symptoms onset. A combination of both DFA and reverse transcriptase-PCR methods for HSCT high-risk patients is the best diagnostic flow for HRSV diagnosis among these patients.

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

Respiratory responses during affective picture viewing

Previous research has demonstrated covariation of physiological responding with judgments of valence and arousal. However, until now links between these affective dimensions and respiratory measures have not been extensively investigated. In this study, eight picture series of different affective valence and arousal level were shown to 30 subjects, while respiration, skin conductance level (SCL), heart rate (HR) and affective judgments were measured. With increasing pleasantness, inspiratory time lengthened, mean inspiratory flow decreased and thoracic breathing increased. With increasing arousal, inspiratory time and total breath duration shortened and mean inspiratory flow, minute ventilation, thoracic breathing and electrodermal activity increased. These findings confirm the importance of arousal in respiratory responding, but also indicate a modulatory role of affective valence.We propose that the arousal effects reflect energy mobilization in preparation to act, and thatthe valence effects might be a manifestation of an attention bias toward negative stimuli. [Authors]

Characterisation of "fou2", a constitutive wound-activated mutant and analysis of the proteome and leaf physiology in the region proximal to wounds in Arabidopsis

Résumé : Les jasmonates (JA), une famille d'hor1none végétale, jouent un rôle central dans la réponse à la blessure, et aux attaques d'insectes et de pathogènes. Les JA sont principalement dérivés d'un acide gras, l'acide linolénique. L'addition par une lipoxygénase d'une molécule d'oxygène à l'acide linolénique initie la synthèse de JA. Cependant les mécanismes régulant l'activation de la biosynthèse de JA ne sont pas encore connus. C'est pour cette raison que dans ce travail, nous avons caractérisé chez Arabidopsis thaliana (l'Arabette des Dames) un mutant fou2 dont l'activité lipoxygénase est plus élevée que celle d'une plante sauvage. Les niveaux de JA sont constitutivement plus élevés et l'activation de la synthèse de JA après blessure est fortement plus induite chez fou2 que chez le type sauvage. En outre, fou2 est plus résistant au pathogène Botrytis cinerea et à la chenille Spodoptera littoralis. Afin de comprendre quel mécanisme chez fou2 génére ce phénotype, nous avons cloné le gène responsable du phénotype de fou2. Le mutant fou2 porte une mutation dans le gène d'un canal à deux pores transportant probablement du potassium, du lumen de la vacuole végétale vers le compartiment cytosolique. L'analyse du protéome de fou2 a permis d'identifier une expression plus élevée de sept protéines régulées par les JA ou le stress. La découverte de l'implication d'un canal dans le phénotype de fou2 renforce l'hypothèse que les flux de cations pourraient être impliqués dans les étapes précoces de la synthèse des JA. Nous avons également étudié le protéome et la physiologie d'une feuille blessée, Pour évaluer les changements d'expression protéique en réponse à la blessure et contrôlés par les JA, nous avons quantifié l'expression de 5937 protéines chez une plante d'Arabidopsis sauvage et chez un mutant incapable de synthétiser des JA. Parmi ces 5937 protéines, nous avons identifié 99 protéines régulées par la blessure chez le type sauvage. Nous avons observé pour 65% des protéines dont l'expression protéique changeait après blessure une bonne corrélation entre la quantité de transcrits et de protéines. Plusieurs enzymes de la voie des chorismates impliquées dans la biosynthèse des acides aminés phénoliques étaient induites par les JA après blessure. Une quantification des acides aminés a montré que les niveaux d'acides aminés phénoliques augmentaient significativement après blessure. La blessure induisait aussi des changements dans l'expression de protéines impliquées dans la réponse au stress et particulièrement au stress oxydatif. Nous avons quantifié l'état réduit et oxydé du glutathion, un tripeptide qui, sous sa forme réduite, est l'antioxydant majeur des cellules. Nous avons trouvé une quantité significativement plus élevée de glutathion oxydé chez le type sauvage blessé que chez la plante aus blessée. Ce résultat suggère que la génération d'un stress oxydatif et la proportion relative de glutathions réduits et oxydés sont contrôlés par les JA après blessure. Abstract : Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri?unsaturated fatty acid a-linolenic-acid (18:3). Addition of an oxygen molecule to 18:3 by 13-lipoxygenases (13-LOX) initiates JA biosynthesis. Actually components regulating the activation of JA biosynthesis are poorly defined. Therefore we characterized in Arabidopsis thaliana the fatty acid Qxygenation upregulated 2 (fou2) mutant, which was previously isolated in a screen for mutants with an enhanced 13-LOX activity. As a consequence of this increased 13-LOX activity, JA levels in fou2 are higher than in wild type (WT) and wounding strongly increased JA biosynthesis compared to WT. fou2 was more resistant to the fungus Botrytis cinerea and the generalist caterpillar Spodaptera littomlis, The fou2 mutant carries a missense mutation in the Two Pore Channel 1 gene (TPCJ), which encodes a vacuolar cation channel transporting probably K* into the cytosol. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA- inducible proteins. The discovery of the implication of a channel in the fou2 phenotype strenghtens the hypothesis that cation fluxes might be implicated in early steps of JA synthesis. We further concentrated on the proteome and leaf physiology in the region proximal to wounds in Arabidopsis using the WT and the aos JA-biosynthesis deficient mutant in order to find JA- induced proteins changes. We used two successive proteomic methods to assess protein changes in response to wounding Arabidopsis leaves, two dimensional electrophoresis (2DE) and linear trap quadrupole ion-trap mass spectrometry. In total 5937 proteins were quantified. We identified 99 wound-regulated proteins in the WT. Most these proteins were also wound-regulated at the transcript level showing a good correlation between transcript and protein abundance. We identified several wound-regulated enzymes involved in amino acid biosynthesis and confirmed this result by amino acid quantification. Proteins involved in stress reponses were upregulated, particularly in redox species regulation. We found a significantly higher quantity of oxidized glutathione in wounded WT relative to wounded aos leaves. This result suggests that levels of reduced glutathione are controlled by JA after wounding.

Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection

Viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (ARIs) for all age groups. The aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with ARIs. Nasopharyngeal aspirates were obtained from 162 individuals between August 2007-August 2009. Twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IIF). Through IIF, 33 (20.4%) specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. Through a multiplex real-time RT-PCR assay, 88 (54.3%) positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (RSV). Six cases of viral co-detection were observed, mainly involving RSV. The use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza A (H1N1) pdm09 virus, human coronavirus (HCoV) NL63 and HCoV HKU1].

Effect of expiratory positive airway pressure on sleep disordered breathing.

STUDY OBJECTIVES: We sought to determine the effect of expiratory positive airway pressure on end expiratory lung volume (EELV) and sleep disordered breathing in obstructive sleep apnea patients. DESIGN: Observational physiology study PARTICIPANTS: We studied 10 OSA patients during sleep wearing a facial mask. We recorded 1 hour of NREM sleep without treatment (baseline) and 1 hour with 10 cm H2O EPAP in random order, while measuring EELV and breathing pattern. RESULTS: The mean EELV change between baseline and EPAP was only 13.3 mL (range 2-25 mL). Expiratory time was significantly increased with EPAP compared to baseline 2.64 +/- 0.54 vs 2.16 +/- 0.64 sec (P = 0.002). Total respiratory time was longer with EPAP than at baseline 4.44 +/- 1.47 sec vs 3.73 +/- 0.88 sec (P = 0.3), and minute ventilation was lower with EPAP vs baseline 7.9 +/- 4.17 L/min vs 9.05 +/- 2.85 L/min (P = 0.3). For baseline (no treatment) and EPAP respectively, the mean apnea+hypopnea index (AHI) was 62.6 +/- 28.7 and 56.8 +/- 30.3 events per hour (P = 0.4). CONCLUSION: In OSA patients during sleep, the application of 10 cm H2O EPAP led to prolongation of expiratory time with only marginal increases in FRC. These findings suggest important mechanisms exist to avoid hyperinflation during sleep.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.

Bronchiolite respiratoire et bronchiolite respiratoire avec pneumopathie interstitielle [Respiratory bronchiolitis and respiratory bronchiolitis-associated interstitial lung disease].

The increasing use of chest CT imaging in medical practice rises the likelihood of the general practitioner to be confronted with cases of interstitial lung disease. Respiratory bronchiolitis (RB) and respiratory bronchiolitis-associated interstitial lung disease (RB-ILD) are two smoking-related lung damages that may have important implications for the patient's management. The authors present in this paper a review of current knowledge of the epidemiology, clinical features, prognosis, and treatment options of RB and RB-ILD.

Epidemiological and clinical data of hospitalizations associated with respiratory syncytial virus infection in children under 5 years of age in Spain: FIVE multicenter study.

BACKGROUND Respiratory syncytial virus (RSV) is an important pathogen in lower respiratory tract infections (LRTI) in infants, but there are limited data concerning patients with underlying conditions and children older than 2 years of age. METHODS We have designed a prospective observational multicenter national study performed in 26 Spanish hospitals (December 2011-March 2012). Investigational cases were defined as children with underlying chronic diseases and were compared with a group of previously healthy children (proportion 1:2). Clinical data were compared between the groups. RESULTS A total of 1763 children hospitalized due to RSV infection during the inclusion period were analyzed. Of them, 225 cases and 460 healthy children were enrolled in the study. Underlying diseases observed were respiratory (64%), cardiovascular (25%), and neurologic (12%), as well as chromosomal abnormalities (7·5%), immunodeficiencies (6·7%), and inborn errors of metabolism (3·5%). Cases were statistically older than previously healthy children (average age: 16·3 versus 5·5 months). Cases experienced hypoxemia more frequently (P < 0·001), but patients with respiratory diseases required oxygen therapy more often (OR: 2·99; 95% CI: 1·03-8·65). Mechanical ventilation was used more in patients with cardiac diseases (OR: 3·0; 95% CI: 1·07-8·44) and in those with inborn errors of metabolism (OR: 12·27; 95% CI: 2·11-71·47). This subgroup showed a higher risk of admission to the PICU (OR: 6·7, 95% CI: 1·18-38·04). Diagnosis of pneumonia was more frequently found in cases (18·2% versus 9·3%; P < 0·01). CONCLUSIONS A significant percentage of children with RSV infection have underlying diseases and the illness severity is higher than in healthy children.

Connexins in lymphatic vessel physiology and disease.

Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (<1kDa) between the extracellular space and the cytosol. Gap junction channels allow diffusion of similar molecules between the cytoplasms of adjacent cells. The expression and function of connexins in blood vessels has been intensely studied in the last few decades. In contrast, only a few studies paid attention to lymphatic vessels; convincing in vivo data with respect to expression patterns of lymphatic connexins and their functional roles have only recently begun to emerge. Interestingly, mutations in connexin genes have been linked to diseases of lymphatic vasculature, most notably primary and secondary lymphedema. This review summarizes the available data regarding lymphatic connexins. More specifically it addresses (i) early studies aimed at presence of gap junction-like structures in lymphatic vessels, (ii) more recent studies focusing on lymphatic connexins using genetically engineered mice, and (iii) results of clinical studies that have reported lymphedema-linked mutations in connexin genes.

Respiratory syncytial virus seasonality in Brazil: implications for the immunisation policy for at-risk populations

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalisation for respiratory diseases among children under 5 years old. The aim of this study was to analyse RSV seasonality in the five distinct regions of Brazil using time series analysis (wavelet and Fourier series) of the following indicators: monthly positivity of the immunofluorescence reaction for RSV identified by virologic surveillance system, and rate of hospitalisations per bronchiolitis and pneumonia due to RSV in children under 5 years old (codes CID-10 J12.1, J20.5, J21.0 and J21.9). A total of 12,501 samples with 11.6% positivity for RSV (95% confidence interval 11 - 12.2), varying between 7.1 and 21.4% in the five Brazilian regions, was analysed. A strong trend for annual cycles with a stable stationary pattern in the five regions was identified through wavelet analysis of the indicators. The timing of RSV activity by Fourier analysis was similar between the two indicators analysed and showed regional differences. This study reinforces the importance of adjusting the immunisation period for high risk population with the monoclonal antibody palivizumab taking into account regional differences in seasonality of RSV.

Nemaline myopathy revealed by respiratory failure in adults

Most forms of myopathy may involve the respiratory muscles and progress to respiratory failure. However, the diagnosis of myopathy is seldom considered in an adult patient with no history of muscle disease and presenting with respiratory failure. Nemaline myopathy (NM) is a rare disorder characterized by symmetrical diffuse muscle weakness and rod-like nemaline bodies in muscle fibers. Respiratory muscle involvement is a major determinant of mortality in congenital NM, but is rare in late onset NM. Here, we report that acute or chronic respiratory failure may be caused by NM in subjects with no known history of muscle disease. Adult-onset NM was diagnosed in a 67-year-old woman with chronic respiratory insufficiency. Late onset childhood NM was revealed by respiratory failure in twin sisters aged 31. The diagnosis was established by muscle biopsy and electron microscopy (and mutations in the nebulin gene in the two sisters). Long-term clinical improvement was obtained with non-invasive ventilation (NIV) in the three patients. In conclusion, respiratory failure in an adult patient with no known history may correspond to NM with diaphragm involvement. Long-term outcome may be favorable with NIV.

Respiratory quotient evolution during normal pregnancy: what nutritional or clinical information can we get out of it?

Food intake increases to a varying extent during pregnancy to provide extra energy for the growing fetus. Measuring the respiratory quotient (RQ) during the course of pregnancy (by quantifying O2 consumption and CO2 production with indirect calorimetry) could be potentially useful since it gives an insight into the evolution of the proportion of carbohydrate vs. fat oxidized during pregnancy and thus allows recommendations on macronutrients for achieving a balanced (or slightly positive) substrate intake. A systematic search of the literature for papers reporting RQ changes during normal pregnancy identified 10 papers reporting original research. The existing evidence supports an increased RQ of varying magnitude in the third trimester of pregnancy, while the discrepant results reported for the first and second trimesters (i.e. no increase in RQ), explained by limited statistical power (small sample size) or fragmentary data, preclude safe conclusions about the evolution of RQ during early pregnancy. From a clinical point of view, measuring RQ during pregnancy requires not only sophisticated and costly indirect calorimeters but appears of limited value outside pure research projects, because of several confounding variables: (1) spontaneous changes in food intake and food composition during the course of pregnancy (which influence RQ); (2) inter-individual differences in weight gain and composition of tissue growth; (3) technical factors, notwithstanding the relatively small contribution of fetal metabolism per se (RQ close to 1.0) to overall metabolism of the pregnant mother.

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.