Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA) gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied). Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc.) of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.

Selection, Recombination and History in a Parasitic Flatworm (Echinococcus) Inferred from Nucleotide Sequences

Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection) drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1). The phylogenetic analyses revealed several ambiguities, indicating that the taxonomic status of the E. granulosus horse strain should be revised

Association of Lifecourse Socioeconomic Status with Chronic Inflammation and Type 2 Diabetes Risk: The Whitehall II Prospective Cohort Study.

BACKGROUND: Socioeconomic adversity in early life has been hypothesized to "program" a vulnerable phenotype with exaggerated inflammatory responses, so increasing the risk of developing type 2 diabetes in adulthood. The aim of this study is to test this hypothesis by assessing the extent to which the association between lifecourse socioeconomic status and type 2 diabetes incidence is explained by chronic inflammation. METHODS AND FINDINGS: We use data from the British Whitehall II study, a prospective occupational cohort of adults established in 1985. The inflammatory markers C-reactive protein and interleukin-6 were measured repeatedly and type 2 diabetes incidence (new cases) was monitored over an 18-year follow-up (from 1991-1993 until 2007-2009). Our analytical sample consisted of 6,387 non-diabetic participants (1,818 women), of whom 731 (207 women) developed type 2 diabetes over the follow-up. Cumulative exposure to low socioeconomic status from childhood to middle age was associated with an increased risk of developing type 2 diabetes in adulthood (hazard ratio [HR] = 1.96, 95% confidence interval: 1.48-2.58 for low cumulative lifecourse socioeconomic score and HR = 1.55, 95% confidence interval: 1.26-1.91 for low-low socioeconomic trajectory). 25% of the excess risk associated with cumulative socioeconomic adversity across the lifecourse and 32% of the excess risk associated with low-low socioeconomic trajectory was attributable to chronically elevated inflammation (95% confidence intervals 16%-58%). CONCLUSIONS: In the present study, chronic inflammation explained a substantial part of the association between lifecourse socioeconomic disadvantage and type 2 diabetes. Further studies should be performed to confirm these findings in population-based samples, as the Whitehall II cohort is not representative of the general population, and to examine the extent to which social inequalities attributable to chronic inflammation are reversible. Please see later in the article for the Editors' Summary.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

A Randomized Trial for the Treatment of Refractory Status Epilepticus.

BACKGROUND: Refractory status epilepticus (RSE) has a mortality of 16-39%; coma induction is advocated for its management, but no comparative study has been performed. We aimed to assess the effectiveness (RSE control, adverse events) of the first course of propofol versus barbiturates in the treatment of RSE. METHODS: In this randomized, single blind, multi-center trial studying adults with RSE not due to cerebral anoxia, medications were titrated toward EEG burst-suppression for 36-48 h and then progressively weaned. The primary endpoint was the proportion of patients with RSE controlled after a first course of study medication; secondary endpoints included tolerability measures. RESULTS: The trial was terminated after 3 years, with only 24 patients recruited of the 150 needed; 14 subjects received propofol, 9 barbiturates. The primary endpoint was reached in 43% in the propofol versus 22% in the barbiturates arm (P = 0.40). Mortality (43 vs. 34%; P = 1.00) and return to baseline clinical conditions at 3 months (36 vs. 44%; P = 1.00) were similar. While infections and arterial hypotension did not differ between groups, barbiturate use was associated with a significantly longer mechanical ventilation (P = 0.03). A non-fatal propofol infusion syndrome was detected in one patient, while one subject died of bowel ischemia after barbiturates. DISCUSSION: Although undersampled, this trial shows significantly longer mechanical ventilation with barbiturates and the occurrence of severe treatment-related complications in both arms. We describe practical issues necessary for the success of future studies needed to improve the current unsatisfactory state of evidence.

The status of the Lutzomyia longipalpis species complex and possible implications for Leishmania transmission

The sand fly Lutzomyia longipalpis sensu latu has been identified as the principal vector of American visceral leishmaniasis, a potentially fatal disease that primarily affects children in several countries of South and Central America. Over the past several years increases have occurred both in the number of reported cases and the population at risk: approximately 1.6 million people reside in highly endemic areas with 16,000 cases reported annually. Several studies have attempted to relate the epidemiology of this disease to variability in Lu. longipalpis that is now recognized to be a complex of at least three sibling species. Morphological variation in this species was first noted by Mangabeira (1969). Since then physiological and biochemical differences have been reported by several investigators. Recent reports in Costa Rica of the presence of Lu. longipalpis in a focus of cutaneous leishmaniasis caused by Leishmania chagasi may be an additional indication of variability in this species. While existing evidence indicates that the morphospecies Lu. longipalpis may represent a complex of sibling species, genetic, epidemiological and ecological distinctions have not been fully resolved. Thus, delimitation of systematic boundaries within the complex and corresponding to geographic distributions and roles in transmission remain unresolved. The purpose of this review is to summarize from the literature observations of polymorphism in this morphospecies and consider what significance this reported variability may have to the epidemiology of visceral leishmaniasis.

Socioeconomic status, structural and functional measures of social support, and mortality: The British Whitehall II Cohort Study, 1985-2009.

The authors examined the associations of social support with socioeconomic status (SES) and with mortality, as well as how SES differences in social support might account for SES differences in mortality. Analyses were based on 9,333 participants from the British Whitehall II Study cohort, a longitudinal cohort established in 1985 among London-based civil servants who were 35-55 years of age at baseline. SES was assessed using participant's employment grades at baseline. Social support was assessed 3 times in the 24.4-year period during which participants were monitored for death. In men, marital status, and to a lesser extent network score (but not low perceived support or high negative aspects of close relationships), predicted both all-cause and cardiovascular mortality. Measures of social support were not associated with cancer mortality. Men in the lowest SES category had an increased risk of death compared with those in the highest category (for all-cause mortality, hazard ratio = 1.59, 95% confidence interval: 1.21, 2.08; for cardiovascular mortality, hazard ratio = 2.48, 95% confidence interval: 1.55, 3.92). Network score and marital status combined explained 27% (95% confidence interval: 14, 43) and 29% (95% confidence interval: 17, 52) of the associations between SES and all-cause and cardiovascular mortality, respectively. In women, there was no consistent association between social support indicators and mortality. The present study suggests that in men, social isolation is not only an important risk factor for mortality but is also likely to contribute to differences in mortality by SES.

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14.

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.

Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma

DNA sequence comparison of 412 base-pairs fragments of the mitochondrial cytochrome B gene was used to infer the genetic structure of nine geographical Triatoma infestans populations and their phylogenetic relationship with T. melanosoma and T. brasiliensis. T. infestans and T. melanosoma were compared by morphometry, allozyme and cytogenetic analyses, as well as subjected to reciprocal crosses, in order to clarify the taxonomic status of the latter. No differences were found to distinguish the two species and the crosses between them yielded progeny. T. infestans populations presented four haplotypes that could be separated in two clusters: one formed by the samples from Bolivia (Andes and Chaco) and the other formed by samples from Argentina and Brazil. Silvatic and domestic T. infestans populations from Bolivia (Andes) were genetically identical.

Prédiction des durées de séjour en réadaptation en fonction du statut fonctionnel. [Usefulness of functional status to predict length of stay in rehabilitation.]

Objectif : En Suisse, la réadaptation est financée en¦partie par l'assureur qui fixe préalablement à l'admission¦un nombre de jours (durée garantie) qu'il s'engage¦à rembourser. Lorsqu'une durée garantie est trop courte,¦une demande de prolongation est nécessaire, induisant¦des démarches administratives. Les objectifs de cette¦étude étaient a) d'étudier le lien entre durées garanties¦et caractéristiques du patient ; b) d'estimer les coûts¦liés aux demandes de prolongation ; c) d'évaluer¦l'impact de l'introduction d'un modèle d'attribution de¦durée garantie basé sur l'état fonctionnel du patient.¦Méthodes : Les corrélations entre état fonctionnel,¦durée effective et durée garantie ont été testées sur¦208 séjours représentatifs. Des durées garanties fictives¦ont été calculées à partir de la médiane de durée de¦séjour de 2 335 patients, groupés selon leur niveau¦fonctionnel (score des activités de base de la vie quotidienne¦(BAVQ) 0-1 vs 2-4 vs 5-6), puis comparées aux¦durées de séjour effectives et garanties.¦Résultats : L'état fonctionnel du patient n'est pas¦corrélé à la durée garantie, et 69 % des séjours nécessitent¦au moins une demande de prolongation, représentant¦2,6 équivalents temps plein en temps administratif¦projeté sur le canton. L'application du modèle proposé¦réduirait de 28 % les demandes de prolongation, et¦n'augmenterait que marginalement la proportion de¦jours garantis en surplus (11,2 % contre 6,5 % actuellement).¦Conclusion : L'utilisation systématique d'un modèle¦d'attribution de durées garanties basées sur l'état¦fonctionnel du patient permettrait de réduire sensiblement¦les coûts administratifs liés aux demandes de¦prolongation, sans entraîner de risque accru d'une augmentation¦de la durée de séjour.

A non-circadian role for clock-genes in sleep homeostasis: a strain comparison.

BACKGROUND: We have previously reported that the expression of circadian clock-genes increases in the cerebral cortex after sleep deprivation (SD) and that the sleep rebound following SD is attenuated in mice deficient for one or more clock-genes. We hypothesized that besides generating circadian rhythms, clock-genes also play a role in the homeostatic regulation of sleep. Here we follow the time course of the forebrain changes in the expression of the clock-genes period (per)-1, per2, and of the clock-controlled gene albumin D-binding protein (dbp) during a 6 h SD and subsequent recovery sleep in three inbred strains of mice for which the homeostatic sleep rebound following SD differs. We reasoned that if clock genes are functionally implicated in sleep homeostasis then the SD-induced changes in gene expression should vary according to the genotypic differences in the sleep rebound. RESULTS: In all three strains per expression was increased when animals were kept awake but the rate of increase during the SD as well as the relative increase in per after 6 h SD were highest in the strain for which the sleep rebound was smallest; i.e., DBA/2J (D2). Moreover, whereas in the other two strains per1 and per2 reverted to control levels with recovery sleep, per2 expression specifically, remained elevated in D2 mice. dbp expression increased during the light period both during baseline and during SD although levels were reduced during the latter condition compared to baseline. In contrast to per2, dbp expression reverted to control levels with recovery sleep in D2 only, whereas in the two other strains expression remained decreased. CONCLUSION: These findings support and extend our previous findings that clock genes in the forebrain are implicated in the homeostatic regulation of sleep and suggest that sustained, high levels of per2 expression may negatively impact recovery sleep.

Biocontrol strain Pseudomonas fluorescens CHA0 and its genetically modified derivative with enhanced biocontrol capability exert comparable effects on the structure of a Sinorhizobium meliloti population in a gnotobiotic system

The impact of biocontrol strain Pseudomonas fluorescens CHA0 and of its genetically modified, antibiotic-overproducing derivative CHA0/pME3424 on a reconstructed population of the plant-beneficial Sinorhizobium meliloti bacteria was assessed in gnotobiotic systems. In sterile soil, the final density of the reconstructed S. meliloti population decreased by more than one order of magnitude in the presence of either of the Pseudomonas strains when compared to a control without addition of P. fluorescens. Moreover, there was a change in the proportion of each individual S. meliloti strain within the population. Plant tests also revealed changes in the nodulating S. meliloti population in the presence of strains CHA0 or CHA0/pME3424. In both treatments one S. meliloti strain, f43, was significantly reduced in its root nodule occupancy. Analysis of alfalfa yields showed a slight but statistically significant increase in shoot dry weight when strain CHA0 was added to the reconstructed S. meliloti population whereas no such effect was observed with CHA0/pME3424.