Effect of adding lay-tutors to the educational part of a back school programme for patients with subacute, non-specific low back pain: A randomized controlled clinical trial with a two-year follow-up.

OBJECTIVE: To evaluate the effect of adding a lay-tutor to the educational sessions of a back school programme for patients with subacute low back pain. METHODS: Patients with subacute low back pain were randomized to a 10-week programme comprising 10 h education and 20 h physical exercise led by a former patient as lay-tutor, or a programme led by a physiotherapist. In the intervention group, former patients served as lay-tutors in the educational sessions, teaching in conjunction with physiotherapists. In the control group, 2 physiotherapists led the entire educational programme. Disability, back pain, leg pain and health status were evaluated blindly at 3 and 24 months. RESULTS: Eighty-seven patients with subacute low back pain referred for treatment at 6 selected physiotherapy clinics were allocated to either an intervention group (n = 42) or a control group (n = 45). No statistically significant difference was found between the 2 groups. Both groups of patients showed a statistically significant improvement in health and pain measurements from the start of the study to the 3- and 24-month follow-up. CONCLUSION: No short- or long-term effect was found of adding a lay-tutor to the educational sessions of a back school programme for patients with subacute low back pain with regards to functional activity, back pain, leg pain or general health. The main limitations are that the potential effect of including lay-tutors in the educational part of a back school programme as an intervention in itself has to be tested, and the programme has to be tested as a complete protocol. Also, no specific testing has been performed to confirm the ideal number of sessions in the programme.

A Third Way for Entomophthoralean Fungi to Survive the Winter: Slow Disease Transmission between Individuals of the Hibernating Host.

In temperate regions, insect pathogenic fungi face the challenge of surviving through the winter. Winter is a time when hosts are immobile, low in number or are present in a stage which is not susceptible to infection. Fungi from Entomophthoromycota have so far been known to survive the winter in two ways: either as (1) thick-walled resting spores released into environment from dead hosts, or as (2) structures inside the dead host (e.g., hyphal bodies). Here we report, from the Danish environment, a third way to survive the winter, namely a slow progression and transmission of Entomophthora schizophorae in adult dipteran Pollenia hosts that hibernate in clusters in unheated attics, sheltered areas outdoors (under bark etc.). Fungus-killed sporulating flies were observed outside very early and very late in the season. By sampling adults at the time of their emergence from hibernation in late winter/early spring we documented that the fungus was naturally prevalent and killed flies after a period of incubation. Experimentally we documented that even at the low temperature of 5 °C, the fungus was able to maintain itself in Pollenia cohorts for up to 90 days. From these observations the full winter cycle of this fungus is elucidated. The three types of winter survival are discussed in relation to fungus epidemic development.

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR.

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex-polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.

Value of host range, morphological, and genetic characteristics within the Entomophthora muscae species complex.

Entomopthora muscae sensu lato is a complex of morphologically similar fungal species pathogenic to evolutionarily advanced flies (Cyclorrhapha). To reach an operational species definition and recognition of species within this complex, the values of host range, morphological and genetic characteristics are reconsidered. Within the E. muscae species complex morphological and nuclear characteristics of the primary conidia are taxonomically important. In this study we compared the dimensions and nuclear numbers of the primary conidia of isolates from their original (natural) hosts and after being transferred to alternative hosts (cross-transmission) in order to check the stability of these characteristics. The conidial characteristics change substantially when produced in alternative host species, but their overall range in variability still fit within the traditional morphological species circumscriptions. The phylogenetic analyses of the ITS II and LSU rRNA gene sequences, revealed three distinct lineages within the complex: E. schizophorae, E. muscae and E. syrphi. Within each of these lineages sequence divergence was seen between isolates originating from different host species. Our studies on the physiological host range showed that several isolates were able to infect alternative dipteran species. Musca domestica was a particularly good receptor. The ecological host range of any individual isolate seems, however, to be limited to one host species evidenced by the occurrence of distinct genotypes within each natural infected host species shown by RAPD. The high host specificity of these fungi emphasizes the importance of identifying the host taxon at species level in the recognition of Entomophthora species. We recommend that morphological characteristics of fungal structures and host taxon, together with molecular data, serve as criteria for species determination in future studies on members of the E. muscae complex.

Accuracy of tympanometric middle ear pressure determination in secretory otitis media: dose-dependent overestimation related to the viscosity and amount of middle ear fluid.

HYPOTHESIS: Tympanometric measurements of middle ear pressure in children with secretory otitis media are overestimated in a dose-response manner because of increased hysteresis explained by the viscosity and amount of middle ear fluid. BACKGROUND: Tympanometric middle ear pressure is important in evaluating children with secretory otitis media. These measurements are influenced by hysteresis appearing as a peak pressure difference in bidirectional tympanometry. This represents an inaccuracy of 0.5 x peak pressure difference, which is only 5 to 25 daPa in normal ears. However, previous experiments found increased hysteresis, suggesting an inaccuracy of 225 daPa in secretory otitis media ears. MATERIALS AND METHODS: In 56 patients with secretory otitis media, bidirectional tympanometry was performed; Type B curves were excluded. The middle ear fluid was semiquantified subsequently at surgery according to viscosity (serous, seromucoid, or mucoid) and amount (small, medium, or large). A control group included 28 normal children. Peak pressure difference was calculated by the difference between middle ear pressure determined by a positive and negative pressure sweep. RESULTS: Mean peak pressure difference was 10 and 69 daPa in the normal and secretory otitis media groups, respectively (p <0.001). However, peak pressure difference ranged to 205 daPa in the secretory otitis media group and showed a significant positive correlation to viscosity and amount of the fluid (both p <0.0001). CONCLUSION: Peak pressure difference is significantly increased in secretory otitis media because of additional damping explained by the viscosity and amount of the fluid. The mean error was 5 daPa in normal ears and 35 daPa in secretory otitis media ears, but ranged to greater than 100 daPa. These results were only a low estimate of the inaccuracy, because patients with Type B tympanograms could not be included, and errors of more than 100 daPa can be anticipated.

Density-dependence and within-host competition in a semelparous parasite of leaf-cutting ants.

BACKGROUND: Parasite heterogeneity and within-host competition are thought to be important factors influencing the dynamics of host-parasite relationships. Yet, while there have been many theoretical investigations of how these factors may act, empirical data is more limited. We investigated the effects of parasite density and heterogeneity on parasite virulence and fitness using four strains of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, and its leaf-cutting ant host Acromyrmex echinatior as the model system. RESULTS: The relationship between parasite density and infection was sigmoidal, with there being an invasion threshold for an infection to occur (an Allee effect). Although spore production was positively density-dependent, parasite fitness decreased with increasing parasite density, indicating within-host scramble competition. The dynamics differed little between the four strains tested. In mixed infections of three strains the infection-growth dynamics were unaffected by parasite heterogeneity. CONCLUSIONS: The strength of within-host competition makes dispersal the best strategy for the parasite. Parasite heterogeneity may not have effected virulence or the infection dynamics either because the most virulent strain outcompeted the others, or because the interaction involved scramble competition that was impervious to parasite heterogeneity. The dynamics observed may be common for virulent parasites, such as Metarhizium, that produce aggregated transmission stages. Such parasites make useful models for investigating infection dynamics and the impact of parasite competition.

PCR-RFLP is used to investigate relations among species in the entomopathogenic genera Eryniopsis and Entomophaga.

The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.

Application of nested-PCR technique to resting spores from the Entomophthora muscae species complex: implications for analyses of host-pathogen population interactions.

We developed new Entomophthora-specific primers for nested-PCR of the ITS II region to be used on in vivo material and combined it with RFLP. Resting spores from Scathophaga stercoraria (3 specimens), Delia radicum (9 specimens), Botanophila fugax (1 specimen), and two syrphid host species, Platycheirus peltatus and Melanostoma mellinum (one specimen of each) were characterized genetically after analysis of RFLP-profiles of the PCR-products. The genetic characterization of the resting spore isolates was compared with twenty isolates of known primary conidial morphology (in vitro and in vivo) from the E. muscae species complex. The analysis allowed for the first time a separation of resting spore isolates into the species level, which is not possible only using morphological characters (diameter). Isolates originating from different specimens of the same host taxa appeared to be strongly clonal even they were sampled at different localities in different years. Isolates morphologically belonging to E. muscae s. str. (e.g., including E. scatophagae) could be separated genetically further into sub-groups entirely depending on the host taxa; each fungal genotype, either present at the conidial stage or at the resting spore stage, is correlated with one host species. Furthermore, E. muscae s. str. originating from D. radicum proved to be much more closely related to E. scatophagae than to E. muscae s. str. originating from M. domestica. None of the resting spore isolates could be assigned to E. schizophorae. The nested-PCR approach accompanied by RFLP proved its usefulness for identification of resting spores and for more detailed studies clarifying host-pathogen specificity and interactions. It seems that different members of the E. muscae species complex are able to complete their life cycle in only one host species and, further, that each pathogen-host system is independent.