Amblyomma americanum is a common pest and vector of infectious diseases for humans and other mammals in the southern and eastern United States. A Coxiella sp. bacterial endosymbiont was highly prevalent in both laboratory-reared and field-collected A. americanum. The Coxiella sp. was demonstrated in all stages of tick and in greatest densities in nymphs and adult females, while a Rickettsia sp. was less prevalent and in lower densities when present.The lone star tick A. americanum by injecting engorged nymphs or engorged, mated females with single doses of an antibiotic (rifampin or tetracycline) or buffer alone. Burdens of the bacteria after molting or after oviposition were estimated by quantitative polymerase chain reaction with primers and probes specific for each bacterial species or, as an internal standard, the host tick. Post-molt adult ticks that had been treated with rifampin or tetracycline had lower numbers of the Coxiella sp. and Rickettsia sp. and generally weighed less than ticks that received buffer alone. Similarly, after oviposition, females treated previously with either antibiotic had lower burdens of both bacterial species in comparison to controls. Treatment of engorged females with either antibiotic was associated with prolonged time to oviposition, lower proportions of ticks that hatched, lower proportions of viable larvae among total larvae, and lower numbers of viable larvae per tick. These fitness estimators were associated with reduced numbers of the Coxiella sp. but not the Rickettsia sp.We manipulated the numbers of both bacterial species in laboratory-reared Coxiella sp. is a primary endosymbiont, perhaps provisioning the obligately hematophagous parasites with essential nutrients. The results also suggest that antibiotics could be incorporated into an integrated pest management plan for control of these and other tick vectors of disease.The findings indicate that the  Francisella, Rickettsia, or Coxiella have been found in both hard (ixodid) and soft (argasid) ticks Rhipicephalus and Haemaphysalis and an argasid tick of the genus OrnithodorosCoxiella but also are vectors of Coxiella burnetii, the Q fever agent Endosymbiotic bacteria inhabit a variety of invertebrates Coxiella sp. in the ixodid tick Amblyomma americanum, also known as the lone star tick Coxiella sp. is an endosymbiont of A. americanumA. americanum ticks examined to date, including field-collected samples from several locations and from laboratory-reared specimens. In contrast, a Rickettsia sp. was not detectable in some of the same sample. (2) The Coxiella sp. was present in eggs, an indication of transovarial transmission. (3) The organism appears to have a reduced genome, a feature of several endosymbionts of invertebrates We recently identified and characterized a hitherto unknown The finding of endosymbiotic bacteria in an arthropod that is obligately hematophagous at all stages of its life cycle led to questions about the functions, if any, of the bacteria for its host's physiology and reproduction. For instance, could the endosymbionts provide one or more essential nutrients that are absent or scant in the tick's blood meal? With this longer term goal in mind, we set out to determine first whether manipulation of the numbers of the bacteria in the ticks could discernibly affect the tick host of the bacteria. Application of antibiotics has been a useful approach for investigating the role of endosymbionts for their invertebrate hosts, for example in filarial worms and weevils Coxiella sp. and Rickettsia sp. bacteria in the ticks Disease prevention was another rationale for assessing the effect of antibiotics on the endosymbiotic bacteria and, indirectly, on the ticks themselves. If antibiotic treatment was associated with both reduced numbers of bacteria and lowered fitness of ticks, this would justify further investigation of the bacteria as targets for vector control efforts. We used the lone star tick as a model, in part because it is easily reared in the laboratory. But, in addition, this tick species is of growing importance as a pest and disease vector in the United States for humans and agricultural animals A. americanum larvae, nymphs, and adults or egg masses were obtained in late 2005 and early 2006 from the Department of Entomology and Plant Pathology of Oklahoma State University, Stillwater, OK. The tick colony was started in 1976 with engorged females collected in Cherokee County, OK. Engorged females are introduced every 2 years in approximately equal numbers to mated colony females, and since 1991 they have been obtained from Payne County, OK. The most recent introduction of field-collected females had been in July 2004. The nymphs had fed on rabbits, and adults ticks had fed on sheep. Ticks were maintained in a Model I-36 VL Percival Scientific environmental chamber  at 24°C, 94–96% humidity, and on a 12:12-hr light:dark cycle Engorged or flat Stock solutions of tetracycline HCl or rifampin  were made in 70% ethanol or in pharmaceutical grade dimethylsulfoxide (Sigma), respectively, at concentrations of 100 mg/ml. The antibiotics were diluted in 137 mM NaCl-2.7 mM KCl-4.3 mM sodium phosphate-1.4 mM potassium phosphate, pH 7.0 (PBS). The doses of each antibiotic were 12.5 ng per mg weight of engorged nymphs and 17 ng per mg weight of engorged, mated female adults. On their fourth day after repletion and dropping from the host mammal, engorged nymphs or females were weighed and then injected under a dissecting microscope with a locally-fabricated injection apparatus and glass capillaries that had been pulled with a Sutter needle puller. The ticks were affixed on their sides to double-sided tape adhered to a glass slide, and the slide was adjusted to a 45° angle to the microscope stage. The tip of the capillary was inserted into the hemocoel through the space between the first and second legs. After injection of 0.25 µl into nymphs or 0.5–0.8 µl into females, the needle was left place for at least 30 sec and then slowly withdrawn. For treatments of engorged nymphs, the adult ticks were sexed and weighed after molting. For experiments with engorged females, unhatched eggs, live larvae, and dead larvae were counted under a dissecting scope 7 d after oviposition was complete for a given female.fusA (elongation factor G) gene of the Coxiella sp. and the gltA (citrate synthase) gene of the Rickettsia sp. gltA sequence of the Rickettsia sp. of the laboratory-reared A. americanum was EF514206. For estimating gene copies in A. americanum, we used the macrophage inhibitory factor (MIF) gene, for which we had both genomic sequence and mRNA sequence and which occurs in single copy per genome Escherichia coli plasmid, and these were serially diluted from 106 to 10−1 gene copies per reaction 2R) for the log regression of the tC values vs. DNA dilutions of 0.99, efficiencies of>92%, and sensitivities of 1–10 gene copies.Ticks were frozen and pulverized in liquid nitrogen, and then DNA was extracted as described Standard parametric statistical analyses were carried out with SYSTAT v. 11 , and significance tests were two-sided. Ratios and skewed data were log-transformed for significance testing. Similarly, means and 95% confidence intervals (CI) were determined for log-transformed data, and then the antilogs were calculated for presentation. The Mann-Whitney-Wilcoxon and Spearman non-parametric tests were exact and carried out with StatXact v. 6.3 . For the Kaplan-Meier survival analysis we used the Mantel log-rank test statistic. Exact logistic regression  was used to evaluate the association between scalar predictor variables and discrete outcomes. Box-and-whisker plots in figures show the median, first and third quartiles, and 1.5X interquartile range.fusA gene of the Coxiella sp. to the MIF gene of the A. americanum in a given tick were used to estimate the burdens of the bacterium in the ticks at different stages of their life cycle. fusA/MIF gene ratio were similar for the eggs and larvae, but the variance of the ratios was substantially greater for the larvae. In contrast, there were an estimated ∼10 Coxiella sp. bacteria for every tick cell by the time nymphs were 2 months post-molt, and these levels persisted through adults 3 mo. post-molt. Moreover, with the exception of nymphs 1 mo. post-molt, the variances were comparatively lower for nymphs and adults.The ratios of the Rickettsia sp. was not as prevalent, and, when present, the bacterial burden per tick was lower than with the Coxiella sp. By the criterion of a gltA to MIF gene ratio of>0.001, the Rickettsia was not detectable in the egg masses (n = 5), 1 mo. nymphs (n = 10), or 3 mo. nymphs (n = 7). Six of 20 larvae had detectable Rickettsia sp., but the median gltA/MIF ratio among positive ticks was only 0.002. Of the 10 nymphs 2 mo. post-molt, only 1 had detectable Rickettsia. For the 1 mo., 2 mo., and 3 mo. post-molt females the prevalences of the Rickettsia sp. were 7 of 10, 7 of 10, and 6 of 10, respectively. Among the positive ticks, the median numbers of Rickettsia sp. bacteria per tick cell increased to 0.44 and 0.27 for 1 mo. and 2 mo. females, respectively, but declined to 0.002 in 3 mo. females.The Coxiella sp., but, as expected from the studies of post-molt females, only 7 of 11 males had detectable Rickettsia sp. Male ticks had lower densities of the Coxiella sp. than did adult females; for the fusA/MIF ratio the median was 0.73 in males, and the mean  was 0.76 (0.39–1.13). In contrast the density of the Rickettsia sp. in infected males was about the same as found in infected post-molt females; for the gltA/MIF ratio the median was 0.68, and the mean was 0.71 (0.53–0.89). We dissected 4 adult females and determined that the mean fusA/MIF ratio was 381 (252–512) in the midgut and 79 (39–298) in the ovaries, but only 12 (11–14) in the salivary glands.Among 11 male adult ticks that were studied, all had the Rickettsia sp. was not as prevalent as the Coxiella sp. in the ticks at any stage, and among ticks with the Rickettsia sp. the burdens of this bacterium were considerably lower than the Coxiella sp.These findings were further evidence that the fusA/MIF gene ratios for the Coxiella sp. were 4.0 (2.7–6.0) for 7 adult females who received PBS alone and 1.2 (0.5–2.9) for 8 females who received tetracycline . The post-molt weights were 4.2 (3.9–4.4) mg for the PBS control group and 3.8 (3.6–4.0) mg for the tetracycline group .In the first experiment nymphs were injected with PBS alone or PBS with tetracycline. After the molt, the means (95% CI) for the fusA/MIF gene ratios for the Coxiella sp. in post-molt adults were 9.3 (5.8–12.9) for the PBS control, 0.9 (0.3–1.6) for rifampin (t test p<0.001), and 1.2 (0.6–1.8) for tetracycline (p<0.001). Corresponding values for the gltA/MIF gene ratios for the Rickettsia sp. were 4.3 (1.5–7.0) for the PBS control, 1.8 (1.4–2.1) for rifampin treatment (p<0.01), and 1.4 (1.1–1.7) for tetracycline treatment (p<0.001). The mean post-molt weights were 5.6 (5.4–5.9) mg for PBS, 5.1 (4.8–5.5) mg for rifampin (p<0.01), and 4.7 (4.4–4.9) mg for tetracycline (p<0.01).In the second, larger experiment engorged nymphs were divided into three groups of 30 each and injected with PBS alone, PBS with rifampin, or PBS with tetracycline (day 0). The mean weights (and standard deviations) for pre-treatment nymphs were similar at 8.6 (2.2), 8.6 (2.2), and 8.5 (2.2) mg for the PBS, rifampin, and tetracycline groups, respectively. The ticks began molting by day 23; molting was complete by day 27. There was not a difference between the three groups in the onset of the molt or its last day. The 19 females in each of the PBS and tetracycline groups and the 17 females in the rifampin group were further studied. The means (95% CI) for the Coxiella endosymbiont in A. americanum were lowest in eggs and rose in number through the nymph stage and remained stable as the tick matured as an adult, suggesting that the greatest impact of symbiont manipulation would be on reproduction. To investigate this we injected 30 fed, mated females in groups of 10 with mean weights of 0.6 g with PBS, rifampin, or tetracycline on day 0. The ticks were maintained individually in an incubation chamber and observed daily for oviposition. The date for the start of oviposition was noted, and marks were made daily on the tube to monitor oviposition progress as the female advanced along the tube. When oviposition ceased, the female was removed, the tubes were weighed, and the tube's tare was subtracted to estimate the egg mass. The egg masses in the tubes were returned to the same incubation conditions, and the start date for hatching of the eggs was recorded. Seven days after hatching commenced, the number of viable larvae, dead larvae, and unhatched eggs were counted in each tube.The study of bacterial burdens by stage showed that overall densities of the p = 0.004).One of the 10 control ticks started oviposition before day 10 but died before its oviposition was complete, and this tick was excluded from subsequent analysis. Coxiella sp. and the Rickettsia sp. in the females after oviposition. In the control group the ratio of the Coxiella sp. fusA gene to the MIF gene was approximately 10-fold higher than in flat female adults , but there was an association between the fitness index with the fusA/MIF ratio  in the post-oviposition female. By the same measure, there was no association between the pre-treatment weight of the female and the fitness index . We also examined the relationship between the ratio of live larvae to dead larvae plus unhatched eggs to estimated burdens of the Coxiella sp. and the Rickettsia sp. by Spearman rank correlation. There was an association  between a higher viable larvae ratio and lower Coxiella sp. burdens but not between the viable larvae ratio and Rickettsia sp. burdens .We used a binary fitness index where “0” was assigned to a tick with no eggs or a ratio live larvae to dead larvae plus unhatched eggs ≤ 1.0, and “1” was assigned to a tick with a ratio of live to dead larvae plus unhatched eggs of>1.0. Using logistic regression, there was no association between the fitness index and the Coxiella sp. in the female after oviposition. The mean fusA to MIF gene ratio was 2.8 (2.2–3.5) for the control group and 1.5 (1.3–1.7) in the rifampin-treated group (t test p = 0.0001). The mean ratio (95% CI) of live larvae to dead larvae plus unhatched eggs was 2.5 (1.5–4.4) for the control group and 0.25 (0.07–0.88) for the rifampin-treated group (p = 0.005). Thus, the lowered reproductive fitness observed again in this second experiment could be not be attributed to the DMSO.Although the final concentration of the intial diluent, dimethylsulfoxide (DMSO), in the rifampin solution for injection was only 10% (v/v) and less than 0.08 µl DMSO was injected, it was possible that this substance could account wholly or in part for the observed effects of rifampin. Therefore, we carried out another experiment in which engorged, mated females were injected with 1 µl PBS/10% DMSO (n = 12) or 1 µl PBS/10% DMSO with 10 µg rifampin (n = 14). A. americanum ticks Coxiella sp. was highly prevalent in all sampled ticks and then documented that it was present in all stages, reaching its highest densities in older nymphs and in adults, as assessed by species-specific quantitative PCR of whole tick extracts with a tick gene as an internal standard. In contrast, the Rickettsia sp. was less prevalent among the ticks, and, when present, in significantly lower densities than the Coxiella sp. While we cannot conclusively identify the Rickettsia sp. in this study, it is probably synonymous to what has been named “Rickettsia amblyommi” Rickettsia sp. in our study was identical to the gltA sequence of “R. amblyommi” from Amblyomma spp. ticks in Brazil and Argentina (AY375163 and DQ517290), as well as the gltA sequences of two unnamed Rickettsia sp. (AF031496 and AY3888929) from A. americanum. Moreover, Mixson et al. reported that “R. amblyommi” was transovarially transmitted and had a prevalence in A. americanum that was similar to what we found in our present and previous studies In this series of experiments we further characterized two bacterial species that occur in both laboratory-reared and field-collected Coxiella and Rickettsia species was present in A. americanum and vertically transmitted. But if a third (or fourth) species was present, unlike the Coxiella and Rickettsia species, it was not represented in a large cDNA library of A. americanum obtained from the same tick rearing facility Dermacentor, have a Francisella-like endosymbiont, but these endosymbionts have not been found in Amblyomma species Borrelia lonestari, which has a prevalence of about 1–2% across the south-central and southeastern regions of the U.S. B. lonestari organisms were originally present in one or more of the introduced females in 2004, they likely would have been lost or further diluted during subsequent generations in the laboratory facility during the ensuing year. In any case, Borrelia spp. are resistant to rifampin Ehrlichia pathogens may have been present in a few of the field-collected ticks that were introduced into the colony in 2004. But E. chaffeensis is not transmitted transovarially in A. americanumIt is possible another bacterial species besides the Coxiella and Rickettsia species, we proceeded to what, to our knowledge, is the first attempt at manipulation of endosymbiont numbers in ticks. For this aim we administered single injections of either of two antibiotics or buffer alone to engorged nymphs or engorged, mated females. Ticks that received tetracycline or rifampin as nymphs generally weighed less as adults than ticks that had received buffer alone. This effect was associated with lower numbers of both the Coxiella sp. and the Rickettsia sp. in post-molt ticks. When examined with estimators of fitness, treatment of engorged females with either of these antibiotics was associated with prolonged time to oviposition, lower proportions of ticks that hatched, lower proportions of viable larvae among total larvae that hatched, and lower numbers of viable larvae per tick.From the descriptive study of the Rhodnius prolixus from acquiring its bacterial symbiont, Rhodococcus rhodnii, and demonstrated lower fecundity in the symbiont-free insects Wigglesworthia glossinidiaBlochmannia floridanusAn association between symbionts and reproductive success has also been noted in other arthropods. An early demonstration was Harington's experiments in which he prevented the triatomine Coxiella burnetii in tissue culture cells at minimum inhibitory concentrations (MIC) of 1 to 4 µg/ml Rickettsia species in similar concentrations Wolbachia pipientis of mosquitoes and growing in tissue culture, tetracycline and rifampin (MIC 0.125 µg/ml) were each more effective than fluoroquinolones, which had MICs of 1–4 µg/ml, or macrolides, which had MICs of 8–64 µg/ml For this study we used two antibiotics: tetracycline and rifampin. Both antibiotics inhibit the growth of Coxiella sp. burdens were not lower than those of the control group following oviposition.Both antibiotics inhibit protein synthesis, either by interference with DNA-dependent RNA polymerase in the case of rifampin or at the level of translation by tetracycline. For many bacteria these antibiotics are bacteriostatic rather than bactericidal. Therefore, the antibiotics may have affected endosymbiont function without necessarily reducing the number of bacteria. The results in the second experiment with adult females and summarized Rickettsia sp. to the effects observed in this study, the evidence to date indicates a greater or exclusive role of the Coxiella sp. The absence of detectable Rickettsia sp. DNA in ∼30% of A. americanum in field-collected samples as well as in a laboratory colony of ticks suggests that the Rickettsia sp. is not essential for tick viability or reproduction. When the Rickettsia sp. was also present in ticks, along with the Coxiella sp., it was in lower densities than the Coxiella sp. and declined in numbers as adults aged. Although both the Coxiella sp. and Rickettsia sp. had lower numbers in antibiotic treated ticks, a direct correlation between bacterial densities and measures of reproductive fitness were observed for the Coxiella sp. but not the Rickettsia sp.While we cannot rule out a contribution of the Acyrthosiphon psium), an endosymbiont of the aphid, and the hymenopteran parasitoid, Aphidius ervi, a major natural enemy of aphids A. americanum and the Coxiella sp., is based on nutritient provisioning by the endosymbiont Herpetomonas roitmaniSince the experiment was carried out in the laboratory and with ticks from a long-established breeding colony, we cannot ascribe the effects of antibiotics on fitness to increasing the vulnerability of endosymbiont-free ticks to parasites, such was the case with pea aphids  the bacteria are transmitted to humans by the adult stage of that tick, (b) the nymphs' vertebrate hosts are reservoirs of the zoonosis, and (c) the bacteria are susceptible to that antibiotic. For example, an oral antibiotic that was administered in bait to a rodent infected with a given pathogen and at the time of nymphal ticks' greatest feeding activity could possibly result in the curing of the rodent of the infection, prevention of transmission of the pathogen to the larval tick, and fewer ticks in the next generation in that environment.


Twenty-two children and adolescents who had received cisplatinum for the treatment of solid tumours underwent audiometry to ascertain the extent of hearing damage. Five patients complained of hearing difficulties, causing difficulty at school in one child. Hearing loss greater than 20 decibels occurred in four patients at 1,000 Hz, seven at 2,000 Hz, 13 at 4,000 Hz and 21 at 8,000 Hz. Median hearing loss was greater at higher frequencies (P less than 0.0001), and with increasing cumulative dose of cisplatinum. However, a 'plateau' phenomenon was observed, with no apparent further deterioration in hearing loss at doses greater than 600 mg m-2. Two children who had received prior aural radiotherapy had severe hearing loss. Severe, mostly asymptomatic, ototoxicity is common in children given cisplatinum. However, there is considerable interpatient variability in the hearing loss suffered.


While hearing loss in HIV-infected people after beginning nucleoside reverse transcriptase inhibitors (NRTIs) has been reported, there have been no prospective studies that measured hearing changes longitudinally in treatment-naïve HIV-infected subjects following initiation of regimens containing NRTIs. The goal of this study was to conduct a prospective assessment of the contribution of zidovudine (ZDV) and didanosine (ddI) to hearing lossA prospective observational pilot study to determine whether ZDV or ddI, alone or in combination, are associated with sensorineural hearing loss in HIV-infected persons. Changes in hearing levels at all frequencies and in low and high frequency pure tone averages were measured at baseline, 16, and 32 weeks after initiating antiretroviral therapy.Treatment with ZDV and ddI did not result in loss of hearing, even after taking into account noise exposure, immune status and age. The results of this prospective pilot study do not support the notion that treatment with nucleoside antiretrovirals damages hearing. Cross-sectional studies and case reports show that hearing loss may be common among HIV-infected people -3. HeariWhile hearing loss in HIV-infected people after beginning nucleoside reverse transcriptase inhibitors (NRTIs) has been reported , there hThirty three antiretroviral-naïve subjects with their most recent peripheral blood CD4+ T-cells > 200/ul performed within the past 3 months were prospectively enrolled in this study between January 1996 and December 1999. Subjects were recruited from one research and one outpatient medical clinic for persons with HIV-1.  All subjects initiated therapy with regimens containing either ZDV or ddI, and although use of only one of these agents was preferred, use of both was allowed. People with prior hearing loss requiring hearing aids, and those with active substance abuse that would interfere with their participation in the trial were excluded. All subjects signed an Institutional Review Board-approved consent prior to participation in this study.Subjects underwent audiometry prior to initiating antiretroviral therapy (entry) and at weeks 16 and 32. At each visit, plasma HIV RNA and CD4+ T-cells were measured.All audiometric testing was completed by a certified audiologist or under their direct supervision. Following otoscopic inspection, tympanometry screening was performed to rule out significant middle ear pathology. Audiometric testing was completed with the use of a clinical diagnostic audiometer  in a sound-treated test booth. Air conduction thresholds were obtained for each ear at 250, 500, 1000, 2000, 3000, 4000, 6000, 8000 and 12000 hertz (Hz). Pure tone bone conduction testing was administered on those persons demonstrating hearing loss by air conduction. Hearing levels were measured at 250, 500, 1000, 2000, 3000, 4000, 6000, 8000 and 12000 Hz by formal audiometry. "Hearing Level" was defined as the intensity of the sound (in dB – decibels) needed to reach threshold (both for air conduction and bone conduction). Normal hearing is generally considered to be 0–25 dB hearing level [10 transformed for analysis.Plasma HIV-1 RNA was measured using the Roche UltraSensitive PCR Amplicor HIV-1 Monitor test . Values below the limit of detection (50 c/ml) were imputed to be 25. All viral loads were logDue to the lack of a standard definition of ototoxicity, we evaluated changes in hearing using several methods . AssociaThe demographics of the 33 subjects included in the analysis, shown in Table At study entry, 22 of 33 subjects had a hearing level greater than 25 dB at one or more frequencies in one or both ears, 16 had at least one hearing level greater than 40 dB and 7 had at least one hearing level greater than 60 dB. Most of the decreased hearing sensitivity was at higher frequencies. No one had a hearing level above 40 dB in both ears at 2000 Hz or lower, whereas 9 subjects had a hearing level above 40 dB in both ears at 12000 Hz, 2 subjects had a hearing level above 40 dB in both ears at 6000 and 8000 Hz, and 3 subjects had a hearing level above 40 dB in both ears at 3000 and 4000 Hz. Only 4 subjects reported a history of occupational sound exposure, and 12 subjects reported a history of recreational sound exposure. Tinnitus was reported at entry in 14/33 (42%) subjects.Low and high frequency PTA at baseline were not significantly associated with baseline viral load or baseline CD4+ T-cell count. Table Pneumocystis jiroveci pneumonia and one with previous esophageal candidiasis). Their mean low frequency PTA was the same as the subjects without an AIDS diagnosis (11 dB). The mean high frequency PTA for subjects with an AIDS diagnosis (28 dB) was higher than subjects without an AIDS diagnosis (20 dB), but the difference was not significant (p = 0.40).Two subjects had a history of an AIDS-defining illness , but peripheral blood CD4+ T-cell count at entry did not differ between the two groups (p = 0.94).Twenty-two of the 33 subjects returned at 16 weeks and nineteen returned at 32 weeks. Four patients had a 16-week visit and no 32-week visit and one patient had a 32-week visit and no 16-week visit. Subjects with follow up had a trend toward worse hearing at baseline, compared to baseline hearing in subjects who did not follow up, but these differences were not significant. Specifically, the baseline low frequency PTA in those with follow-up was 5.1 dB higher than in those without follow-up (p = .19) and the baseline high frequency PTA in those with follow-up was 6.5 dB higher than in those without follow-up (p = .22). The difference in baseline high and low frequency PTAs between those with and without a 16-week visit is similar to the difference between those with and without a 32-week visit, and both are similar to the difference between those with any follow up and those with no follow up. Those with follow up had lower plasma HIV RNA concentration at entry . Similarly, the change in PTAs did not differ between the two groups (data not shown). While there was a trend to decreased tinnitus reported at week 16,  and week 32 , compared to 42% at baseline, these changes did not reach statistical significance. .We conducted this longitudinal study to evaluate changes in hearing following the initiation of therapy with ZDV, ddI, or both drugs in antiretroviral therapy (ART)-naïve subjects. There were no significant changes in hearing at 16 and 32 weeks, even taking into account noise exposure, CD4+ T-cell count and plasma viral load. Our previous cross-sectional study, which was larger (99 versus 24 subjects), showed an interaction between antiretroviral use and age [Prior cross-sectional studies and case reports have shown an association between hearing loss and NRTI therapy -3. The rThere have been two case reports of hearing loss in subjects receiving ART regimens that included NRTIs and a second class of antiretrovirals; one with a non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine) and one with a protease inhibitor (PI) (lopinavir/ritonavir) each combined with NRTIs, . One case reported sudden hearing loss two weeks after the person completed one month of post-exposure prophylaxis which resulted in long-term hearing loss . The othOur finding of little impact on hearing after beginning ZDV or ddI is reassuring and suggests that, if such agents do cause hearing loss, it is uncommon. Alternatively, baseline hearing impairment due to HIV itself may improve with improved immune status as a result of antiretroviral treatment, masking any potential ototoxicity. This hypothesis could be tested in a future study by comparing hearing changes in subjects who do and do not experience improved immune status after beginning NRTIs.ART- antiretroviral therapydB- decibelsddI- didanosineGEE- generalized estimating equationsHL- hearing levelHz- hertzNNRTI- non-nucleoside reverse transcriptase inhibitorNRTI- nucleoside reverse transcriptase inhibitorPI- protease inhibitorPTA- pure tone averageRNA- ribonucleic acidZDV- zidovudineThe author(s) declare they have no competing interests.JTS, DWL, TSR, ACC, and CMM have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; have been involved in drafting the manuscript or revising it critically for important intellectual content; and have given final approval of the version to be published. The principal investigator, CMM, and JTS had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.The funding agencies were not in control of the design and conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval of the manuscript.The pre-publication history for this paper can be accessed here:


The first chiropractic adjustment given in 1895 was reported to have cured deafness. This study examined the effects of a single, initial chiropractic visit on the central nervous system by documenting clinical changes of audiometry in patients after chiropractic care.Fifteen patients are presented  with a mean age of 54.3 (range 34–71). A Welch Allyn AudioScope 3 was used to screen frequencies of 1000, 2000, 4000 and 500 Hz respectively at three standard decibel levels 20 decibels (dB), 25 dB and 40 dB, respectively, before and immediately after the first chiropractic intervention. Several criteria were used to determine hearing impairment. Ventry & Weinstein criteria of missing one or more tones in either ear at 40 dB and Speech-frequency criteria of missing one or more tones in either ear at 25 dB.All patients were classified as hearing impaired though greater on the right. At 40 dB using the Ventry & Weinstein criteria, 6 had hearing restored, 7 improved and 2 had no change. At 25 dB using the Speech-frequency criteria, none were restored, 11 improved, 4 had no change and 3 missed a tone.A percentage of patients presenting to the chiropractor have a mild to moderate hearing loss, most notably in the right ear. The clinical progress documented in this report suggests that manipulation delivered to the neuromusculoskeletal system may create central plastic changes in the auditory system. The broad category of hearing loss is the third most prevalent chronic condition in older Americans, following hypertension and arthritis . BetweenThe diminished ability to hear and to communicate is frustrating in and of itself, but the strong association hearing loss with depression and functional decline adds further to the burden on older individuals . The onsChiropractic has long been associated with hearing. The first chiropractic adjustment given in 1895 was reported to have cured deafness. Wagner and Fend , from GeHulse , also frSvatko , from RuThis current study examined hearing impairment in a chiropractic patient population and the effects of a single, initial chiropractic visit on changes in audiometry in these patients after chiropractic care.A sample of convenience consisting of fifteen consenting patients  with a mean age of 54 years range 34–71), obtained from a panel of 200 patients presenting for chiropractic care in Vicenza, Italy during one year (June 2000 to June 2001) was the basis for this case series study. As seen in the patient characteristics in Table 1, obtainExamination and palpatory findings were used to define areas of joint dysfunction and each patient received a high velocity, low amplitude thrust in the thoracic, lumbar spine and locomotor system including extremities. No "specific" adjustment was given to solely restore hearing.In the patient group with hearing impairment, the total number of tones heard on initial exam was fewer in the right ear (55 tones) than the left (83 tones). The normal patient group heard approximately 120 tones in each ear on the initial visit. After a single chiropractic intervention, the total tones heard increased to 104 on the right and 111 on the left (an increase of 49 and 28 respectively)  presumes that changes in one sensory modality may create a convergence upon other areas of the cortex that integrate that information into a polysensory event. Some authors have pointed to the site of this neuronal plasticity as characteristic of the non-primary auditory thalamus and cortex . CorticaRecent electrophysiological evidence has changed the traditional view that language and memory being primarily in the cortex to focus on the role of subcortical structures . Loss ofIn a study of 500 participants, Carrick  examinedThe changes in a persons' ability to hear tones at speech threshold would fall under the classification of central adaptive changes or plasticity. There is no doubt that central plastic changes occur in the brainstem, specifically at the level of the vestibular nerve. Central plastic changes and recovery in vestibular nuclei adapt so rapidly that complete unilateral labyrinthectomy (complete damage to one labyrinth) should create extreme vertigo and imbalance. However, patients can become asymptomatic in less than two weeks . SpontanAnother possible site for confluence of somatic and acoustic input is the vestibulo-cochlear system within the brain stem. Unilateral hearing loss is frequently noted in persons with vertigo -30. In fA percentage of patients seeking chiropractic care have a mild to moderate hearing loss, identified by audiometry. In accordance with other reports, the clinical progress documented here suggests chiropractic care may benefit hearing loss and that chiropractic adjustments to various areas of the spinal column and locomotor system may have an effect on central auditory processing, though alternative explanations can not be disregarded. There is a difference in the unilateral aspect of the hearing deficit noted in the right ear of patients in this current study as reported in others. The observations documented in this case series provide limited support to previous works indicating that, when hearing is tested immediately after a single chiropractic adjusting visit, hearing may be improved in both ears. Further research in this area is required, in the form of a well designed randomised controlled trial.The author, Joseph O Di Duro declares no competing interests, financial or non-financial.


The neurophysiological mechanisms underlying tinnitus perception are not well understood. Surprisingly, there have been no group studies comparing abnormalities in ongoing, spontaneous neuronal activity in individuals with and without tinnitus perception.n = 17) is characterised by a marked reduction in alpha (8–12 Hz) power together with an enhancement in delta (1.5–4 Hz) as compared to a normal hearing control group (n = 16). This pattern was especially pronounced for temporal regions. Moreover, correlations with tinnitus-related distress revealed strong associations with this abnormal spontaneous activity pattern, particularly in right temporal and left frontal areas. Overall, effects were stronger for the alpha than for the delta frequency band. A data stream of 5 min, recorded with a whole-head neuromagnetometer under a resting condition, was sufficient to extract the marked differences.Here, we show that the spontaneous neuronal activity of a group of individuals with tinnitus (Despite some limitations, there are arguments that the regional pattern of abnormal spontaneous activity we found could reflect a tinnitus-related cortical network. This finding, which suggests that a neurofeedback approach could reduce the adverse effects of this disturbing condition, could have important implications for the treatment of tinnitus. Regional patterns of abnormal brain activity identified in people with tinnitus suggest the presence of a tinnitus- related cortical network that it may be possible to modify by neurofeedback. Tinnitus is characterised by the perception of sounds  in the absence of any objective physical sound source. For the affected individual, this condition often causes a considerable amount of distress. Although it is now widely accepted that the generation of tinnitus has a central basis –3, the nSurprisingly, even though spontaneous activity has been a frequent research target in animal models of tinnitus, studies in humans have been rare. Data from a group study for abnormal activity of the cochlear nerve were reported by Martin . An abnoThe generation of tinnitus, in most cases, can be linked to damage to the auditory system, usually to receptors of the inner ear –3, probaThe data recorded by MEG represent the summed activity of tangential sources on a two-dimensional (sensor space) level. By applying various inverse solution strategies , it is pSeventeen individuals with chronic tinnitus —16 with high-frequency hearing loss and one with deafness due to transection of the hearing nerve—and 16 normal hearing control individuals  participated in the study. Five minutes of resting MEG were measured and analysed with regard to the spectral content  to test for the presence of main and interaction effects (group × frequency band) in the overall average for each frequency. Since the result of this analysis was significant, we executed this analysis for specified regions (see above) separately. Associations with tinnitus-related distress were tested using product-moment correlations. In a first step, the average over all dipoles for each frequency band was correlated with the tinnitus questionnaire. As this analysis yielded significant associations that were specific and consistent in direction for the alpha and delta band, values from each region were correlated with the scales of the questionnaire in order to help interpret effects gained in the first step.F2,62 = 4.47, p < 0.02, power 0.78). Differences between the values of each frequency range  were calculated for each individual, and the results were subjected to a between-subjects analysis of variance. This analysis shows that the control group exhibited a significantly higher alpha than theta power , whereas the tinnitus group has stronger delta activity than alpha activity . No differences were found between the groups when theta was compared with delta . These results statistically confirm the impression of a reduced alpha and enhanced delta peak in the tinnitus group. This interaction pattern was statistically significant for all regions, with the exception of right frontal and anterior parietal regions of both hemispheres. The foci of the altered spontaneous activity pattern in the tinnitus group are clearly found bilaterally in temporal areas , which can be seen in The explorative analysis of the frequency spectrum of the recorded magnetic fields revealed characteristic changes in participants with tinnitus from the usual pattern seen in normal hearing controls . The alpr ranging between 0.5 and 0.7 . Amount and slope of hearing loss were also not significantly correlated with neuromagnetic data (0.12 < r < 0.25).For both alpha and delta power, significant correlations were found with the Tinnitus Questionnaire , with  and 0.7 . These cTo our knowledge, this is the first group study on tinnitus in humans  to sHowever, we can not exclude with certainty the possibility that the effects are not specific to tinnitus, a limitation of our study. One interesting finding is that compared to the normal hearing control group, the tinnitus group also had a high-frequency hearing loss. So, theoretically, the ideal control group would have exactly the same type of hearing loss without a tinnitus sensation. However, to find such a group of an appropriate sample size would be very difficult as both phenomena are strongly associated. For example, in an attempt to investigate the influence of high-frequency hearing loss (similar to that of our study) on cortical reorganisation, Dietrich et al.  noticed The association of our neurophysiologic data with distress suggests that the right temporal and left frontal cortex might be involved in a tinnitus-related cortical network, in which the temporal region is associated more with perceptual issues , and the left frontal region more with affective distress and motivational attention of tinnitus . Without reference to lateralisation, Jastreboff describes the prefrontal cortex as a “candidate for the integration of sensory and emotional aspects of tinnitus” . Our datIn future studies, we hope to elucidate the function of the (left) frontal area, as it may point us to the role of top-down influences that presumably play a role in the perception and perhaps even generation of tinnitus. An important aspect of these results is that they potentially have clinical implications, which are currently being tested by our group. Reducing the abnormal spontaneous activity pattern reported in this study via neurofeedback might cause concomitant reductions in tinnitus distress and/or intensity . BesideFive minutes of MEG under a resting condition was recorded  using a 148-channel neuromagnetometer . The participant was requested to keep eyes open and to maintain gaze on a fixation mark at the ceiling of the recording chamber. Eye movements were monitored with four electrodes attached to the left and right outer canthi and above and below the right eye.In the first step of data analysis, sampling points were reduced by a factor of ten. After noise reduction, eye-movement correction was undertaken using the algorithm proposed by Berg and Scherg . As an eThe second step consisted of the investigation of the underlying source activity via application of the minimum norm estimate ,54 to thTinnitus—hearing sounds such as hissing or roaring that are not really present—is a common condition and can be very distressing. Little is know about why tinnitus starts. Though people who have it often have some hearing loss, the hearing loss does not cause the tinnitus. There are few good treatments for tinnitus.They measured spontaneous activity in the brains of 17 people with tinnitus and 16 without. The found that one type of brain activity—alpha—was decreased and another—delta—was increased in people with tinnitus, particularly in one part of the brain, the temporal region, where the brain processes sounds. The changes were particularly pronounced in people who were most distressed by the tinnitus.It seems that tinnitus is associated with one type of abnormal spontaneous activity in the brain. One way of treating tinnitus therefore might be to try to alter the activity by providing feedback about the ongoing brain activity and training the individual to interfere with this activity, i.e., by attempting to enhance alpha activity and reduce delta activity.http://www.nlm.nih.gov/medlineplus/tinnitus.htmlMedlinePlus has a Web page on tinnitus: http://omni.ac.uk/browse/mesh/D014012.htmlThe Welcome Gateways has a selection of Web sites on tinnitus: http://www.ata.orgThe American Tinnitus Association also offers information on tinnitus:


Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A+/+) and SP-A-deficient (SP-A−/−) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680  Pseudomonas aeruginosa is resistant to SP-A-mediated membrane disruption.Everyday, normal breathing deposits numerous microorganisms on the surfactant membrane that lines the air-exchanging surfaces of the lung. Surfactant protein SP-A, a component of the surfactant membrane, helps to maintain the lung in a germ-free state by aggregating inhaled microorganisms and facilitating their ingestion by immune cells, and by increasing the permeability of their cell membranes. However, the bacterial pathogen P. aeruginosa genes, pch and ptsP, that are required to resist SP-A-mediated membrane permeabilization. Molecular analyses indicate that the pch gene encodes an enzyme that synthesizes salicylate, a compound utilized by bacteria to acquire essential metal ions. The ptsP gene encodes an enzyme called phosphoenolpyruvate-protein-phosphotransferase. The loss of salicylate and phosphoenolpyruvate-protein-phosphotransferase weakens the P. aeruginosa cell membrane, which allows SP-A to poke holes on the membrane and kill the bacteria.Using a genetic tool called comparative signature-tagged mutagenesis, the authors have identified two This is the first known report of the roles played by salicylate and phosphoenolpyruvate-protein-phosphotransferase in maintenance of bacterial membrane, and consequently, protecting bacteria from killing by SP-A, through disruption of membrane integrity. Pulmonary surfactant is a protein-phospholipid complex that lines the air-liquid interface of alveoli, forming the first point of contact with inhaled microbial pathogens ,2. A mulPseudomonas factors that are specifically required to protect P. aeruginosa from killing by SP-A were identified and characterized.Although the collectins inhibit microbial growth, the factors that govern the specificity and the mechanisms of membrane disruption by the collectins are not understood. In this study, we employed comparative signature-tagged mutagenesis (STM) as a tool to identify bacterial factors required to resist SP-A-mediated clearance, in vivo. STM relies on the ability of the pathogen in question to replicate in vivo as a mixed population and allows for the identification of the mutants whose phenotypes cannot be trans-complemented by other virulent strains present in the same inoculum . Two Pse−/− mice exhibit delayed clearance of a clinical P. aeruginosa strain from the lung, suggesting that SP-A plays a role in pulmonary innate immunity against this organism. We also found that the inbred C3H/HeN SP-A−/− mice were more susceptible to P. aeruginosa infection, in this case by strain PA01. SP-A+/+ mice cleared 63-fold (1.8 log) more of intranasally inoculated PA01 by 16 h . When infected with PA01 however, SP-A+/+ mice developed broncho-pneumonia with mild pulmonary infiltrates  agar plates were harvested from each lung for bacterial genomic DNA extractions and oligo-tag amplification. We screened for differential clearance of individual STM mutants using PCR. If the PCR product of a mutated gene was absent on the output gel in SP-A+/+ mice, but present in SP-A−/− mice  were recovered, for a 0.12% recovery rate. We have cloned and sequenced both STM mutants by plasmid rescue [http://www.pseudomonas.com). The output from Pool 4  is shown in pch mutant, which is disrupted in a gene required for salicylate biosynthesis, was unable to survive in the SP-A+/+ lung . The results were confirmed by PCR analysis of the output pool of two additional mice  gene  assays. TLC analysis of pyochelin extracted from bacteria cultured in M63 minimal medium confirmed that the pch mutant is unable to synthesize the green-colored pyochelin (pch lane). In contrast, the parental wild-type PA01 produced pyochelin . The expression of the genes within the downstream operon (PA4232 and PA4233), which is transcribed in opposite orientation to the pchADCBA operon, was found to be unaffected by transposon insertion as assessed by mRNA expression analyses (unpublished data). Collectively, these data suggest that susceptibility to SP-A-mediated clearance is caused by the loss of pch gene function, most probably by affecting the mRNA stability of the pchDCBA operon.The 31) gene C. The pcyochelin –14. Beca5Km2 inserted in between the 2,213th and 2,214th nucleotides of the ptsP (PA0337) gene  [5Km2 insertion into ptsP is predicted to cause polarity and interrupt the transcription of PA0338.The second SP-A-susceptible STM mutant had the transposon pUTminiTn37) gene C. The pt (EINtr) . The tra5Km2 insertion after hybridization with the transposon vector (unpublished data). DNA fragments that flanked both ends of the transposon integrations in pchA and ptsP genes were cloned from the wild-type PA01. They were used to confirm gene interruption by the integrating transposon, by comparing restriction fragment length polymorphisms (RFLP) between wild-type PA01 and the STM mutants. As shown in pch and ptsP mutants than their parental strain PA01, indicating transposon insertion.Both STM mutants were judged to harbor a single transposon pUTminiTn+/+ mice. Specifically, mutant strains pch and ptsP were only 53% and 46% as competitive as PA01, respectively. In contrast, both mutants performed equally well as, if not better than, the wild-type strain in SP-A−/− mice  and 25-fold (1.4 log) higher, respectively, in SP-A−/− mice than in SP-A+/+ mice -expressing bacteria were exposed to mouse alveolar macrophages in the presence of SP-A, the uptake of PA01, its pch, or ptsP mutant was increased by 1.8, 1.3, and 1.6 fold, respectively, over that which occurred in the absence of SP-A ((p<0.01). The magnitude of the increase in the uptake of PA01 in the presence of SP-A was similar to previously published reports [pch and ptsP mutants from SP-A+/+ mice.Previous studies have suggested that SP-A opsonizes rophages ,18. To d of SP-A A. Howeve reports ,18. Althpch and ptsP. As shown in pch and ptsP mutants was not increased in the presence of SP-A. In fact, SP-A-opsonized PA01 bacteria were phagocytosed less efficiently by neutrophils than untreated bacteria. Interestingly, only wild-type PA01 bacteria were susceptible to killing by neutrophils, with approximately 73.7% killed in the absence of SP-A, and 57.2% killed in the presence of SP-A . In addition, in contrast to the robust SP-A-induced aggregation of E. coli K12 , or the inability to synthesize salicylate and phosphoenolpyruvate-protein-phosphotransferase, renders the cells vulnerable to membrane permeabilization and killing by SP-A. Furthermore, bacterial aggregation does not play a role in preferential clearance of the pch and ptsP mutants.We next determined if SP-A mediated permeability kills STM mutants. Live/dead staining, based on exclusion of propidium iodide from the live cells, was performed on bacterial cells following membrane permeability assays. Viable cells are stained green while dead cells are stained red. As shown in e killed B within coli K12 A, SP-A dpch mutant is disrupted in a gene required for salicylate biosynthesis, we performed complementation analysis by growing the pch mutant bacteria in the presence of 1 mM sodium salicylate. As shown in pch mutant bacteria grown in LB were susceptible to SP-A-mediated membrane permeabilization. In contrast, the pch mutant bacteria grown in LB supplemented with 1 mM sodium salicylate were fully resistant to membrane permeabilization by SP-A. We further examined whether the increased susceptibility to SP-A-mediated membrane permeability was due to the inability pch bacteria to synthesize the siderophore pyochelin. The pch bacteria cultured in pyochelin-supplemented LB or iron-deficient minimal medium were still membrane permeabilized by SP-A (unpublished data). Collectively, these results suggest that the increased susceptibility to SP-A-mediated membrane permeability in the pch bacteria is due to their inability to synthesize salicylate, or both salicylate and pyochelin. However, we cannot completely rule out the possibility that pyochelin may be involved in resistance to SP-A-mediated membrane permeability until we have exhausted all the growth conditions and optimal pyochelin concentrations for the assays.The susceptibility of STM mutants to SP-A mediated membrane permeabilization can be exploited for complementation analyses. As the , we also tested ptsP mutant bacteria cultured in the presence or absence of 1 mM sodium salicylate. The ptsP mutant bacteria were permeabilized to the same extent, regardless of whether they were grown in the presence or absence of 1 mM sodium salicylate  did not restore the resistance of ptsP STM mutant to SP-A, nor did it alter the resistance or susceptibility of the parental wild-type PA01 (PA01-gfp) to permeabilization by SP-A . However, the two major LPS core bands of the ptsP mutant showed a reversal in relative abundance compared with the PA01 (unpublished data). Thus, there may be changes in LPS, at least in terms of the proportion of two different cores made. More detailed molecular analyses will be required to determine if LPS modification occurs within pch and ptsP mutants.Previous studies in E. coli  and Bordertussis  have demThe genome-wide mutagenesis scheme called STM was originally developed to identify novel vaccine and antibiotic targets . STM relP. aeruginosa strain PA01 to confer resistance to SP-A. Both mutants were susceptible to SP-A-mediated membrane permeabilization, confirming that disruption of membrane integrity is one of the mechanisms by which SP-A mediates bacterial clearance. The degree of bacterial membrane permeabilization of the STM mutants by SP-A was comparable to that of E. coli K12, which has rough LPS [pch and ptsP mutants. However, we cannot exclude the possibility that phagocytosis contributes to the differential clearance of these two mutants in vivo.In this study, we have employed STM technology to identify bacterial factors that are required for ough LPS . Based ohttp://www.pseudomonas.com). Additional screenings are being carried out to catalog other P. aeruginosa factors conferring resistance to SP-A.Thus far, we have not recovered STM mutants that are susceptible to SP-A-mediated opsonization, or mutants that are disrupted in the genes required for LPS biosynthesis, which play critical roles in resistance to SP-A-mediated membrane permeability. The lack of recovery of opsonization-sensitive and LPS mutants are not particularly surprising, considering that we screened only approximately 30% of the 5,570 mutants required for 1 × genome coverage  and calcium [P. aeruginosa requires salicylate and its downstream product pyochelin to acquire cations. Divalent cations, especially Mg2+, are required to structurally stabilize LPS on the outer membrane. The recently resolved structure of SP-A protein indicates that there are two metal binding domains within SP-A, which are most likely occupied by calcium [pch mutant. Furthermore, inability to acquire cations may potentially alter the expression, stability, and modification of various transmembrane proteins and glycolipids by enzymes that require cations as cofactors. Thus, we speculate that the pch mutation may have contributed to its increased sensitivity to SP-A-mediated membrane permeabilization through direct or indirect effects on LPS integrity and membrane stability.The mechanisms by which mutations in yochelin –14. In aerophore . Salicyl calcium . Thus, i calcium . Thus, SptsP gene encodes phosphoenolpyruvate-protein phosphotransferase EINtr [rfaH gene, which is required for increased production of the O-antigen of LPS in Salmonella enterica serovar Typhi during the stationary phase of growth [PtsP also coordinates carbon and nitrogen metabolism and is required for virulence in P. aeruginosa, by an unknown mechanism [Azotobacter vinelandii ptsP ortholog affects lipidic poly-hydroxybutyrate accumulation within its cytoplasm as a carbon source for long-term starvation survival [E. coli complexed to calcium polyphosphate, and forms divalent cation-selective channels [ptsP mutants may be caused by its reduced ability to transport divalent cations necessary to maintain LPS and outer membrane integrity.The se EINtr , a transse EINtr . RpoN isf growth . The addechanism . Mutatiosurvival . Intereschannels . Thus, iP. aeruginosa PA01 and STM mutants is more robust in vivo than in vitro. One potential explanation for this discrepancy is that our in vitro membrane permeability conditions do not accurately model the environment in the lung. In addition, it is possible that multiple factors contribute to the killing of P. aeruginosa including SP-A, SP-D, lactoferrin, β-defensins, and other antimicrobial peptides and proteins [P. aeruginosa.Our results suggest that the killing of proteins ,30. We aP. aeruginosa proteases may also explain our failure to develop informative in vitro STM screens (unpublished data).Decreased SP-A levels have been found in several respiratory diseases including bacterial pneumonia, adult respiratory distress syndrome ,32, and P. aeruginosa is an emerging clinical problem that can lead to denial for lung transplantation and death [Pseudomonal peptides to combat life-threatening infections with this organism. The discovery of factors governing resistance or susceptibility to SP-A may ultimately have therapeutic value.Antibiotic-resistant nd death . Thus, tE. coli DH5-α [P. aeruginosa strain PA01 [E. coli or P. aeruginosa using the following concentrations: carbenicillin (50 or 300 μg/ml), kanamycin (50 or 250 μg/ml), and tetracycline (5 or 15–30 μg/ml).Shaking cultures of li DH5-α , SM17λpili DH5-α , and P. ain PA01  were gro−/− mice were developed as previously described [+/+) mice were purchased from Charles River Laboratory . All infections were performed intranasally with age-matched, 6-wk-old mice. Animals were housed in positively ventilated microisolator cages with automatic recirculating water, located in a room with laminar, high-efficiency, particle accumulation-filtered air. The animals received autoclaved food, water, and bedding. Mice used in experimental procedures were handled in accordance with protocols approved by the Institutional Animal Care and Use Committee at University of Cincinnati College of Medicine.The C3H/HeN SP-Aescribed ,7. C3H/HP. aeruginosa PA01 STM library and screening methods were previously described [+/+ mouse lungs infected with bacteria (1 × 107 cells) from a representative pool of STM library (unpublished data). We chose the 16 h time point to screen the STM library because it gave us the most reproducible PCR amplification of DNA tags from mutants, with ≥ 10,000 colony forming units (CFU) still recoverable from the SP-A+/+ mice (unpublished data). In contrast, prolonged infection (≥ 36 h) with 1 × 107 bacteria from STM pool frequently killed SP-A−/− mice (unpublished data). Briefly, SP-A+/+ and SP-A−/− mice [7 cells) of each individual STM pool. After 16 h, lungs were removed from sacrificed mice, and homogenized tissues were plated on LB agar plates. At least 10,000 bacterial colonies recovered after in vivo selection were used for multiplex PCR as described previously [The escribed . An adapescribed . In earlia 1 × 10 cells freviously . Twenty eviously  were als7 in 50 μl) composed of a 1:1 ratio of wild-type PA01 and its isogenic STM mutants. Infected lungs were recovered 16 h after infection for bacterial load determination. The competitive index (CI) is defined as the output ratio of mutant to wild-type bacteria divided by the input ratio of mutant to wild-type bacteria [+/+ and SP-A−/− mice (group of six). Attenuation was defined as the log10 difference in CFU between wild-type and mutant bacteria recovered from lung tissue 16 h after inoculation.For competition assays, mouse lungs were inoculated intranasally with bacterial cells . Complementation was assessed by resistance to SP-A-mediated membrane permeabilization.Non-polar, in-frame deletion of  pEX18Ap  to produ8 operon C was PCRd pUCP19  to obtaipch mutant was performed by culturing the bacteria into stationary phase in LB supplemented with 1 mM sodium salicylate.Complementation of the P. aeruginosa was analyzed using the method previously described [P. aeruginosa strains and incubated at 37 °C for 24 h with shaking. A fresh 30 ml of M63 supplemented with 0.5% w/v casamino acids was then inoculated with 0.3 ml of starter culture, and incubated for 36 h at 37 °C with shaking. The culture was then centrifuged at 13,000 rpm for 10 min in 1.5 ml aliquots in a microcentrifuge. The supernatants were removed into a 50 ml polypropylene screw-capped centrifuge tube, pooled, adjusted to (pH 2.0) with 1M HCl, using universal indicator paper to estimate pH, and filter sterilized through a 0.22 μM membrane. Pyochelin was extracted by the addition of 0.4 volumes of ethyl acetate, followed by vigorous vortexing. The two phases were separated by centrifugation at 2,000 rpm for 5 min in a bench top centrifuge, and the upper, organic phase was removed to a separate tube. Pyochelin extract was concentrated by rotary evaporation at 40 °C in 100 ml round bottomed flasks, and the residue was dissolved in 300 μl of methanol. Pyochelin extracts (10 μl) was separated by TLC , using acetone:methanol:0.2M acetic acid (5:2:1) as a development solvent. The chromatography tank was left to stand for several minutes with its lid on to allow the air inside to become saturated with solvent vapor. Pyochelin, which is fluorescent, naturally occurs as two stereoisomers. When visualized under a UV transilluminator, it is revealed as two green fluorescent bands. Pyochelin standard was generously provided by Dr. J. M. Meyer (Université Louis Pasteur).Pyochelin production in escribed . M63 minHuman SP-A (hSP-A) was purified from lung washings of patients with the lung disease alveolar proteinosis, as previously described . SP-A prP. aeruginosa cell wall integrity was assessed by determining permeability to a phosphatase activity substrate, Enzyme-Labeled Fluorescence 97  as described [8 bacterial cells/ml in 100 μl of 5 mM Tris, 150 mM NaCl for 15 min at 37 °C, and 100 μM ELF97 phosphatase substrate was added. Fluorescence was measured at excitation and emission wavelengths of 355 and 535 nm, respectively, for a period of 60–90 min.The effect of the SP-A on 5 from 3 SPA+/+ mice) were cultured on chamber plastic culture slides, coated with 5% poly-D-lysine with 200 μl RPMI  and used immediately. Macrophages were allowed to adhere for 2 h at 37 °C in 5% CO2. Live GFP-expressing PA01 or isogenic STM mutants in RPMI were opsonized with or without 25 μg/ml human SP-A for 1 h at 37 °C with rotation. The medium was removed from each well and replaced with 250 μl of opsonized PA01 or STM mutants, and incubated for 1 h at 37 °C in 5% CO2 at a ratio of 100 bacteria to 1 macrophage. Chamber slides were washed three times with PBS containing 1 mM CaCl2. Extracellular bacteria were quenched with crystal violet (0.8 mg/ml). Following two additional washes, cells were fixed with 1% paraformaldehyde in PBS plus 1 mM CaCl2 for 10 min, and stained with Evans blue for 2 min. The percentage of macrophages with engulfed bacteria was quantified under a phase contrast fluorescence microscope. At least 200 macrophages were counted. Human neutrophils were purified and cultured as previously described [Freshly lavaged mouse macrophages . Briefly, stationary phase E. coli or P. aeruginosa bacteria were washed three times with 5 mM Tris, 150 mM NaCl, and adjusted to an of OD600 = 1.0. A 100-μl aliquot of the cells was added to culture tubes containing 100 μl of SP-A (100 μg/ml) or 100 μl of 5 mM Tris, 150 mM NaCl, as a control. The mixtures were incubated at 37 °C, 300 rpm for 1 h, and stained according to the instructions provided by the supplier. The viability of the cells was checked under a fluorescence microscope. At least 1, 000 cells were counted.The viability of SP-A exposed bacteria was determined by LIVE/DEAD BaclightP. aeruginosa by acetone/phenol extraction [LPS was isolated from traction . LPS samt-test and one-way analyses of variance (ANOVA). A significant difference was considered to be p < 0.05.Statistical analysis was performed using the Student's


Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr/Thr 11 form of SP-D is associated with low serum levels and assembles predominantly as trimers as opposed to the more common multimeric forms of SP-D.Preliminary experiments were done to establish the effects of different monoclonal antibodies against SP-D on ability of SP-D to bind to or neutralize the virus. We then purified natural human trimeric and multimeric forms of SP-D from amniotic fluid and tested ability of these preparations to bind to IAV, to inhibit infectivity and hemagglutination activity of IAV in vitro.In initial experiments mAbs directed against different areas on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs) in viral neutralization assays as compared to multimeric SP-D.These studies indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP-D can be useful for dissecting out different functional properties of the protein. IAV infections are a major cause of morbidity and mortality, causing ~40,000 deaths per year in the United States . Recent SP-D has strong antiviral activity and mediates several other important functions some of which may be important during infection, including maintenance of surfactant homeostasis in the lung, clearance of apoptotic cells, enhancement or inhibition of uptake of various organisms by phagocytes, and inhibition of inflammatory reactions in the lung ,14-19. Sin vivo and in recombinant preparations predominantly as trimers, lacking the usual higher multimeric forms observed for other polymorphic forms of SP-D [SP-D is a collagenous lectin that assembles as a trimer with a globular carbohydrate recognition domain (CRD) and an extended collagenous domain. These trimers can further multimerize into assemblies of 4 or more trimers linked at the N-terminus by disulfide bonds . There a of SP-D . The Thr of SP-D  and to H of SP-D . BindingMycobacterium tuberculosis infection [Additional studies in other populations have found a similar frequency of the Thr/Thr 11 variant of SP-D  suggestinfection . Conversnfection . We have8 plaque forming units/ml.IAV was grown in the chorioallantoic fluid of ten day old chicken eggs and purified on a discontinuous sucrose gradient as previously described . The virRecombinant human SP-D (RhSP-D) was produced in stably transfected CHO-K1 cells as previously described . The recThe innate defense protein preparations used in this report were tested for degree of contamination with endotoxin using a quantitative endotoxin assay . The final concentrations of endotoxin in protein samples containing the highest concentrations of collectins were ~20–100 pg/ml . HNP-1 and HNP-2 were purchased from Bachem Bioscience . The levels of endotoxin in the HNP preparations were 15 and 25 pg/μg of protein, respectively for HNP-1 and 2.MAbs 246-04 and 246-07 were raised against SP-D by inoculating mice with 10 μg/ml of human SP-D as previously described . The 2452SO4). The optical density was measured on an ELISA plate reader at 450 nm wavelength. Each individual data point was performed in duplicate. In some experiments SP-D was pre-incubated with mAbs  for 3 hrs. These IAV-coated plates were then incubated with SP-D preparations, followed by addition of the 246-04 monoclonal antibody (mAb) against SP-D as indicated. Secondary HRP-labeled donkey anti-mouse antibodies were then added. TMB substrate  was then added and the reaction was stopped using 1N sulfuric acid  using PBS as a diluent. After adding 25 μl of IAV, giving a final concentration of 40 HA units per ml or 4 HA units/well, the IAV/protein mixture was incubated for 15 min. at room temperature, followed by addition of 50 μl of a type O human erythrocyte suspension. The minimum concentration of protein required to fully inhibit the hemagglutinating activity of the viral suspension was determined by noting the highest dilution of protein that still inhibited hemagglutination. Inhibition of HA activity in a given well is demonstrated by absence of formation of an erythrocyte pellet. If no inhibition of HA activity was observed at the highest protein concentration used then the value is expressed as > the maximal protein concentration.2 in DMEM. The monolayers were subsequently washed three times with PBS and fixed with 80% acetone (volume/volume) for 10 minutes at 4°C. The monolayers were then labeled by incubating with monoclonal antibody directed against the influenza A viral nucleoprotein  in reagent A  for 30 min. at 4°C. The monolayers were washed three times in PBS and incubated with FITC-labeled goat anti-mouse IgG. The fluorescent foci were counted directly under fluorescent microscopy. Initially, various dilutions of virus were used to find the dose yielding approximately 50 fluorescent foci per high powered (40×) field. These foci appeared to be single infected cells in general. In most experiments IAV was pre-incubated for 30 min. at 37°C with SP-D and/or HNPs or control buffer, followed by addition of these viral samples to the MDCK cells.MDCK monolayers were prepared in 96 well plates and grown to confluency. These layers were then infected with diluted IAV preparations for 30 min. at 37°C in PBS, followed by washing of the monolayer three times in serum free Dulbecco's Modified Eagle Medium (DMEM) containing 1% penicillin and streptomycin. The monolayers were then incubated for 7 hours at 37°C with 5% COt test or ANOVA with post hoc test (Tukey's).Statistical comparisons were made using Student's paired, two-tailed In our prior report we showed that natural human SP-D multimers bind more strongly to IAV using polyclonal anti-SP-D antibodies. We wanted to confirm this finding using mAbs. In addition, we wanted to determine if mAbs alter functional interactions of SP-D with IAV. As shown in figure We next tested the effects of the mAbs in an ELISA assay for binding of SP-D to IAV. In the first set of experiments we incubated IAV with recombinant human SP-D dodecamers and then added mAbs. Bound antibodies were detected with HRP labeled goat anti-mouse antibodies. As shown in panel B of figure In initial experiments we coated natural SP-D multimers and trimers on ELISA plates and tested for detection with MAb 246-04. This mAb was found to detect natural human SP-D trimers to a similar, if not greater, extent than it detected natural SP-D multimers (data not shown). Since the 246-04 mAb also did not alter functional interactions of SP-D with IAV, we used this mAb to detect binding of the natural and recombinant human trimers and multimers to IAV  is critical for antiviral activity. Further studies of epitope mapping of the SP-D CRD coupled with studies on the ability of mAbs to interfere with functional activities will be of interest.Overall our findings suggest that the Thr/Thr11 variant may be associated with increased susceptibility to IAV infection and that studies correlating SP-D genotype with IAV susceptibility should be pursued. Since SP-D is one of a number of innate factors involved in defense against IAV, such studies may be most fruitfully carried out in high risk populations  where effects of SP-D would be more likely to manifest themselves. The trimers also had different interactions with HNPs. The findings could have implications for host defense in some circumstances .Mycobacterium tuberculosis associated meningitis [in vitro and murine in vivo studies [It is unclear what evolutionary pressure may have led to retention of an apparently less active form of SP-D. Mannose-binding lectin (MBL) deficiency is also present in a percentage of many populations and is associated with increased severity of a variety of infections. However, MBL deficiency also seems to play a deleterious pro-inflammatory role in some circumstances and subjects with MBL deficiency are protected against leishmaniasis and ningitis . One pos studies . It is i studies . We spec studies . It may  studies . These dStudies in SP-D knockout mice show that the absence of SP-D is associated with increased replication of IAV and RSV and increased inflammatory responses and illness ,48. In tPolymorphic variations in SP-D result in differences in serum levels and oligomeric structure of the protein. Most importantly, the common Thr/Thr11 polymorphism is associated with assembly predominantly as trimers, whereas the more common forms of SP-D are assembled into dodecamers or higher molecular weight multimers. Natural trimeric SP-D has reduced ability to bind to and neutralize IAV as well as distinct interactions with HNPs as compared to natural SPD multimers. These findings suggest that polymorphisms of SP-D could contribute to susceptibility of certain individuals to IAV infection. In addition, mAbs directed against different areas on the CRD of SP-D have differing effects on antiviral activity and may be useful in future studies to dissect out, or interfere with, various functional properties of SP-D.The author(s) declare that they have no competing interests.KLH planned experiments and prepared the manuscript.MRW carried out viral preparation and most of the experiments involving viral binding and inhibition and reviewed manuscript.TT assisted in viral preparation and in some of the viral binding assays and reviewed the manuscript.IT purified SP-D fractions from amniotic fluid and in preparation of mAbs and reviewed the manuscript.GLS assisted in purification of SP-D fractions from amniotic fluid and in preparation of mAbs and reviewed the manuscript.ECC prepared recombinant human SP-D and polyclonal antibodies and participated in developing experimental plans and revision of manuscript.UH originally developed mAbs to SP-D and the techniques involved in purification of SP-D fractions from amniotic fluid and participated in developing experimental plans and revision of manuscript.


Because of the increasing costs and anticipated shortage of Ear Nose and Throat (ENT) specialists in the care for hearing-impaired persons, an integrated care pathway that includes direct hearing aid provision was developed. While this direct pathway is still under investigation, in a survey we examined expectations and potential barriers and facilitators towards this direct pathway, of patients and professionals involved in the pathway.Two study populations were assessed: members of the health professions involved in the care pathway for hearing-impaired persons , hearing aid dispensers, ENT-specialists and clinical audiologists) and persons with hearing complaints. We developed a comprehensive semi-structured questionnaire for the professionals, regarding expectations, barriers, facilitators and conditions for implementation. We developed two questionnaires for persons with hearing complaints, both regarding evaluations and preferences, and administered them after they had experienced two key elements of the direct pathway: the triage and the hearing aid fitting.On average GPs and hearing aid dispensers had positive expectations towards the direct pathway, while ENT-specialists and clinical audiologists had negative expectations. Professionals stated both barriers and facilitators towards the direct pathway. Most professionals either supported implementation of the direct pathway, provided that a number of conditions were satisfied, or did not support implementation, unless roughly the same conditions were satisfied. Professionals generally agreed on which conditions need to be satisfied. Persons with hearing complaints evaluated the present referral pathway and the new direct pathway equally. Many, especially older, participants stated however that they would still visit the GP and ENT-specialist, even when this would not be necessary for reimbursement of the hearing aid, and found it important that the ENT-specialist or Audiological Centre evaluated their hearing aid.This study identified professional concerns about the direct pathway for hearing-impaired persons. Gaps exist in expectations amongst professions. Also gaps exist between users of the pathway, especially between age groups and regions. Professionals are united in the conditions that need to be fulfilled for a successful implementation of the direct pathway. Implementation on a regional level is recommended to best satisfy these conditions. An important step in an implementation process is to determine the barriers that will impede implementation, and the facilitators that will enhance implementation, within different subgroups -5. Insigreferral pathway.Most hearing-impaired adults have an age-related sensorineural hearing loss, where hearing aids are the only option for rehabilitation. Until recently, in the Netherlands all hearing-impaired persons who seek help first had to consult their general practitioner (GP) for referral to an Ear Nose and Throat (ENT) specialist or Audiological Centre (AC). In an AC a multidisciplinary team under the direction of a clinical audiologist offers specialized care for the more serious or complicated hearing problems. Only ENT-specialists and ACs were entitled to prescribe hearing aids. Generally, a hearing aid dispenser performed the actual hearing aid fitting. Once a satisfying hearing aid was fitted, the hearing-impaired persons returned to the ENT-specialist or AC for approval of the hearing aid, which entitled them to  reimbursement by their medical insurance. We will refer to this pathway as the care pathway for hearing-impaired persons by referral Figure , in shorSince 2002 however a number of health insurance companies in the Netherlands permit hearing aid reimbursement without a referral, prescription or approval. This allows for direct hearing aid provision by the hearing aid dispenser. One reason for this new care pathway was to reduce health care costs. Approximately 10% of the general population of western countries is hearing impaired, and this prevalence heavily increases with age ,7. As a direct pathway , divided over the four professions.The second study population consisted of persons who consulted for hearing complaints. We included persons of 50 years and older with hearing complaints, who consulted ENT departments and ACs or hearing aid dispensers in the three regions. Persons who had consulted for hearing complaints in the preceding year were considered to be under treatment and were therefore excluded.Data for this exploratory survey were collected as part of a multi-centre non-controlled prospective evaluation study examining the direct pathway versus the referral pathway . The stuThe study focused on two key elements of the direct care pathway: distinguishing persons who need medical or specialized audiological care (the patients) from persons who do not need medical or specialized audiological care (the clients), and the quality of the hearing aid provision. For the purpose of the study, participants first visited the hearing aid dispenser for a triage visit  were asked to complete a comprehensive semi-structured questionnaire regarding the direct pathway [see Additional file Two questionnaires for persons with hearing complaints, designed for self-completion, were developed. The questionnaires were administered after the participants had experienced the two key elements of the direct pathway: the triage and the hearing aid fitting. The first questionnaire was administered directly after the ENT/AC-examination [see Additional file As our study population consisted of persons with hearing complaints, and not only hearing aid applicants, not all participants had a hearing aid fitted. The subset of persons with hearing complaints who were fitted with a hearing aid within the study period completed the final questionnaire after the control visit at the AC [see Additional file We checked the results for differences in sex  and age group (below versus above median age) using Mann-Whitney U tests. Differences between the regions  were examined with Kruskal Wallis tests, and Mann-Whitney U tests for post-hoc pairwise comparisons.A total of 60 health care professionals completed the questionnaire regarding their expectations towards the direct pathway. This group consisted of 14 GPs, 16 hearing aid dispensers, 16 ENT-specialists and 14 clinical audiologists.The professional groups gave significantly different answers on all aspects: diagnosing hearing impairment; indicating a hearing aid; paying attention to medical, audiological and psychological complications; giving objective information about the impairment, treatment options and hearing aids; choice and fine-tuning of the hearing aid; and the quality of the ear moulds . On average GPs and hearing aid dispensers expected the direct pathway to have positive consequences for all aspects Figure . The ENTAll professionals considered the direct pathway an improvement for a subpopulation of hearing-impaired persons Table . ProfessThe majority of professionals (78%) on the other side considered the direct pathway a deterioration for persons in need of medical care, 'younger' persons, and persons who are dissatisfied with their hearing aid. The majority of professionals (83%) mentioned the risk of undetected pathology, wrongful indication for a hearing aid or commercialization of care as potential risks of the direct pathway. In addition, GPs emphasized that their role as gatekeeper should be maintained. They reported that a medical cause of the hearing problems should be ruled out first and that diagnostics and treatment should not go hand in hand. By this they meant that the hearing aid dispenser should not both diagnose hearing impairment and provide the hearing aid. Clinical audiologists additionally reported that both the GP and the hearing aid dispenser are not capable of distinguishing adequately between patients and clients.Ten out of 14 GPs stated that they supported the direct pathway, provided that specific conditions were met Figure . These cTwo GPs completely supported implementation. One GP reported not to support implementation, unless GP and hearing aid dispenser would work together, and one GP reported absolutely not to support implementation.Twelve out of 16 hearing aid dispensers reported that they supported implementation, but only when adequate training, strict requirements for hearing aid dispensers, clear criteria for referral, excellent communication between professionals, and good arrangements regarding task division were provided. Four hearing aid dispensers reported to support the implementation completely.Eight out of 16 ENT-specialists stated to support implementation of the direct pathway, provided that the quality of audiometry and otoscopy would be sufficient, that hearing aid dispensers would not prescribe hearing aids for commercial reasons only, that the professional education for hearing aid dispenser would improve, and that clear criteria for referral, a good evaluation system and a complaints service would be present. Five ENT-specialists reported not to support implementation, unless some of the above mentioned conditions were satisfied. Two ENT-specialists did not support implementation and one ENT-specialist reported to completely support implementation of the direct pathway.Six out of 14 clinical audiologists stated that they did support implementation, provided that quality would be guaranteed, that there would be a second-opinion possibility, that task division, responsibilities and criteria for referral would be clear, and that hearing aid dispensers would be adequately trained for their new tasks. Four clinical audiologists did not support implementation, except when hearing aid dispenser and GP would recognize all pathology, hearing aid dispensers were sufficiently trained, and a random evaluation of the care pathway would exist. Three clinical audiologists did absolutely not support implementation, in contrast to one who completely supported implementation.Between April 2004 and August 2005, a total of 337 persons with hearing complaints were included in the study and were asked to complete the first questionnaire after the ENT/AC-examination. A total of 332 participants (99%) completed the questionnaire after the ENT/AC-examination Figure . Their mOf the 337 participants in the study, 181 (54%) had a hearing aid fitted. Of them, 88 (49%) attended the AC for the control visit after the hearing aid trial. Ninety-three participants (51%) who had a hearing aid fitted did not show up at the control visit. These participants had not finished their hearing aid trial before the end of the study (n = 35), or had a hearing aid fitted at a different dispenser that not participated in the study (n = 58) and therefore dropped out of the study. Of the 88 participants who did attend to the control visit, 85 (97%) completed the final questionnaire. These 85 participants had a mean age of 70.6 years  and 72% were male. The participants were included in Amsterdam (n = 14), Maastricht (n = 53) and Rotterdam (n = 18).After the ENT/AC-examination, 89% of the persons with hearing complaints reported to have confidence in the trained hearing aid dispenser Table . Even moThe subset of persons with hearing complaints who had finished their hearing aid trial and therefore completed the whole pathway (n = 85), completed the final questionnaire after the AC control visit. They graded the referral pathway and the direct pathway equally (7.6 and 7.7 respectively). Both scores ranged from 2 to 10, with a median of 8 for both pathways. The grades were not significantly different .In the first questionnaire, a quarter of the persons with hearing complaints 24%) reported that they would be inclined to obtain a hearing aid sooner when they didn't have to visit their GP, an ENT-specialist or AC first , and found it important that the ENT-specialist or AC evaluated the hearing aid fitting (64%).Additionally, we checked the results for differences in sex , age group and region . The age groups were defined below and above the median age, being 70 for the group with hearing complaints that completed the first questionnaire, and 71 for the group with a hearing aid fitting that completed the second questionnaire. This resulted in a comparison of persons younger than 70 versus 70 years and older for the first questionnaire, and a comparison of persons younger than 71 versus 71 years and older for the second questionnaire.No statistically significant differences were found between men and women. There were differences in preferences between the age groups. Of the group of persons aged 70 years and older, 69% would still visit the ENT-specialist or AC, even if this was not necessary for reimbursement of the hearing aid, versus 58% of those younger than 70 years. This difference is statistically significant . Also, statistically significantly more persons of 71 years and older (79%) than persons younger than 71 (49%) found it important that an ENT-specialist or AC evaluated their hearing aid fitting .We also found differences in preferences for the regions, specifically in whether respondents would still visit their GP when this was not necessary . Pairwise comparison showed that in Maastricht (60%) and Amsterdam (57%) statistically significantly more persons stated that they would still visit the GP for their hearing complaints than in Rotterdam (37%) .This exploratory study provides insight in the barriers and facilitators of implementing the direct care pathway for hearing-impaired persons. Hearing aid dispensers and GPs had on average positive expectations towards the direct pathway, while ENT-specialists and clinical audiologists had on average negative expectations. Also more ENT-specialists and clinical audiologists found the direct pathway a deterioration and stated possible risks, and they were somewhat more reluctant to implement the direct pathway. Besides concerns about safety and quality, a possible explanation for this is that in the direct pathway, ENT-specialists and clinical audiologists will lose domain in the health care for hearing-impaired persons. They have to hand over part of their work to the GP and hearing aid dispenser. They may feel that their professional authority will decline if they are relegated more to the role of specialist, seeing patients only on referral from hearing aid dispensers and GPs. Hearing aid dispensers on the other hand experience an expansion of their tasks in the care for hearing-impaired persons, as they will have greater autonomy in the direct pathway. This makes hearing aid dispensers a so-called 'encroaching profession'. Also in other health care settings established professions develop strategies to protect their boundaries, while encroaching professions try to expand their work areas -23.In 2005 an updated report regarding the labour market of ENT-specialists was published. This report concluded that the predicted shortage of ENT-specialists was tackled, and that a sufficient number of ENT-specialists will be available in the near future . These fAlthough negative expectations exist, the results of this study show that most health care professionals either supported implementation of the direct pathway, provided that a number of conditions are satisfied, or did not support implementation, unless roughly the same conditions are satisfied. The professionals generally agreed on which conditions need to be satisfied. In general, these conditions are: good communication between the professionals involved in the direct care pathway, adequate training for the hearing aid dispensers, frequent evaluation, clear criteria for referral, clear division of tasks, a second opinion possibility, and a complaints service.Before implementing the direct care pathway, all parties involved  should reach a consensus on the criteria for referral and the division of tasks. Regardless of these criteria and the division of tasks, it should always be possible for hearing-impaired persons to visit the ENT-specialist or AC for a second opinion. Furthermore, the parties must agree on clear requirements regarding the training of the hearing aid dispensers, and these requirements must concur with the referral criteria and task description of the hearing aid dispensers. Next, a complaints service should be facilitated, possibly by the patients' association. Especially in the beginning the direct care pathway should be evaluated carefully. Good communication between professionals is of great importance, but this is probably not easy to realize. Regional implementation may be part of the solution, as in small regions it is less difficult to create a basis of trust among the professionals involved. When the professionals and hearing-impaired persons make agreements on a regional level, they will get to know each other better. As a result this might improve their communication and their confidence in the other professionals' capabilities.All four groups of professionals stated that the direct pathway had risks as well as benefits. When we apply the framework of Cabana et al  to the bThe professionals did not perceive any external barriers, but we examined these separately by asking persons with hearing complaints for their evaluations and preferences. Due to the study design, approximately half of the participants fitted with a hearing aid could not be asked to complete the final questionnaire. We checked whether age and region in this group were different from the group that did complete the final questionnaire, as these characteristics were found to influence preferences. Since there was no statistically significant difference, we had no reason to believe that the low response rate of the final questionnaire influenced the results.Although participants evaluated both pathways equally and had confidence in the hearing aid dispenser, many participants stated that they would still visit the GP and ENT-specialist, even when this would not be necessary for reimbursement. This means that after implementation of the direct pathway, clients may still choose to visit the ENT-specialist, bypassing the direct pathway. It is therefore important that persons with hearing complaints are well informed about the direct pathway and are involved in the implementation process.A larger proportion of older participants stated that they would visit the ENT-specialist or AC, even when this was not necessary for reimbursement of the hearing aid, and found it important that the ENT-specialist or AC evaluated their hearing aid fitting. This indicates that older persons are more attached to the opinion of the medical specialist. This difference was not caused by differences in hearing aid experience. The implication of this difference is that especially older clients may bypass the direct care pathway by still visiting the ENT-specialist or AC for their examination and control visit. Older persons therefore may need special attention in the implementation process. Differences in preferences were found between the regions, which is probably due to the variety in care pathways for hearing-impaired persons that currently exist among regions. Since 2002 a number of health insurance companies already permit some form of direct hearing aid provision without consultation of the involved professionals. This makes the current care for hearing-impaired persons diffuse. The differences between the regions and the current diffuse forms of care for hearing-impaired persons argue for implementation on a regional level. The different types of hospitals may have caused the difference in participant numbers included in the study. Maastricht included more participants than Rotterdam and Amsterdam, possibly because the hospital in Maastricht also has a community function and sees more persons that are not referred by another hospital.Since multidisciplinary collaboration and task substitution play an important role in many other integrated or shared care pathways ,23,25,26While the direct care pathway is still under investigation, we have already obtained valuable information regarding support of the direct care pathway and potential barriers and facilitators. Barriers and facilitators to change are often examined after or during implementation ,27-29. BThis exploratory study identified professional concerns about the direct care pathway for hearing-impaired persons. It is clear that gaps in expectations amongst GPs, hearing aid dispensers, ENT-specialists and clinical audiologists exist. It is also clear that gaps in expectations exist among the users of the care pathway, especially with older persons demonstrating a preference for the traditional pathway. Also, persons from different regions where currently different pathways exist demonstrated different preferences. Despite these differences and despite the fact that implementation of the direct pathway is not yet broadly based, professionals are united in the conditions that need to be fulfilled for a successful implementation of the direct pathway. It is important that these conditions are met before implementing the direct pathway in the Netherlands. Implementation on a regional level is recommended to best satisfy the stated conditions.The author(s) declare that they have no competing interests.All authors have participated in designing the study. JG contributed to the acquisition of the data, drafted the manuscript, performed the statistical analyses and interpreted the data. FVDH, MJ and LA contributed to the acquisition and interpretation of the data and were involved in drafting the manuscript. WD and HV contributed to the acquisition of the data and provided critical edits to the manuscript. All authors have given final approval of the submitted manuscript.The pre-publication history for this paper can be accessed here:Questionnaire for professionalsClick here for fileFirst questionnaire for persons with hearing complaintsClick here for fileFinal questionnaire for persons with hearing complaintsClick here for file