Few questionnaires on outpatients' satisfaction with hospital exist. All have been constructed without giving enough room for the patient's point of view in the validation procedure. The main objective was to develop, according to psychometric standards, a self-administered generic outpatient questionnaire exploring opinion on quality of hospital care.First, a qualitative phase was conducted to generate items and identify domains using critical analysis incident technique and literature review. A list of easily comprehensible non-redundant items was defined using Delphi technique and a pilot study on outpatients. This phase involved outpatients, patient association representatives and experts. The second step was a quantitative validation phase comprised a multicenter study in 3 hospitals, 10 departments and 1007 outpatients. It was designed to select items, identify dimensions, measure reliability, internal and concurrent validity. Patients were randomized according to the place of questionnaire completion  (participation rate = 65%). Third, a mail-back study on 2 departments and 248 outpatients was conducted to replicate the validation (participation rate = 57%).A 27-item questionnaire comprising 4 subscales (appointment making, reception facilities, waiting time and consultation with the doctor). The factorial structure was satisfactory . Interscale correlations ranged from 0.42 to 0.59, Cronbach α coefficients ranged from 0.79 to 0.94. All Item-scale correlations were higher than 0.40. Test-retest intraclass coefficients ranged from 0.69 to 0.85. A unidimensional 9-item version was produced by selection of one third of the items within each subscale with the strongest loading on the principal component and the best item-scale correlation corrected for overlap. Factors related to satisfaction level independent from departments were age, previous consultations in the department and satisfaction with life. Completion at hospital immediately after consultation led to an overestimation of satisfaction. No satisfaction score differences existed between spontaneous respondents and patients responding after reminder(s).Good estimation of patient opinion on hospital consultation performance was obtained with these questionnaires. When comparing performances between departments or the same department over time scores need to be adjusted on 3 variables that influence satisfaction independently from department. Completion of the questionnaire at home is preferable to completion in the consultation facility and reminders are not necessary to produce non-biased data. Medical care aims not only to improve health status but also to respond to patient needs and wishes and to ensure their satisfaction with care . LikewisFrom a conceptual point of view, the construct of patient satisfaction as been defined by Ware as an "attempt to capture the personal evaluation of care that cannot be known by observing care directly" and to consider opinion of patients as a multidimensional subjective indicator of quality of care . The modIn France, measuring satisfaction has been mandatory since 1996 and several questionnaires have been developed to evaluate inpatient care -12. MostFew questionnaires have been developed for hospital consultations. Of these, some were specific to one type of consultation like oncology , rheumatThe main objective was to develop, according to psychometric standards, a generic outpatient satisfaction questionnaire that could be used to compare hospital outpatient departments one with another or the same department over time. The questionnaire needed to be brief, understandable and easy to complete for outpatients aged18 years or older in medicine, surgery and psychiatric hospital consultations. It was designed to be self-administered. The French final version is being adapted in English, German, Italian, Spanish and Hebrew.The secondary objective was to define administration procedures in routine study that minimize non-response bias. Three situations were tested: i) questionnaire issued and completed at hospital, immediately after consultation; ii) questionnaire mailed and completed at home before any reminder; iii) questionnaire mailed and completed at home only after reminder(s). The groups were compared for satisfaction.It comprised 2 phases. First, a qualitative phase for item generation and construction of a first version of the questionnaire (41-item version). Secondly, a quantitative phase comprising 2 steps. A first validation phase that provided a shortened version of the questionnaire (27-item version). Second, a replication validation phase to corroborate results from the previous steps. Finally a very short-form version (9-item) was constructed. All versions are presented in the Appendix .A steering committee supervised the questionnaire development procedure, comprising methodologists, hospital practitioners and persons from patient associations defending health care user rights. All analyses were performed using SAS software (version 8).A psychologist conducted 25 individual semi-structured interviews with recent outpatients, using the critical analysis incident technique . SubjectA literature review was carried out on validated satisfaction questionnaires -26,28-30This procedure also identified other factors related to outpatient satisfaction with consultation (patient and physician profiles), relevant for inclusion in the questionnaire. The aim was to select the variables linked to satisfaction, independent from place of consultation (department), for the final questionnaire. These variables constitute background adjustment factors needed to avoid bias in comparing departments one with another or the same department over time (age of the patient for example) .A list of satisfaction items (n = 131) was constructed classified into the following domains: administrative procedures, appointment making, receptionist and nurses, waiting time, facilities, duration and privacy of the consultation with the doctor, human relationships with the doctor, information provided by the doctor and shared decision-making, doctor's technical competence, coordination and continuity of care, and global satisfaction. The source of items (interview v. literature) was indicated.Using the Delphi technique , the steA focus group (one two-hour meeting) coordinated by two of the authors (IG, SV) including two patient association representatives and three patients, with previous individual access to the list of items, checked acceptability of item wording and exhaustiveness of the list.A pilot study was conducted on 55 outpatients from different outpatient departments using a preliminary questionnaire comprising the selected items, to check comprehensibility and acceptability of items and response patterns. Confusing items were removed, rewritten or replaced. The list of the items extracted from this qualitative phase is shown on the appendix.The questionnaire obtained from the qualitative phase and tested in the first study comprised 41 negatively and positively worded satisfaction items . The traditional approach was chosen, in which the item is structured as a statement of opinion. A Likert five-point response balanced scale was chosen (in French : 'yes certainly', 'yes probably', 'neither yes nor no', 'probably not, 'certainly not') because it seems to be the best format ,33 and tSeveral other items on general satisfaction were also included in this questionnaire: one overall satisfaction item, using a seven-point scale  and two items on intended behavior (to recommend, to consult again), using a four-point scale ('yes certainly', 'yes probably', 'probably not', 'certainly not') and one open-ended question. These items were included to test concurrent validity.The questionnaire also comprised data on sociodemographic profile, medical status, visit background and characteristics. and an overall satisfaction with life . This last variable was included because of the relationship between affective disposition and the expression of satisfaction ,35 and bTo select items, a first study was conducted in 2001–2002 in 10 wards of 3 short-stay public teaching hospitals of Paris area . Data was collected in 7 medical departments  and 3 surgical outpatient departments .All consecutive eligible ambulatory patients over 18 years in scheduled consultation with a physician were included, to obtain approximately 100 subjects per department. Patients hospitalized before or immediately after the consultation were excluded. Research assistants approached outpatients immediately after consultation and invited them to participate. Outpatients were randomized prior to being approached. Outpatients randomized in group 1 completed the questionnaire alone immediately after consultation and left it in a box. Patients of group 2 received the questionnaire by mail at home for completion. They were asked to complete and return it by post in a prepaid envelope carrying a neutral address. Non-respondents were sent up to 3 more questionnaires at one-week intervals. To assess reliability over time a sample of 38 respondents from the second group was sent a second questionnaire to return completed, without any reminder.Finally of the 1548 outpatients approached, 70.9% agreed to participate (n = 1097) and 65.1% completed the questionnaire (n = 1007). Response rates were 57.0% in group 1 and 73.7% in group 2 (40.2% before any reminder, 63 % after one reminder, 69.7% after two and 73.7% after three). Reasons for non-participation were refusal or lack of time , language barrier (8.5%), inability for medical reasons (7.2%), other reason (0.6%) and agreement but no return of the questionnaire after 3 reminders . Compared to respondents, the non-respondent group comprised older subjects (60.2% v. 52.6% aged over 50 years, p < 0.001), more foreigners (12.5% v. 29.1%, p < 0.001) and more patients consulting for the first time in the department (28.0% v. 22.4%, p = 0.02). Response rates also differed according to the department (p < 0.001) and the hospital (p < 0.001).To confirm the results of the previous study, a second study was conducted in the year 2002 in two departments  in one short-stay public teaching hospital. All consecutive outpatients of 18 years and over  were included to obtain 100 participants per department. The questionnaires were posted with a prepaid envelope. One reminder was sent 10 days after the first mailing to non-respondents. Participation rate was 33.9% before reminder and 56.5% after (n = 248).A first selection of items was made from descriptive response distribution for each item. The criteria used to guide item selection/deletion were: high rates of non-response and 'not applicable' response (≥ 20%) except for items where high rates in this response category were expected, ceiling and floor effects (≥ 50%), and unacceptable test-retest reliability (weighted kappa coefficient<0.60). Pragmatic considerations also tempered selection: interest of the item in itself, number of items covering the same domain, redundancy.Results showed that the proportion of missing responses per item was low. As predictable, for the two items relating to accessibility of the service in case of emergency  the number of 'does not apply' responses was high (30.7% and 45.0%). A ceiling effect was observed for all items (from 54.4% to 79.6%), except for those on facilities and waiting time (items 10 to 13).Test-retest reliability was good for 20 items (weighted kappa ≥ 0.7 for 10 items and from 0.6 to 0.69 for 10 items). For 5 items, the coefficient ranged from 0.45 to 0.56. The item on doctors' warnings on side effects of treatment (item 22) had a very low weighted kappa (k = 0.17). At this stage 12 items were discarded. Item 22 was retained for its clinical relevance  with 'varimax' rotation and the 26 items with substantial loading ≥ 0.40) on only one factor were retained . Another PCFA was computed on the 26 remaining items to determine the structure of the instrument. The screeplot revealed a predominant eigenvalue with nevertheless a four-dimensional structure . Hence the proposal is to consider a four-dimension structure with the possibility of an overall score. One dimension grouped the 13 items relating to consultation with the physician. The second dimension grouped the 6 variables relating to appointment-making. The third and fourth related respectively to waiting time or facilities (4 items) and reception (3 items).None of the 26 items loaded on more than one factor. Only item 26 ('doctor in touch with attending physician') had a borderline loading (0.37), but it was kept because coordination of care in hospital care is important.The stability of the 4 factors was ascertained with PCFA on subgroups and with 'oblique' rotation .Scores for each scale were based on the standardized sum of the items, giving a range from 0 (low satisfaction) to 100 (high satisfaction). Scores were computed when at least half the items in a scale were completed. Because of a ceiling effect, mean scale scores are relatively high except for the 'waiting time and facilities' scale  and one item had a low correlation (r = 0.33 for item 26 'doctor in touch with attending physician'). All items had a higher correlation to their hypothesized scales than to other scales.Reliability was good, meeting both Cronbach alpha and intraclass correlation coefficient requirements Table .A very strong association between the overall scale, intended behaviors, comments and global satisfaction question was noted, suggesting good concurrent validity Table .A modified version of the questionnaire was constructed at the end of the previous step. To avoid the ceiling effect highlighted in the previous stage, responses choices were modified . One satisfaction item on waiting time was added and one item on the facility was reworded to improve the chance of revealing a 'waiting time' subscale and a 'reception-facilities' subscale) and because reliability of the 'reception' subscale was borderline. Patient demographic variables identified at the previous stage as having a relationship with satisfaction scores, one item on intended behavior and an open-ended comment field were also added to the questionnaire.The number of items with ceiling effect decreased. Item completion rates were good Table . PCFA waFor item-scale correlations, item 9 also correlated to these two scales ('reception-facilities' factor and 'contact-appointment'). It was decided to attribute it to the factor that maximized internal consistency . All items met the requirement of being highly correlated to their own scale, all interscale correlations were satisfying, as well as internal consistency Table . The scaAs the factorial analysis of both the first validation and replication phases revealed a predominant factor that could be split into four underlying dimensions, it was decided to construct a unidimensional form of the questionnaire, that could produce an overall global outcome that could be very useful in case of evaluation study. Within each dimension, one third of the items were selected according to two criteria: items without 'not applicable' response choice, and items having strong loading on the principal component in PCFA. Thus 9 items were selected, 4 items from the 'consultation with the doctor' scale, 2 from the 'contact-appointment' scale, 2 from 'reception-facilities' and 1 from 'waiting time' .Final PCFA on these 9 items showed scale unidimensionality. Item loading on this factor ranged from 0.56 to 0.78. Item-scale correlation corrected for overlap ranged from 0.47 to 0.65. Internal consistency was good (Cronbach α = 0.86).First study showed that compared to the satisfaction score obtained with completion at home, mean scores for all hospital-completed satisfaction scales were very significantly higher. In the group that completed the questionnaire at home, comparison between respondents before any reminder and respondents after reminder(s) showed no difference in satisfaction scores, whatever the scale considered . Satisfaction scores ranged from 79.3 to 91.7 for 'consultation with the doctor' scale, from 72.8 to 94.2 for 'appointment making' scale, from 83.4 to 91.3 for 'reception' scale, from 57.3 to 80.5 for 'waiting time-facilities' and from 77.8 to 89.3 for the overall scale).Older age, good satisfaction with life and numerous previous consultations in the department were all associated with high levels of satisfaction, independently from the department Table .The 27-item and 9-item versions of the questionnaire developed here appear sufficiently concise, valid and reliable to provide a non-biased subjective evaluation of outpatient viewpoint on the quality of care and services in hospital consultations. The questionnaire demonstrated very good internal consistency and good reliability over time. The construction strategy presented here follows most of the recommendations for "good practice" in validation of measurement tools of patient satisfaction with care ,23,26,37The predominant role given to patients in the early development stages, the literature review and the implication of various experts ensure good content, construct and face validity. This first qualitative step, often insufficiently detailed and structured in satisfaction questionnaire construction, is indeed crucial .The quantitative phase  used not only statistical and psychometric results to reach decisions, but also the "intrinsic" and "clinical" relevance of items. This is a very important point. First, because satisfaction studies aim not only to measure quality from user viewpoint, but also to highlight practical elements that can be modified to improve quality. Second, questionnaires that are perceived to have content validity are needed to generate interest in results among health professionals and provide incentive for changes in approach to their jobs. Third, the tendency of health professionals to develop "home-made" questionnaires and their reluctance to use validated questionnaires developed elsewhere can be countered if questionnaire items are perceived as relevant.Each dimension comprises items exploring both technical aspects of care (i.e. equipment, competence, accessibility, continuity, compliance, pain management, waiting and consultation time...) and interpersonal aspects of care (i.e. information, decision sharing, attitude...). These aspects are both predictors of patient opinion on care and services ,23,37 beThe most important dimension explaining outpatient opinion of hospital quality is the actual consultation with the physician, representing half the items in the tool. This is consistent with other generic patient questionnaires on satisfaction with ambulatory care, also comprising a majority of items related to the medical intervention ,26,29,37No independent subscales regarding specific aspects of the patient-physician encounter  were identified here. They have been regularly identified by authors developing GP satisfaction questionnaires ,37,39,40The three other dimensions ('contact-appointments', 'reception-facilities' and 'waiting time') are all related to organizational non-medical aspects of care. These dimensions are classically identified in other generic questionnaires ,29,40,42It was shown that satisfaction scores were strongly related to consultation department, regardless of outpatient, physician and care-provision characteristics. These results suggest that this measure is more sensitive to levels of department performance than to patient profile or to modes of consultation, as shown elsewhere . TherefoAs in numerous studies, it was observed that older patients have a higher opinion of care provided than others ,23,48. FThe same was observed for patients with multiple contacts with a department . This coThe strong relationship between overall satisfaction with life and opinion on care expresses the influence of the individual affective disposition trait  which influences job satisfaction, a concept very close to patient satisfaction with care . Other sThe influence of these three background factors suggests the need to adjust patient satisfaction scores on these three variables (i.e. patient age, number of contacts and satisfaction with life) when comparing performances between departments or measuring performance over time within departments .For patients completing the questionnaire immediately after consultation in the hospital, satisfaction estimates were higher than in case of home completion, in spite of procedures to preserve anonymity and confidentiality at hospital. Little data exists on the impact of place of completion for self-administered questionnaires on satisfaction with consultation: two studies conclude that patients express less satisfaction when the questionnaire is completed at home rather than in the medical facility ,50 and oIn the present study, no difference was observed between respondents without reminder and respondents only after reminder(s). This result is in agreement with other studies assessing inpatient satisfaction ,52. It cThis work entails several limitations. First, overall response rates only reached 65% despite reminders sent to patients receiving mailed questionnaires at home. However, unlike other studies, the response rates calculated did not exclude patients unable to respond for medical reason (i.e. who were very ill or did not understand French) and homeless patients giving an invalid address (shelter...).Second, non-respondents differed from respondents regarding two background factors influencing satisfaction levels, with over-sampling of less-satisfied subjects in the respondent group (young patients and first consultants). There are also differences in participation rates between departments that could lead to over-estimating real differences between departments, because the more satisfied outpatients within each department may have been excluded.Third, validation is a continuous process and further studies are required to confirm these first results. The experimental nature of these studies may have induced bias in questionnaire responses. So there is a need to replicate findings using confirmatory statistical methods  using the data from non experimental, routine studies.Good estimation of patient opinion on hospital consultation can be obtained with these two questionnaires. When comparing performances between departments or the same department over time scores need to be adjusted on the three variables that influence satisfaction independently from department . Mail-back completion at home of the questionnaire seemed preferable to completion in the consultation facility immediately after the consultation. Reminders are not necessary to produce non-biased data.IG – initiation of the research, supervision of the project and drafting the manuscript; SV – coordination of the 2 studies, participation in the interpretation of the results and revision of the draft paper; CDS – performing statistical analyses; PD and PR- participation in the conception, design and coordination of the research; BF – participation in the interpretation of the results, supervision of the statistical analysis and revision of the draft paper.


Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals.To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR.Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species. A gene superfamily of heme-proteins, the cytochromes P450 (CYP), encodes the main enzymatic system for metabolism of structurally diverse lipophilic substrates . A subseFugu rubripes [Danio rerio [Caenorhabditis elegans [Strikingly, in contrast to the two xenobiotic-sensing nuclear receptors in mammals, only one xenosensor has been found in non-mammalian species, e.g. chicken , fish (frubripes  and zebrio rerio ) or the  elegans . The ami elegans . Moreove elegans .In this report, we studied the evolutionary aspects of these findings. Despite using various methods and techniques, we were unable to isolate further genes that encode chicken xenobiotic-sensing nuclear receptors confirming the hypothesis that non-mammalian genomes only have one xenosensor gene. Since PXR and CAR exhibit different typical features concerning their activation, localization and regulation ,19, we eC. elegans are about equally related to the mammalian PXRs and CARs , that are related to the xenobiotic-sensing nuclear receptors, the BXRs are pharmacologically distinct from PXR and CAR and do not respond to xenobiotics [3 receptor (VDR, NR1I1) in addition to PXR and CAR, intron-exon junctions are highly conserved [Orthologs of PXR and CAR have been isolated from man, monkey, pig, dog, rabbit, mouse and rat . In non-ARs Fig. . Of the obiotics ,20. No donserved . Human aonserved . Using aTo further test this hypothesis, we used different experimental approaches in order to isolate additional chicken xenobiotic-sensing nuclear receptors. Neither high- and low-stringency screening of a chicken liver cDNA library using CXR, CAR and PXR fragments as probes nor PCR-based strategies with degenerate primers designed on CAR and PXR alignments or degenerate primers based on generic nuclear receptor DNA-binding domains  resultedIf CXR in fact is the only chicken xenobiotic-sensing nuclear receptor, ablation of CXR expression or function is predicted to drastically reduce drug-induction of CYPs and other target genes. To reduce CXR expression, we designed RNAi oligonucleotides targeting CXR and stably expressed those in the chicken hepatoma cell line leghorn male hepatoma (LMH). LMH cells express endogenous CXR and retain induction of genes by PB-type inducer compounds and other drugs . As showThus, we used an alternative method that aimed at reducing CXR activity by designing dominant-negative CXR alleles. These CXR mutants were then tested in reporter gene assays on drug-responding enhancer elements. In our case, we generated three different CXR alleles Figure : first, First, the three CXR mutants were tested for their ability to bind to and activate a 264-bp PBRU isolated from the 5'-flanking region of chicken CYP2H1 ,33. As sLLLLTEL) is also found in the CXR sequence between leucine 356 and leucine 362. Thus, to assess the relatedness of CXR to PXR and CAR in terms of cellular localization, we engineered different CXR-green fluorescent protein (GFP) fusion proteins. These were subsequently tested for functionality in CV-1 cell transactivation assays using the 264-bp PBRU as drug-sensitive enhancer. CXR with N-terminal, but not C-terminal GFP is activated by 400 μM PB and 10 μM clotrimazole after 16 hours of incubation , unsuccessful attempts to isolate further xenosenors in chicken and functional assays showing that ablation of CXR function drastically reduces drug-inducibility in a chicken hepatoma cell line. Our findings presented here and those of other laboratories imply that PXR and CAR origin from one ancestral gene which diverged into two genes in mammals. This ancestral gene, in chicken coding for CXR, is a promiscuous, PXR-like receptor. Thus, CXR and related receptors from fish are activated by a variety of different compounds ,15. InteEvolution of drug-metabolizing CYPs and xenobiotic-sensing nuclear receptors is influenced by diet and exposure to other environmental chemicals. Accordingly, drug-induction is very species specific. This is reflected in the unusually divergent ligand-binding domains of PXRs and CARs orthologs . When coCulture and transfection of LMH cells with FUGENE 6 Transfection Reagent  were performed as published ,33. BefoChicken BAC filters (UK Human Genome Mapping Project Resource Center, UK) were hybridised with a probe encoding for CXR. Positive clones were purchased, digested with different restriction enzymes and Southern blots obtained using the same probe. Bands hybridising with the CXR probe were isolated, subcloned and CXR genomic information obtained by PCR using primers designed after the CXR mRNA sequence.Mutagenesis was carried out using overlapping primers as described . Mutatedin vitro transcription/translation kit  before being subjected to non-denaturing SDS-polyacrylamide gel electrophoresis with [32P]-radiolabeled CYP2H1 264-bp PBRU.Electrophoretic mobility shift assays have been described in detail . ProteinGGATGGGGCTCTGGCCGGCTTCAAGAGAGCCGGCCAGAGCCCCATCCTTTTTGGAAA) and the anti-sense strand (AGCTTTTCCAAAAAGGATGGGGCTCTGGCCGGCTCTCTTGAAGCCGGCCAGAGCCCCATCCGGG) and subsequent ligation into pSUPER cut with BglII and HindIII (underlined letters refer to CXR-specific targeting sequence). After verification of the ligation product the pSUPER-CXR-RNAi expression cassette was cut out using BamHI and XhoI and subcloned into BglII/XhoI-digested pcDNA3 (Invitrogen, Carlsbad, USA). The ScaI-linearised construct was transfected into LMH cells using FUGENE 6 . Stable transfectants were selected by addition of 175 μg/ml G418 (PAA Laboratories, Pasching, Austria) to the cell culture medium. A control cell line was selected in parallel which was stably transfected with pcDNA3 carrying the empty pSUPER expression cassette. Reporter gene assays in LMH cells using the CXR-RNAi clones were performed using reporter constructs for CYP2H1, CYP3A37, CYP2C45 and ALAS-1 described previously [Expression of CXR in LMH cells was repressed by RNAi as described . In brieeviously -26.LMH cells were cultivated on glass cover slips and subsequently transfected with pEGFP-C1 or pEGFP-N1 expression plasmids (Clontech, Allschwil, Switzerland) before cells were either treated with vehicle, 400 μM PB or 0.1 μM okadaic acid for 16 hours. Cells were washed with PBS, fixed in 3% formaldehyde for 30 minutes, washed again with PBS, stained with 300 nM DAPI and subsequently mounted on glass slides. Digital images were captured using a Leica DC 300F camera (Leica, Nidau, Switzerland) mounted on a Leitz DMRB microscope with the Leica IM50 Image Manager program version 1.20. Figures were assembled with Adobe Photoshop version 5.0.CXR ligand-binding domain was expressed in bacteria, purified and injected into rabbits for antibody production according to standard procedures. Anti-CXR-ligand-binding domain antibody from rabbit serum was subsequently used in Western blots. LMH cells were grown under standard conditions and treated with vehicle or 400 μM PB overnight. Cells were subsequently washed with PBS and protein extracts prepared using RIPA buffer. As control, CV-1 cells were transfected with empty pSG5 expression vector or vector expressing CXR and subsequently lysed with RIPA buffer. Nuclear extracts were prepared as published .LMH cells were treated with the indicated compounds for 16 hours before total RNA was isolated using the TRIZOL Reagent (Life Technologies, Basel, Switzerland). Twenty μg of total RNA were subjected to electrophoresis and analysed in Northern hybridisations as described ,33.CYP, cytochrome P450; PB, phenobarbital; PXR, pregnane X receptor; CAR, constitutive androstane receptor; CXR, chicken xenobiotic receptor; PBRU, phenobarbital-responsive enhancer unit; ALAS-1, δ-aminolevulinate synthase; AF-1/2, activation function-1/2; LXR, liver X receptor; PB, phenobarbital; XRS, xenochemical response signal; GFP, green fluorescent protein; PPAR, peroxisome-proliferator activated receptor; FXR, farnesoid X receptor.The authors declare that they have no competing interests.CH carried out the cellular localization assays, cloned the various CXR mutants, performed the reporter gene and the electrophoretic-mobility shift assays as well as the transcriptional regulation studies. SB did the various screens for further chicken xenobiotic-sensing nuclear receptors. AR performed the RNAi experiments. RL and MO isolated the CXR antibody and carried out the protein stabilization and localization assays. MRK helped with the RNAi experiments. MP and CG helped with the CV-1 cell transactivation assays. UAM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Pseudomonas aeruginosa and Acinetobacter baumanniii has led to the re-use of polymyxins. Our objective was to study the toxicity of prolonged intravenous administration of colistin (polymyxin E).The intravenous use of polymyxins has been considered to be associated with considerable nephrotoxicity and neurotoxicity. For this reason, the systemic administration of polymyxins had been abandoned for about 20 years in most areas of the world. However, the problem of infections due to multidrug-resistant (MDR) Gram-negative bacteria such us An observational study of a retrospective cohort at "Henry Dunant" Hospital, a 450-bed tertiary care center in Athens, Greece, was undertaken.Patients who received intravenous colistin for more than 4 weeks for the treatment of multidrug resistant Gram-negative infections were included in the study. Serum creatinine, blood urea, liver function tests, symptoms and signs of neurotoxicity were the main outcomes studied.We analyzed data for 19 courses of prolonged intravenous colistin [mean duration of administration (± SD) 43.4 (± 14.6) days, mean daily dosage (± SD) 4.4 (± 2.1) million IU, mean cumulative dosage (± SD) 190.4 (± 91.0) million IU] in 17 patients. The median creatinine value increased by 0.25 mg/dl during the treatment compared to the baseline (p < 0.001) but returned close to the baseline at the end of treatment (higher by 0.1 mg/dl, p = 0.67). No apnea or other evidence of neuromuscular blockade was noted in any of these patients who received prolonged treatment with colistin.No serious toxicity was observed in this group of patients who received prolonged intravenous colistin. Colistin should be considered as a therapeutic option in patients with infections due to multidrug resistant Gram-negative bacteria. Pseudomonas aeruginosa and Acinetobacter baumannii resistant to most classes of antimicrobial agents has made the medical community to rethink of colistin, an old, basically abandoned for the last two decades antibiotic. Intravenous colistin (polymyxin E) has been used in the industrialized countries during the last years mainly in patients with cystic fibrosis infected with Pseudomonas aeruginosa resistant to other available antimicrobial classes [The worldwide spread of the problem of infections due to Gram-negative bacteria such us  classes . Excludi classes .Several reports during the early years of use of the medication, mainly in the decade 1960 to 1969 left the medical community with the impression that the medication is very toxic ,4. Howev®, Kent, UK or Colistin, Norma®, Athens, Greece). The study was approved by the Institutional Review Board (IRB) of the hospital.Patients who received intravenous colistin from 1/October/2000 to 31/January/2004 at "Henry Dunant" Hospital, a 450-bed tertiary care center in Athens, Greece, were identified by the pharmacy electronic databases. All patients who were found to have received intravenous colistin for more than four weeks continuously, or in two courses of colistin but with no more than seven days interruption between the courses were included in this study. Administration of intravenous colistin in two courses for more than four weeks in each course were studied as two different units in the analysis of toxicity, if there was more than one month colistin-free period between the two courses. One milligram of the colistin formulations used is approximately equal to 12.500 IU (Colomycin, Forest Laboratories3) or leukocytosis (= 12.000 WBC/mm3)], and at least two of the following: new onset of purulent sputum or change in character of sputum, increased respiratory secretions or increased suctioning requirements, new onset or worsening of cough or dyspnea or tachypnea, rales or bronchial breath sounds, or worsening gas exchange [Diagnosis of pneumonia required two or more serial chest radiographs with at least one of the following: new or progressive and persistent infiltrate, consolidation, cavitation, or pleural effusion. In addition, patients must have had fever >38°C with no other recognized cause, or abnormal white blood cell count [leukopenia (< 4000 WBC/mmexchange .and at least one of the following: a) common skin contaminant (e.g., diphtheroids, Bacillus sp., Propionibacterium sp., coagulasenegative staphylococci, or micrococci) grown from two or more blood cultures drawn on separate occasions or b) common skin contaminant (e.g., diphtheroids, Bacillus sp., Propionibactαium sp., coagulase-negative staphylococci, or micrococci) grown from at least one blood culture from a patient with an intravascular line and physician- instituted antimicrobial therapy [Bacteremia required either growth of a recognized pathogen from one or more blood specimen cultures or at least one of the following signs or symptoms: fever (>38°C), chills, or hypotension  therapy .Infections at other body sites or fluids, such as urinary tract infections and surgical site infections were defined based on guidelines from the Centers for Disease Control and Prevention . ClinicaData for several variables including demographic and clinical information as well as results of laboratory and imaging tests were collected and entered in a computer database. All available results of renal function tests , liver function tests , creatine phosphokinase (CPK) during the course of colistin treatment and at hospital discharge were collected.Creatinine values at the beginning of colistin treatment were compared with the maximum value of creatinine during therapy as well as with the creatinine value at the end of treatment using a non-parametric test (Wilcoxon). The data analysis was performed using SPSS and S-plus software.During the period from 1/October/2000 to 31/January/2004, 152 patients received intravenous colistin. Data about the efficacy of the medication were analysed and reported elsewhere [Pseudomonas aeruginosa strains, the minimum inhibitory concentrations (MICs) were not available. In the remaining 10 Pseudomonas aeruginosa isolates, the MICs to colistin ranged from = 0.5 mg/l to 2 mg/l. In vitro susceptibility testing for the 5 isolated Acinetobacter baumannii strains revealed MICs to colistin = 0.5 mg/l, for the 2 Klebsiella pneumoniae strains MICs were 0.5 mg/l and 2 mg/l, and for the Enterobacter cloacae strain MIC was 1 mg/l.The analysis of the responsible pathogens was based on 20 isolates from 19 episodes of infection in 17 patients (two pathogens were isolated in one episode). In 2 episodes of infection, both caused by The all-cause in-hospital mortality was 41,2% (7 out of 17 patients). Clinical cure and improvement, defined as resolution and partial resolution, respectively, of presenting symptoms and signs of the infection by the end of colistin treatment was observed in 14 of the 19 episodes of infection (73.7%) [cure 10/19 episodes (52.6%), improvement 4/19 episodes (21.1%)]. Unresponsiveness, defined as persistence or worsening of presenting symptoms and/or signs of the infection during intravenous colistin administration, was observed in 5 of the 19 episodes of infection (26.3%).All patients had been admitted to the ICU at some time during their hospitalisation. In 12 courses out of the 19 analyzed the whole colistin therapy was administered in the ICU, in 1 course it was administered in a hospital ward, and in 6 courses it was given both in the ICU and a hospital ward.Figure At the initiation of colistin administration the median value of baseline blood urea for the 18 courses was 42 mg/dl. During the 18 prolonged courses of colistin administration there was a slight increase in blood urea values; the median maximum value was 60 mg/dl. However, at the completion of colistin therapy median blood urea value returned to 41 mg/dl.During the 19 courses of colistin administration, other potentially nephrotoxic drugs were given. Specifically, aminoglycosides were co-administered in 12 courses [median duration of co-administration (± SD) in these 12 courses 12,5 (± 16,8) days], teicoplanin in 10 [median duration of co-administration 16,0 (± 9,0) days], vancomycin in 8 [median duration of co-administration 23,0 (± 11,2) days], amphotericin in 4 [median duration of co-administration 8,0 (± 9,0) days].Four patients had history of chronic renal dysfunction, with only one of them receiving hemodialysis treatment prior to admission. This patient continued the hemodialysis during his treatment with colistin and died due to sepsis. For the other 3 patients the baseline, the maximum, and the end-of-therapy creatinine values (mg/dl), were 1,8/2,1/2,1 for the first patient, 3,7/3,7/2,4 for the second patient and 2,4/3,8/2,3 for the third patient.Acinetobacter baumannii pneumonia, sensitive only to colistin and gentamicin. Apart from a slight elevation of serum creatinine (from 2,3 mg/dl to 2,7 mg/dl) on the 5th day of treatment no other sign of nephrotoxicity was observed during a 34-day administration of colistin (the 9th day creatinine returned to 0,7 mg/dl).Only one patient had acute renal failure prior to colistin use, which was due to severe haemorrhage during surgery. This patient received 13 courses of hemodialysis. One day after the last course, colistin therapy had to be initiated for the management of th day of colistin treatment and became worse on the 27th day. From then on, and although colistin was continued for 11 more days, the symptoms gradually subsided. No confirmatory electromyographic testing was performed. The second patient was transferred from another ICU where he was diagnosed as having myopathy due to sepsis. Fifteen days after his admission, and while his myopathy was improving, therapy with colistin was initiated; the treatment did not affect the gradual improvement. An electromyography performed on his 21st day in our ICU, showed mild axial sensory-motor polyneuropathy and myopathy. He received colistin for a total of 52 days and made full recovery from his neuropathy/myopathy during the course of colistin administration. The third patient developed ICU polyneuropathy one week prior to colistin administration and, again, recovered gradually from it while he was on colistin treatment. The fourth patient was transferred from another ICU, where he had developed ICU polyneuropathy. Only this patient had moderate worsening of the neuropathy while he was receiving colistin. Despite that, he received colistin for a total of 35 days for a respiratory infection with persisting Pseudomonas aeruginosa sensitive only to colistin. His neuropathy improved gradually after the end of colistin treatment. Thus, our assessment regarding these 4 patients who experienced polyneuropathy and/or myopathy is that colistin therapy was probably associated with the development of neurotoxicity only in one of the 4 patients.No apnea or other evidence of neuromuscular blockade was noted in any of these patients who received prolonged treatment with colistin. Four patients were clinically diagnosed to have polyneuropathy and/or myopathy before or during colistin treatment. In the first patient the polyneuropathy symptoms appeared while she was on her 25Data for the possible hepatobiliary toxicity of colistin were collected. Both laboratory and clinical findings were taken into consideration. In subgroup analyses of patients for whom data were available, no substantial changes on liver function tests was found, as described in Figure The main finding of the study is that no significant deterioration of renal function was found in patients with baseline normal serum creatinine value during prolonged administration of intravenous colistin. In addition, in the group of patients with pre-existing chronic renal dysfunction  the administration of the medication for a long duration did not influence further their renal function. The observed slight increase in the serum creatinine values at the end of treatment  cannot be attributed only to the prolonged colistin administration as other factors such as concomitantly administered potential nephrotoxic agents, and sepsis per ce may have had an effect. In addition, apnea or other evidence of neuromuscular blockade was not observed in any of the 17 patients who received prolonged treatment with colistin.Pseudomonas aeruginosa and E. coli strains from the excised specimen. This patient developed acute tubular necrosis (confirmed by autopsy) and apnea requiring intubation and mechanical ventilation. However, in this case the dosage of colistin used was 30 mg/kg of body weight every six hours (120 mg/kg/day) whereas the recommended dose is 2,5 – 5 mg/kg/day. One year later, several reports showed high renal adverse reaction rates after colistin administration [Early experience with colistin revealed a high incidence of toxicity, mainly nephrotoxicity. In 1969, Ryan et al. reported the fatal case of a previously healthy 4-year-old child with persistent fever after appendectomy who died due to cardiac arrest . The chistration . In manyAcinetobacter baumannii, who received either intravenous colistin (21 patients) or imipenem (14 patients), showed an almost double increase in nephrotoxicity rates among the patients in the imipenem group compared to colistin group [However, recent data from published reports do not corroborate this finding . Notablyin group . In addiin group .The conclusion in the majority of the published studies during 1960 – 1970 was that colistin must be used in serious infections where other antimicrobial agents have failed, always with close monitor of renal function, and with precaution in the co-administration of other possible nephrotoxic or neutotoxic agents ,4. SubseOur study is not without limitations. It is a retrospective study with the inherited problems related to this study design, including difficulties in the determination of colistin associated sensory neurotoxicity. In addition, the number of studied patients is relatively small and there is no control group. However, our study offers insight related to interesting experience from a group of patients who received prolonged treatment with colistin for various infections, mainly as a salvage treatment of persisting and serious infections.st century.In conclusion, intravenous colistin seems to be a safe therapeutic intervention for serious infections due to multidrug-resistant Gram-negative bacteria even when it is administered for a prolonged duration (more than four weeks). More studies will be needed to further clarify the correct use of this old antibiotic in the 21The author(s) declare that they have no competing interests.MEF and AM conceived the idea; MR, IAB, KR, and SKK collected the data; all authors contributed in the writing and preparation of the manuscript.The pre-publication history for this paper can be accessed here:


The director of the United States Centers for Disease Control and Prevention gives a personal view of how the world should tackle the HIV pandemic As an intern, I took care of the first patients with HIV/AIDS at San Francisco General Hospital, and so I grew up with AIDS in the early days of my medical career. We struggled through the confusion about what was making people so sick, and each new day brought a new discovery about the disease and its consequences. I went through that evolutionary process along with everybody else, and it shaped me in many profound ways. Before long, I recognized that this wasn't a disease of “those people over there.” This was a disease that could strike anyone, anytime. And as physicians, we had to adjust our thinking about our own vulnerability to occupational risk, and to emphasize prevention, because there wasn't going to be a cure for a long, long while. And not only physicians had to rethink things—AIDS has reshaped society's very notions of the most basic human behaviors.I was in Uganda in 1985, early in the AIDS epidemic there. We knew then where that epidemic was going to go, absent an effective vaccine or cure, but few of us could have imagined that it would evolve so quickly without an end in sight. While the people of Africa have achieved a huge amount in tackling HIV/AIDS, particularly in Uganda, the epidemic is far from being under control on that continent and is spreading through other parts of the world with alarming speed.The theme of this year's International AIDS Conference in Bangkok was “Access for All.” Over the past few years, it has become increasingly apparent that a critical component of assuring access to care and treatment is human capital. Like fiscal capital, human resources are essential to ending the AIDS pandemic. I visited Africa with US Health and Human Services Secretary Tommy Thompson and many AIDS experts last December, and we saw evidence of this critical need in every country we visited. The miracles of modern science are meaningless without systems and people to deliver them to those in need.The World Health Organization estimates that of the 40 million people worldwide infected with HIV, 6 million need immediate, life-sustaining antiretroviral therapy. Fewer than 400,000 people in developing countries have access to such treatment  1,2]. T,2. T1,2]Only 360 of the 1,200 doctors trained in Zimbabwe during the 1990s continue to practice within the country.Two-thirds of University of Zimbabwe medical students intend to leave the country after graduating, and one of the country's major 1,000-bed teaching hospitals lacks a single qualified pharmacist.In Zambia, only 50 of the 600 doctors trained locally since independence have remained in the country.In Ghana, 320 nurses are recorded to have been lost in 1999, roughly equivalent to that country's annual output of nurses; losses for the year 2000 totaled 600.It has now been shown, beyond any doubt, that even in resource-poor countries with the most basic health infrastructure, people get the same benefit from treatment and prevention interventions as those in the rich world . In factWe hear the numbers—the millions upon millions infected—and we grow numb. That is why we must go to the front lines—the households and communities—and start focusing on each individual living with HIV. I was at the dedication of an AIDS clinic in Kenya. It was raining, and we were waiting outside with our umbrellas. A 12-year-old girl in front of me turned around and leaned her head against my belly and said, “Could you take me to America? I need drugs.” If you take that girl's face and multiply it a thousand times—that is the memory I bring home from Africa: the faces of the children and their asking, “Why are so many of our parents dying? Why are we dying?”We visited a US Centers for Disease Control and Prevention (CDC) program in the very remote areas of Uganda where there are no roads and it is impossible for people to come into population centers to receive HIV testing and other services. Young staff from the CDC are working with Ugandans and community organizations in that area to deliver antiretroviral therapy. Some may think that the difficulties of delivering antiretroviral therapy into such a remote area are overwhelming—and some may question whether this is a sustainable intervention. But once you see firsthand what miracles are possible, your world view changes almost overnight.What we saw was the success of a wonderful home-based treatment and care program for people who don't have access through other means. And when I say “home-based care,” picture a hut without running water or electricity, where only motorcycles are available to deliver medications. The first step of the program is to provide clean water. Coliforms and other pathogens in the water supply for the household are removed through an inexpensive water vessel fitted with a filter and through a chlorination process. In addition, a cotrimoxazole tablet is given every day, which, in one patient's words, changed his life because he began to feel well almost immediately. Not only do the cotrimoxazole prophylaxis and the water treatment improve diarrheal illness, but malarial parasitemia also drops. So that is a very positive, unexpected consequence of just two very simple and inexpensive interventions. Many patients with HIV/AIDS in Africa also have tuberculosis and are put on tuberculosis therapy in addition to cotrimoxazole. As a result, they begin to feel better even before they begin antiretroviral therapy.We spent time with one of the patients in the home-based care program. As she began to participate in these programs, tests became available to measure her CD4 count. She explained to me what her CD4 count was, what it meant, and how it improved when she started the cotrimoxazole and tuberculosis therapy. She had begun taking three antiretroviral drugs and held up her pill box to explain her regimen in detail. Every week a Ugandan health aide delivered her supply of pills on a CDC motorcycle and monitored her adherence to the treatment. Not only was she extremely reliable in taking her medications, but she also knew more about them and their side effects than most of the patients I treated at San Francisco General. She was also an expert in HIV prevention. I asked her, “What do you do to protect your three young sons from this infection?” She replied, “Every day I take them by the hand, and I go out of the house and I say, ‘Do you see that mound of dirt? That is your father’s grave. Your father acquired this fatal infection through sex. Be careful.'” And then she talks to them about the “ABCs” (“A” for abstinence, “B” for being faithful, “C” for condoms).So when you see a story like that unfold in the middle of Africa, it's impossible not to be hopeful. And yet, it's also very sobering because we are reminded of our responsibility. The question is not what the international health community is accomplishing in these countries now, but what we could accomplish if we joined together to really fight this war on AIDS. Such a story also inspires hope because you can see the multiplier effect that comes from taking on one problem and can see the way that effort can expand to encompass and address a much greater set of problems.When we think about successful prevention models in Uganda, “ABC” certainly stands out . HoweverThe innovative programs and ideas emerging in Africa can change the picture of the AIDS epidemic. The purchase of antiretroviral drugs for Africans is not the big challenge. Access to drugs will improve in Africa. The real challenges are delivering drugs in a safe and effective way, monitoring therapy, and sustaining the pipeline of drugs so that ongoing treatment can be guaranteed. In the example of the home-based program in Uganda, we have seen that these challenges can be overcome. Expanding access to prevention, care, and treatment services isn't going to be easy, but it is certainly possible. It will take unprecedented commitment by people in the public sector, the private sector, faith communities, and community organizations, and perhaps most importantly, individual volunteers who make up their minds to contribute in any way they can.Last fall, the US Peace Corps announced that it was activating programs in some countries that allow volunteers to help communities fight the AIDS epidemic, but this is just one of many steps that are being taken. The US president's Emergency Plan for AIDS Relief will provide $15 billion, including almost $10 billion in new funds, over five years for international AIDS assistance , and I a


PLoS Biology, biochemist Peter Walter and his colleagues from the University of California at San Francisco demonstrate two new signaling mechanisms that appear to give the yeast unfolded protein response the means for amplitude adjustment.Machinery within the endoplasmic reticulum (ER) of eukaryotic cells modifies, folds, and assembles proteins as needed to suit their functions at or past the cell membrane. When this system is hampered or overtaxed, a buildup of unfolded or misfolded proteins within the ER triggers the “unfolded protein response,” which alerts the nucleus to boost production of protein-processing machinery that helps proteins fold. This system for adjusting manufacturing capacity is similar in organisms from yeast to human. If the unfolded protein response cannot be turned on when needed, cells die. Prior study suggested that, in yeast cells, the response to unfolded protein buildup is binary: either off or on. In this month's HAC1 messenger RNA waits in readiness for ER emergencies, each molecule locked against translation into protein by intronic RNA sequences that interrupt mRNA translation. Accumulation of unfolded or misfolded proteins in the ER releases this translational block, triggering production of Hac1p and activating the unfolded protein response. Previously, this binary HAC1 signal was the only known regulator of the unfolded protein response in yeast. In two complementary papers, Walter and colleagues now present evidence that the repertoire includes new factors and regulators that amplify the unfolded protein response under conditions of ER stress.In yeast, a transcription regulator called Hac1p activates the genes required for the unfolded protein response. A cytoplasmic pool of HAC1 severalfold. Primed with high levels of HAC1 mRNA, the cells were ready to produce a bumper crop of Hac1p and to induce a supercharged unfolded protein response. In the accompanying paper by Patil et al., the authors show that Hac1p is not working alone. A second regulator of transcription called Gcn4p is required to activate most of the genes associated with the unfolded protein response. The regulatory elements of these genes now appear far more diverse than previously appreciated. The authors propose that cells adjust the levels of Hac1p and Gcn4p to drive a continuum of transcriptional programs equipped to deal with incoming challenges. Together, these two papers demonstrate that the control of the unfolded protein response is far from a simple on/off mechanism, but exhibits complex fine-tuning through a network of signaling pathways that interpret and respond to the cell's needs.Leber et al. stressed yeast cells by exposing them to substances that cause protein misfolding or buildup in the ER. In response, the cells ratcheted up transcription levels of


Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections.Whereas there was a trade-off between transmission success and virulence as defined by host mortality, contradictory clone-specific patterns occurred when defining virulence by anaemia. A negative relationship between anaemia and transmission success was found for one of the parasite clones, whereas there was no relationship for the other. Notably the two parasite clones also differed in a transmission phenotype (gametocyte sex ratio) that has previously been shown to respond adaptively to a changing blood environment. In addition, as predicted by evolutionary theory, mixed infections resulted in increased anaemia. The increased anaemia was, however, not correlated with any discernable parasite trait (e.g. parasite density) or with increased transmission.We found some evidence supporting the hypothesis that there is an adaptive basis correlating virulence  and transmission success in malaria parasites. This confirms the validity of applying evolutionary virulence theory to biomedical research and adds support to the prediction that partially effective vaccines may select for increasingly virulent malaria parasite strains. By contrast, there was no consistent correlation between transmission and sub-lethal anaemia, a more common outcome of malaria infection. However, overall, the data are not inconsistent with the recent proposal that sub-lethal effects may impose an upper limit on virulence. Moreover, clone specific differences in transmission phenotypes linked to anaemia do suggest that there is considerable adaptive potential relating anaemia and transmission that may lead to uncertain consequences following intervention strategies. Contrery rate ,4. Thus,ery rate -8. Incorery rate . Empiricery rate . Identifsmission . Howeversmission . In thessmission .Theoretical advances in virulence theory have revealed the importance of additional properties of host-pathogen systems, such as pathogen population structure and sub-lethal  effects, which can significantly alter the conclusions arising from the simple virulence/transmission trade-off hypothesis ,15. InfeMalaria parasites replicate asexually within their vertebrate hosts, but must produce sexual stages  to be transmitted to mosquitoes. Asexual parasites provide the source for gametocyte production and so increasing asexual parasite density is expected to correlate with transmission success. Therefore, the adaptive trade-off hypothesis would predict that faster asexual replication leads both to higher virulence and to higher transmission success . AlthougP. gallinaceum in the chicken host. In this specific system, anaemia and parasite transmission are linked physiologically, where the host's response to anaemia (reticulocyte production) triggers an increase in the proportion of gametocytes that are male rather than female [a priori to result in host death or control by the host, enabling independent study of parasite transmission to mosquitoes with respect to either mortality or anaemia. Under both infection conditions, we examine the impact of mixed clone infections on overall virulence and transmission. Thus we assess to what extent there is an adaptive basis relating virulence and transmission under infection conditions of theoretical significance.In this study we examine the trade-offs between transmission and virulence defined by mortality and anaemia in single and mixed clone infections, using the avian malaria system n female . This han female -37. Expe6 infected red blood cells (iRBC)  of either of two clones of P. gallinaceum (the Thai or SL clone) or an equal mix of the two. The peak densities of asexual parasites did not differ between Thai and SL clone infections (χ21 = 0.3 P = 0.629), but were both significantly higher than those in mixed clone infections (combined single clones vs. mixed clone χ21 = 6.3 P = 0.012). The peak gametocyte density was, however, significantly higher in Thai than in SL clone infections (χ21 = 4.3 P = 0.038), and higher in both the single clone groups than in the mixed infections (χ21 = 4.05 P = 0.044) . This effect of co-infection was confirmed with a comparison of the mixed clone with the 2 single clone type infections grouped together . The gametocyte sex ratios  were found to be different among groups (χ22 = 33.5, P < 0.001), where the Thai clone had a higher sex ratio than the SL clone infections. Closer inspection and analysis of the data revealed similar sex ratios in the SL clone and mixed clone infections . As previously observed after infections by injection with high inocula [We first considered infection by injection of 10 inocula , the proThese data strongly suggest that the clones had a negative impact on each other, thus resulting in lower densities of asexual parasites and hence gametocytes. The anaemia  was greater in the mixed clone infections, as previously observed in mouse models of malaria  and natuThai clone were significantly higher (χ21 = 4.73, P = 0.029) than the SL and mixed clone infections (4+4 or 8+8), which did not differ from one another. Gametocyte densities followed the patterns of asexual parasite densities, except for the high mixed clone infection (8+8), whose peak gametocyte production paralleled that of the Thai clone . Data inspection and model simplification regrouping the treatments into single vs. mixed clone infections revealed that mixed clone infections resulted in greater anaemia for a given parasite density . This increased anaemia was particularly evident for the 4+4 mixed infection group and was reflected in the significantly stronger reticulocyte response in this group (mixed vs. single clone infections χ21 = 6.3, P = 0.012). Once again, the gametocyte sex ratio varied with group and anaemia independently   than SL/4+4 types and both types responded similarly to haematocrit (χ21 = 17.7, P < 0.001) . These sporozoite-induced infections were initiated by gorging a fixed number of infected mosquitoes on the individual birds: 8 mosquitoes per bird for the single clone infections and either 8+8 or 4+4 for the mixed clone infections. Thus we controlled for total or clone-specific dose in the mixed infections. The experiment was performed twice, with a different batch of infected mosquitoes used for each experiment. The resulting infections were very consistent and no differences were found between the two experimental replicates for any of the analyses. Parasite densities of the per se.Thus, again the data support the notion that there is competition between the clones. The greater anaemia in mixed infections was again observed. In addition, the level of anaemia and the reticulocyte response to infection were greatest for the lowest inoculation dose. The extent of anaemia did not, however, seem to affect parasite densities in the mixed infections, where the parasitological parameters measured  aligned with those of either one of the 2 clones. Although the clones do appear to compete aggressively, sub-lethal virulence was more strongly influenced by infecting dose. Thus the sub-lethal measure of virulence (i.e. anaemia) appears to be more strongly dependent on the host's response to infection than on parasite density 21 = 5.6, P = 0.017; percentage infected: χ21 = 4.6, P = 0.032)  was not found to correlate with transmission success . In addition, neither sex ratio  nor gametocyte density  was correlated with transmission success. This is evidence that virulence, as defined by mortality, is positively correlated with transmission success.For the iRBC-induced infections, one set of mosquito transmission studies was carried out on individuals with comparable parasite and gametocyte densities 3 days after inoculation, when parasite densities were low. Transmission success to mosquitoes at this early infection time point, as measured by both geometric mean oocyst load and the percentage of mosquitoes that were oocyst positive, was proportional to the subsequent rate of death (oocyst load: χ2) Figs.  &5b. Inf21 = 4.54, P = 0.032), however the oocyst load was not (χ21 = 3.36, P = 0.067). By contrast, infection type was not found to correlate with the percentage infected (χ23 = 5.56, P = 0.135), but did with the oocyst load (χ23 = 9.71, P = 0.021); this latter result is most likely the consequence of the small sample size (n = 8), three individuals of which were infected with the Thai clone, died on days 4 and 5 and produced very similar oocyst loads in the mosquitoes. The significant correlation between rate of death and the percentage of mosquitoes infected in both iRBC- and sporozoite-induced infections, despite their small sample sizes, provides consistent evidence that virulence, as defined by mortality, is positively correlated with transmission success.For the sporozoite-induced infections Figs.  &6b, we 21 = 1.91, P = 0.16) or the total oocyst load (χ21 = 0.2, P = 0.63) between those individuals that died at any time and those that lived. However, those individuals that survived for the 4-day acute transmission period, irrespective of whether they subsequently died, infected a higher percentage of mosquitoes (percentage infected: χ21 = 12.9, P < 0.001) and with a higher mean oocyst density mosquitoes (χ21 = 18.2, P < 0.001) than those 3 individuals that died rapidly. Excluding these 3 early deaths, we compared the transmission success of those that lived and died following the acute transmission period. There was no significant effect of host mortality on either the percentage of mosquitoes infected (χ21 = 2.37, P = 0.12) or on mean oocyst loads (χ21 = 1.07, P = 0.33). These results suggest that although parasite-induced mortality may incur a transmission cost by reducing the duration of transmission, there is no additional effect of mortality on transmission success. This absence of a positive relationship between virulence and transmission success contradicts the results in the previous paragraphs where the rate of mortality was correlated with a single day measure of transmission success. This may, however, be the consequence of the small sample size  and complexity of infection types.For the sporozoite-induced infections, we then examined the relationship between mortality and parasite lifetime transmission success, as defined by total mean geometric oocyst load and total percentage of mosquitoes infected. For these analyses, individuals were first grouped according to whether they lived or died, and secondly, if they died, whether it was before or after the 4-day acute transmission period. There was no significant difference in either the percentage of mosquitoes infected (χ21 = 12.2, P = 0.0005) and the total mean oocyst load (χ21 = 10.21, P = 0.0015). In the Thai clone infections, anaemia at infection peak was negatively correlated to total mean oocyst load (χ21 = 7.28, P = 0.007) and the percentage of mosquitoes that were infected (χ21 = 8.4, P = 0.004); in the SL clone infections, however, there was no correlation between anaemia and either oocyst load (χ21 = 0.85, P = 0.36) or the percentage of infected mosquitoes (χ21 = 0.56, P = 0.46). When examining the data for all infection types, although the 8+8 mixed clone infections aligned with the Thai clone infections when considering the oocyst load, this was lost when considering the percentage of mosquitoes infected  attempt to achieve maximal transmission. For the case of malaria, the production of transmission stages (gametocytes) depends on the asexual replication of the parasite clone within the host's blood system. Thus, faster asexual replication would be expected to result in more transmission before the host's immune responses control the infection, but also to cause greater host damage and to increase the risk of the host's death. Hitherto, experimental studies have either used gametocyte density as a proxy for transmission success , or take section ,12. Irre section .per se incurred no cost to transmission. In addition, more virulent infections were generally more infectious prior to host death. These results conform to standard virulence theory where there is a relationship between transmission and virulence, such that intermediate levels of virulence evolve. By contrast, the relationship between transmission success and sub-lethal virulence (anaemia) was less clear. On the one hand, infections with one of the parasite clones (Thai) confirm the suggestion from the mouse models [SL has been shown to adaptively alter its gametocyte sex ratio according to the host response to anaemia [Thai clone was different from that of the SL clone, though its response to anaemia appeared similar . If the two clones differ in a transmission trait (sex ratio) that is apparently sensitive to host anaemia, they may be expected to vary in other, less evident traits implicated in transmission. Such variation in transmission traits would result in between-clone variability in the anaemia/transmission relationship. Further exploration of the covariance in sex ratio and the anaemia/transmission relationship is clearly required to ascertain whether sex ratio can be used as a marker phenotype for the complex interactions between the parasite and the host that determine parasite transmission success. In conclusion, the data are not inconsistent with the proposal that sub-lethal effects may impose an upper limit on virulence [Previous work in experimental mouse models found there to be positive genetic correlations between virulence, asexual density and transmission . Howevere models  that sube models , sub-let anaemia . Notablyirulence , but a mvs. single clone infections [The final consideration concerns how the complexity of infection may alter the virulence/transmission trade-off. Competition between clones in multiple infections would generally be expected to favour the more virulent clone , althougfections . Anaemiafections . If parafections . Thus, tThai clone reached a higher peak density. Thus we found dose-dependent differences that may not only alter the potential competitive outcome of a mixed infection, but may also alter the relative virulent nature of the 2 clones: i.e. relative differences in maximum parasite densities of clones may be more influenced by infecting dose than by clone identity. Our data from two clones are clearly limited, but if infecting dose is important, there may be additional subtle consequences of local heterogeneity in transmission intensity  in addition to increasing R0 [An additional surprising outcome of the mixed infections was that the identity of the dominant clone depended upon infecting dose. Infecting dose has previously been shown to influence disease severity in mouse models, but importantly there were consistent clone-specific differences in disease severity across a wide-range of doses . Here, iasing R0 ,45 and gasing R0 .Plasmodium-host interactions. Therefore, with careful consideration of the biological details of each system, extrapolation from laboratory model to field data may be justified. Where field data fail to fit predictions, it is likely that we are missing important biological processes underlying the system in question [vs. re-infections is markedly different and it is likely that the host haematological response alters as well. The proportion of individuals confronting parasites for the first time (i.e. primary infections) depends on the transmission intensity, which also determines the parasite population structure and the parasite dose. Thus, interpreting field patterns with respect to evolutionary predictions necessitates stratification according to the epidemiology of the populations in question. However, despite such complexity, that both laboratory and field populations of malaria parasites generally conform to evolutionary theories concerning life history traits  and virulence is encouraging, and suggests that evolutionary theory can play an important role in predicting consequences of public health intervention strategies [Research using animal models is generating increasing evidence supporting an adaptive basis to life history traits and virulence of malaria parasites ,24,42. Tquestion . One majquestion ,50, the rategies . It remaP. gallinaceum were used: Strain 8A  and a new Thai strain . In this paper, 8A strain is designated as SL and the Thai strain as Thai. Both strains were cloned with limiting dilution in 5-day old chick hosts  (INRA, France). The clones were maintained in vivo by inoculation of 1 ml infected blood (20–45% parasitaemia, percent red blood cells (rbcs) infected) into naïve chicken hosts, with frequent passage through the mosquito vector, Aedes aegypti (Liverpool Blackeye strain). For experimentation, different, healthy 3-week old White Leghorn chickens were used for each experiment. All experimental animals were maintained according to European Union guidelines. Parasitaemias were obtained with Giemsa staining of daily blood smears. Parasitaemias and reticulocytes were calculated as percentages observed in a minimum of 100 rbcs; gametocytaemias observed in 10,000 rbcs. Haematocrit (number of rbcs per unit volume) was measured daily for each host using a haemocytometer. Thus, parasite and gametocyte densities are the number of rbcs infected with any parasites or gametocytes per unit volume. Mature male and female gametocytes are distinguishable after Giemsa staining: males stain a pale rose with no distinct nucleus, females stain blue with a distinct red nucleus [Two strains of the chicken malaria parasite  nucleus ,52. Sex Ae. aegypti mosquitoes were used in all transmission studies. Mosquitoes were maintained under standard conditions (80% humidity and 26°C). Transmission success was measured by (i) the percentage of mosquitoes positive for oocyst stage parasites and (ii) the mean oocyst density in gorged mosquitoes. Oocysts are the zygote stages of the parasite found on the mosquito stomach wall and which are those parasites that have developed successfully from fertilised female parasite gametes . Oocyst stage parasite counts were made 7 days post mosquito infection on midguts dissected from 30 gravid females and then stained with 0.5% mercurochrome in 1× Phosphate Buffer solution. Mean oocyst number per mosquito was chosen as an additional measure of transmission success to the number of infected mosquitoes for several reasons. Firstly, oocyst number is a more sensitive measure and is related to the percentage of infected mosquitoes by a simple negative binomial relationship [tionship . Secondltionship  for rele6 parasites: 6 were inoculated with the SL clone, 6 with the Thai clone and 6 with 5 × 105 of each clone. Ae. aegypti mosquitoes were gorged upon 3 infected chickens from each group, chosen for their matching parasite densities on the day P. gallinaceum was detectable by blood smear (>0.1% parasitaemia). Transmission success (mean number of oocysts per mosquito) on day 3 post inoculation, when parasites were visible in the blood smear (>0.1%), was subsequently related to the day at which that individual died.a) Infection was initiated by injection of parasitized blood to generate an uncontrolled infection (in this paper, these infections are denoted iRBC-induced). Chickens were inoculated by intra-muscular injection of 10SL clone (ii) 8 infective bites per host using the Thai clone (iii) 4 infective bites from the Thai clone and 4 infective bites from the SL clone per host (iv) 8 infective bites from the Thai clone and 8 infective bites from the SL clone per host. The presence of sporozoites in each of these mosquitoes was verified subsequently by dissection of the salivary glands. (Note: the clones do not differ in the gene sequences of the molecular markers available for this species in Genbank (NCBI), so that the clones could not be differentiated and identified, unpubl. data). This experiment was repeated using different batches of mosquitoes infected with either parasite clone and healthy, uninfected chickens. Within each experiment for each parasite clone, each individual was infected by mosquitoes coming from the same cage.b) Infection was performed using infectious mosquitoes to mimic natural low intensity infections (sporozoite-induced infections). Experimental infections were induced by gorging infected mosquitoes on the chicken hosts. 4 experimental groups (3 chickens per group) were considered: (i) 8 infective bites per host using the Ae. aegypti mosquitoes were gorged upon all infected chickens on the 1st day P. gallinaceum became patent in the blood (<0.1% parasitaemia), predictably day 7 , and for the next 3 days at which point the infection reaches its zenith; this is the acute stage of infection. Post-peak infection the parasite is rapidly cleared by the chicken and resurges only intermittently at very low densities. This chronic stage of infection represents the infectious reservoir yielding irregular low levels of transmission to mosquitoes [squitoes . The iniStatistical analyses were conducted using the statistical package Genstat 6.1. Because each individual chicken was included in the data set many times, we corrected for repeated measures by fitting a generalised linear mixed model (GLMM procedure), nesting day within individual chicken in the random model. Parasite densities and blood cell counts were analysed specifying a Poisson error structure.Transmission success: the percentage of mosquitoes that were infected was analyzed using logistic regression specifying a binomial error structure with a logit link function and the mean oocyst densities were analyzed with a logistic regression specifying a Poisson error structure with a logarithmic link function . For th2 distribution. When sex ratio was used as an explanatory variable it was arcsine-transformed.For the sex ratio and transmission analyses the data were over-dispersed and so were corrected for by estimating a dispersion parameter for each analysis. Statistical significance was presented as Wald statistics, which have a χRELP carried out the experimental studies, TL assisted in iRBC-induced studies, CMG assisted in experimental design, NS isolated the Thai strain parasite, PTB assisted in experimental design and edited the manuscript and JCK in elaboration of the manuscript.