MDR1 and CYP3A5. The correlation between genetic polymorphism and sedative effects was assessed. Regarding MDR1 gene, the plasma concentration of midazolam was greater in patients with CGC haplotype (P = 0.012), while it was lower in patients with CAC haplotype (P = 0.005) than in those with other haplotypes. However, genetic polymorphism of neither MDR1 nor CYP3A5 correlated with the plasma concentration of 1-hydroxymidazolam. CGT haplotype of MDR1 was significantly correlated with sedation grade after midazolam administration (P = 0.042). In contrast, genetic polymorphism of CYP3A5 was not correlated with sedation grade. There was no association between genetic polymorphism of MDR1 or CYP3A5 and selected adverse reactions related to midazolam. Genetic polymorphism of MDR1 influences the concentration of midazolam and the sedation grade. However, it is not associated with adverse reactions such as paradoxical response and retrograde amnesia.Genetic polymorphism can result in abnormal pharmacodynamics that subsequently leads to the individual variance in sedative effects and adverse reactions. The aim of this study was to elucidate the association between midazolam-related genetic polymorphism and sedative effects, including adverse reactions, under conscious sedation during upper gastrointestinal endoscopy. We prospectively enrolled 100 eligible patients undergoing upper gastrointestinal endoscopy. The efficacy of the sedation, adverse reactions, plasma concentration of midazolam and 1-hydroxymidazolam were investigated as well as the genetic polymorphism of  The advancement of endoscopic technique and expansion of its application area was largely attributable not only to the development of the endoscopic device itself, but also to the improvement of sedation methods and drugs3. Conscious sedation in GI endoscopy was introduced to promote the patient tolerance and cooperation during the procedure4. Sedation can reduce the patient’s fear of the procedure through anxiolytic effect and transient amnesia which allows the patients to undergo an endoscopy in a comfortable state.Upper gastrointestinal (GI) endoscopy is a common and essential procedure performed for diagnostic or therapeutic purposes in patients with GI symptoms5. This is due to its several advantages such as a short half-life, fast onset of sedation, and strong anterograde amnesia effect6. However, as the effective dose range is considerably variable among individuals, this might cause difficulties in achieving stable sedation during the procedure as well as oversedation-related problems, such as respiratory depression or resedation.To achieve periprocedural safety and patient compliance, sedative drugs should lead to rapid recovery after endoscopic procedures, minimizing the occurrence of adverse reactions. Among various types of sedative drugs available, midazolam is the most widely used drug for conscious sedation in GI endoscopy7. Furthermore, there is an absence of a well-defined set of practice guidelines for the determination of patient suitability for successful conscious sedation in GI endoscopy5. A recent study reported that patients with genetic variability in the gamma-aminobutyric acid (GABA) receptor may result in an abnormal pharmacodynamic response including a differing affinity for midazolam8.Predictive factors of adverse reactions to midazolam during GI endoscopy, including paradoxical response and retrograde amnesia, are unclearTherefore, we hypothesized that genetic polymorphism can alter the pharmacodynamics of midazolam, eventually inducing variances in sedative effects and adverse reactions. The aim of this study was to elucidate the association between midazolam-related genetic polymorphism and sedative effects, including adverse reactions, under conscious sedation during upper GI endoscopy.This was a prospective observational study, and patients aged 18–70 years who underwent upper GI endoscopy at Chung-Ang University Yong-san Hospital were screened over a 1-year period. A total of 100 eligible patients who agreed to participate in this study were consecutively included. The exclusion criteria were as follows: American Society of Anesthesiologists status III-V; history of an upper respiratory infection within the previous 4 weeks; having neurologic symptoms causing poor cooperation during endoscopy; psychological disorders; respiratory diseases requiring oxygen therapy, such as bronchial asthma, chronic obstructive respiratory disease, or sleep apnea; pregnancy; Child-Pugh class B or C cirrhosis; chronic kidney disease; receiving treatment with psychoactive medications; an illicit drug user or a heavy alcoholics; a history of allergic reaction to drugs to be administered; other severe medical conditions not suitable for sedative endoscopy; undergoing endoscopic procedures in an emergency setting. In addition, we also excluded patients taking medications which can interact with drugs in the benzodiazepine class, or midazolam specifically, such as erythromycin, verapamil, diltiazem, itraconazole, and ketoconazole. The study protocol was approved by the Institutional Review Board of Chung-Ang University College of Medicine (No. 2006-013-08-1). This study was performed in accordance with the guidelines and regulations of the above mentioned Ethics Committees, and written informed consent was obtained from all study participants.As premedication for endoscopic examination, the patients were given 5 mg of cimetropium bromide by intramuscular injection, 10 mL of simethicone bromide orally 20 minutes before the procedure. For topical anesthesia of pharynx, the patients were asked to gargle with 10 mL of lidocaine viscous oral solution 2% for 5 minutes before swallowing it. The patients received oxygen insufflation at a rate of 2 L/min via nasal cannula throughout the procedure. Sedation was performed with intravenous bolus midazolam at a dose of 0.06 mg/kg. Prior to the administration of midazolam, consciousness was assessed in all the patients. To achieve conscious sedation in the patients, the target level of sedation was set to moderate degree, which means that patients can respond purposefully to verbal commands, either alone or accompanied by light tactile stimulation.Blood pressure, heart rate, respiration rate, arterial oxygen saturation, and the electrocardiogram  were monitored continuously throughout the procedure. For measurement of plasma midazolam concentration and genotype testing, 5 mL of venous blood was collected 20 minutes after the administration of midazolam. The blood was stored in a vacuumed ethylenediaminetetraacetic acid tube at −30 °C. Upper GI endoscopy was performed by one experienced gastroenterologist (J.K.) in a standardized environment using videoendoscopy equipment .To monitor and assess the level of sedation, the Ramsay Sedation Scale Table  was recoThe same observer also reported any other adverse events that occurred secondary to sedation. The paradoxical response was defined as a condition where midazolam precipitated hostility, rage, and physical violence, necessitating the restraint of the patient until the effects diminished spontaneously. Retrograde amnesia was evaluated by delayed recall of three easily recognizable, commonly used words. Patients were asked to memorize each word at the time of verbal and visual presentation right before the intravenous administration of midazolam. Memorization and verbal recall were confirmed immediately after the initial presentation of the words. To minimize the effect of decreased cognitive function by midazolam, the observer assessed patients’ recall of words only after the patient was leaving the recovery room. If the patient failed to remember any of the three words, retrograde amnesia was considered present. The patients were discharged once they had achieved ≥9 on the Modified Aldrete Scoring System and had reported no pain or any other type of discomfort. Every patient completed a satisfaction questionnaire before leaving the facility. The observer responsible for the monitoring contacted the patients to administer a questionnaire that evaluated patients’ satisfaction with the procedure, adverse events, and the resumption of domestic activities. Patient satisfaction was evaluated with the VNRS system . Similarly, the VNRS was also applied to the endoscopists to assess the level of satisfaction regarding the patient cooperation and ease of endoscopic procedures.Blood samples were centrifuged for genotype testing and genomic DNA was extracted by QIAamp DNA Mini kit  using the leukocyte layer. Blood samples were then centrifuged at 2500 rpm for 10 minutes followed by separation of the leukocyte layer. In the leukocyte layer, 200 μl was collected, mixed with 20 μl QIAGEN protease and 200 μl Buffer AL, and then shaken in a vortex mixer for 15 seconds. After incubation at 56 °C for 10 minutes, the mixture was centrifuged. A total of 200 μl of 96–100% ethanol was added and the sample was vortexed again for 15 seconds, after which it was centrifuged again at 2500 rpm for 10 minutes. The mixture was transferred into a QIAamp Spin Column and centrifuged at 13000 rpm for 1 minute. The DNA was then extracted according to the manufacturer’s recommendation. The A260/A280 ratio of the extracted DNA samples was 1.7–1.9, confirming that they were relatively pure.et al.9 was adopted for DNA amplification by polymerase chain reaction (PCR). The total PCR reagent reaction volume was 25 μl, comprising 2 μl g-DNA, 0.1 μl Taq polymerase , 2 μl (100 µM) deoxynucleotide, 2.5 μl 10 × buffer, 17.4 μl AC D/W, and 0.5 μl sense and antisense primers. The PCR conditions were as follows: 5 minutes of pre-denaturation at 94 °C, followed by 30 cycles of 30 seconds of denaturation at 94 °C, 56 annealing at 60 °C for 30 seconds, synthesis for 1 minute at 72 °C, and extension for 10 minutes at 72 °C. The genotypes of MDR1 1236C > T (assay ID: C__7586662_10), ABCG2 421C > A (assay ID: C__15854163_70), and ABCC2 1249G > A (assay ID: C__22272980_20) single nucleotide polymorphism (SNP) were analyzed by the established TaqMan Genotyping Assays .The method of Lindberg P < 0.05.Data were expressed as number or percentage (%) and analyzed statistically using the Statistical Package for Social Sciences program . Alleles of modified SNP and genotype frequency were evaluated by Chi-square test with deviation from Hardy-Weinberg equilibrium. Correlation between genetic polymorphism and plasma concentration of midazolam and 1-hydroxymidazolam was analyzed with Student’s t-test. A chi-squared test or Fisher’s exact test was used to analyze the correlation between genetic polymorphism and the grade of sedation assessed with the Ramsay Sedation Scale, and adverse events after administering midazolam. Results were considered statistically significant at 2, respectively. Following midazolam administration, 13 patients (13%) exhibited a paradoxical response and 32 patients (32%) showed retrograde amnesia. One patient complained of drowsiness after midazolam administration. During endoscopy, two patients experienced hypoxemia and three patients exhibited hypotension, which were temporary and quickly recovered with conservative management. The mean Ramsay Sedation Score was 2.8 ± 0.1. The mean Modified Aldrete Score was 9.02 ± 0.75, 8.99 ± 0.89, and 9.07 ± 0.84 at 0, 5, and 15 minutes after the arrival in the recovery room, respectively. The satisfaction scores evaluated with VNRS (0–10) for the patients and the endoscopist were 8.59 ± 1.91 and 8.25 ± 2.11, respectively.A total of 100 patients participated in the study, and their baseline characteristics are shown in Table MDR1 and CYP3A5. For haplotype estimation in five genotypes  in the MDR1 gene, TTT, TGC, CGC, CAC, TTC, CGT, and TAT were predominately generated. The frequency of each genetic variation is shown in Fig. CYP3A5, the 3*/3* type was the most frequently detected . On the contrary, plasma concentration of midazolam in patients with CAC haplotype was lower than that in patients with other haplotypes . Meanwhile, this association was not detected with genetic polymorphism of CYP3A5. With regards to adverse events, there was no association between genetic polymorphism of MDR1 or CYP3A5 and paradoxical response or retrograde amnesia , which is encoded by the MDR1 gene16. Consequently, polymorphism of the MDR1 gene can cause reduced response, refractoriness or resistance to drugs17. This individual variability might subsequently result in differences in the level of sedation and adverse reactions of sedative medication. Based on this, it can be assumed that a patient’s response to midazolam may be related with MDR1 gene polymorphism. Therefore, this study investigated the quality of sedation related to midazolam administration from a viewpoint of genetic polymorphism.Although midazolam has many advantageous characteristics, a number of adverse reactions such as over-sedation and paradoxical excitation can occurMDR1 gene was both associated with increased plasma concentration of midazolam. These haplotypes may be linked to the P-gp substrate that reacts with midazolam5. Due to the association between P-gp and CYP3A, that reduces drug absorption, the metabolism of midazolam to 1-hydroxymidazolam should be modulated by P-gp18. P-gp inhibition resulted in a modest increase in midazolam metabolism at higher midazolam concentrations18. However, the concentration of midazolam did not correlate with adverse reactions including paradoxical response and retrograde amnesia. Since adverse reactions occur in dose-dependent manner7, another factor besides plasma midazolam concentration may be attributable to occurrence of these adverse reactions.We found that the presence of CGC haplotype and absence of CAC haplotype in the MDR1 gene was associated with higher sedation grade after administrating midazolam (P = 0.042). This suggests that the CGT haplotype may be associated with the binding of midazolam to the P-gp substrate. Therefore, variations in the phenotypic activity of P-gp due to genetic polymorphisms or competitive inhibition may determine a patient’s susceptibility to sedative effects and side effects of midazolam.CGT haplotype of MDR1 nor CYP3A5 genetic polymorphism correlated with a paradoxical response after administrating midazolam in the present study. Although the exact mechanism of such paradoxical reactions is unknown, several risk factors for this reaction have been reported including midazolam dose, age, gender, psychological background, alcohol abuse, and genetic background7. In particular, genetic variability in the benzodiazepine receptors of GABAA channels, resulting from the multiple allelic forms, may lead to abnormal pharmacodynamic responses in some patients19. Although the mechanism behind the midazolam and retrograde amnesia relationship is unknown, retrograde amnesia post administration of midazolam is dose dependent20. However, there was no significant association between genetic polymorphism and retrograde amnesia following administrating midazolam in the present study. These results suggest that paradoxical response and retrograde amnesia may be a P-gp-independent reaction.However, neither There were a few limitations in this study. First, some patients with insufficient sedation might have shown aggressiveness, violent behavior, or hyperactivity during the procedures. However, it is impossible to perfectly distinguish between insufficient sedation and paradoxical response. Besides, the mean Ramsay Sedation Score was 2.8 in the present study. Moderate sedation, regarded as the appropriate state in conscious sedation, corresponds to a score of three on the Ramsay Sedation Scale (exhibiting brisk response to light glabellar tap or loud auditory stimulus). The satisfaction scores reported by patients and endoscopists were also consistently high. These results indicate that the quality of sedation in this study was generally adequate from the viewpoint of both the patients and endoscopists. Second, it was difficult to confirm the correlation among genetic polymorphism and adverse reactions related with midazolam in relatively small number of patients. In addition, it was difficult to completely rule out the possibility of transient cognitive impairment induced by midazolam might have affected the test results for retrograde amnesia. To minimize the possible effect of impaired cognitive function, the test was done when the patients were leaving the recovery room after confirming the modified Aldrete Score met the criteria. Nevertheless, the true frequency of retrograde amnesia could be lower than the results of this study. Third, as this study investigated the pharmacologic and clinical perspectives of midazolam only, one should be cautious in generalizing the results to the clinical circumstances where mixed sedative agents are concurrently administered. Nevertheless, midazolam has been commonly used as a single sedative agent and still being frequently used in sedation endoscopy in many countries, which makes the results of this study valuable. Fourth, we used Ramsay sedation scale for monitoring the sedation depth during the procedure, which was further dichotomized into two groups in the analysis. This approach to the evaluation and assessment of sedation might be regarded rather crude, compared to EEG monitoring or a combination of clinical sedation scales. However, these advanced monitoring methods are neither easily applicable nor routinely used in diagnostic upper endoscopy sessions. Despite the objectivity issue, the Ramsay sedation scale still has advantages of easy applicability in real clinical practice.MDR1 gene was associated with plasma midazolam concentration and sedation grade after midazolam administration. Although genetic polymorphism was not associated with adverse reactions, patients’ responses and conscious states varied following the administration of midazolam. Therefore, close monitoring until discharge is mandatory in upper GI endoscopy under sedation.In conclusion, genetic polymorphism in


Salmonella senses and responds to harsh environments within the host. Persistence in a bile-rich environment is important for Salmonella to infect the small intestine or gallbladder and the multidrug efflux system AcrAB-TolC is required for bile resistance. The genes encoding this system are mainly regulated by the ramRA locus, which is composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, whose encoding gene is itself transcriptionally repressed by RamR. RamR recognizes multiple drugs; however, the identity of the environmental signals to which it responds is unclear. Here, we describe the crystal structures of RamR in complexes with bile components, including cholic acid and chenodeoxycholic acid, determined at resolutions of 2.0 and 1.8 Å, respectively. Both cholic and chenodeoxycholic acids form four hydrogen bonds with Tyr59, Thr85, Ser137 and Asp152 of RamR, instead of π–π interactions with Phe155, a residue that is important for the recognition of multiple compounds including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6 G. Binding of these compounds to RamR reduces its DNA-binding affinity, resulting in the increased transcription of ramA and acrAB-tolC. Our results reveal that Salmonella senses bile acid components through RamR and then upregulates the expression of RamA, which can lead to induction of acrAB-tolC expression with resulting tolerance to bile-rich environments.During infection,  Salmonella is a bacterial pathogen that causes a variety of foodborne illnesses in humans. During the course of its infection of the intestinal tract and gallbladder, Salmonella is exposed to bile acids. These are detergent-like biological substances that are synthesized in the liver from cholesterol and stored in the gallbladder. These bile acids possess strong antimicrobial activity, disrupt cell membranes, denature proteins and trigger DNA damage2. Enteric bacteria such as Salmonella must tolerate the presence of bile acids in order to survive in the gastrointestinal transit and gallbladder4. Enteric bacteria have developed an intrinsic resistance to the toxic effects of bile acids, due to the low permeability of their outer membranes to lipophilic solutes and the presence of active efflux mechanisms6.Salmonella enterica serovar Typhimurium (S. Typhimurium) contains at least nine multidrug efflux systems7. Among these, the AcrAB-TolC system, whose AcrB transporter belongs to the resistance–nodulation–cell division family, is particularly effective in generating bile acid resistance8. Bile induces the expression of acrAB8, and this induction is mediated by the transcriptional regulators RamA and RamR10. The global transcriptional activator RamA, which belongs to the AraC/XylS family of regulatory proteins, activates the expression of acrAB and tolC genes9. The local transcriptional repressor RamR, which is located directly upstream of ramA, belongs to the TetR family of regulatory proteins and represses the expression of both ramA and ramR , and a secondary bile acid (deoxycholic acid) on the expression level of ramA in the wild-type S. Typhimurium strain and its ramR deletion mutant  and in the ramR deletion mutant (2.7-fold increase).It has previously been shown that ant Fig. . We confant Fig. . Among tramA in a RamR-dependent manner, we hypothesized that these compounds are recognized by RamR. To test the possibility that cholic and chenodeoxycholic acids may bind to RamR, we employed surface plasmon resonance (SPR) analysis using a Biacore T200 instrument . Cholic and chenodeoxycholic acids were separately passed over RamR immobilized on a CM5 sensor chip and the SPR responses indicated that both bound directly to RamR, with the responses increasing in a concentration-dependent manner . Both bile acids inhibited the binding of RamR to the DNA, in a concentration-dependent manner  indicated a much weaker ability of this bile acid to reduce the RamR DNA-binding affinity  of the acrAB and tolC genes in response to bile. We previously reported that bile induces AcrAB efflux pump expression in Salmonella through a specific regulatory protein, RamA, and, additionally, that cholic acid appears able to bind to this protein9. More recently, we also identified a different induction mechanism of acrAB in response to bile, whereby the bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs via transcriptional derepression of the ramA activator gene, likely via the RamR repressor protein controlling expression of ramA10. Although the relative importance of both mechanisms has not yet been investigated, we suspect that these two different modes of regulation may coexist. A possible scenario is that bile first binds to and activates RamA and then, when the concentration of bile increases, proceeds to bind to RamR and stimulates RamA expression and the subsequent overproduction of the AcrAB-TolC efflux system. This scenario, however, requires further careful investigation.The transcription of ramA expression in a RamR-dependent manner. We tried to crystalize RamR bound to deoxycholic acid but failed, probably because it lacks the 7α-hydroxyl group that is important in forming a hydrogen bond with Asp152 of RamR. Indeed, the inhibitory effect of deoxycholic acid on RamR-binding to the ramA promoter region is much weaker than that of cholic and chenodeoxycholic acids 22. Bacterial strains were grown at 37 °C in Luria–Bertani broth supplemented, when appropriate, with ampicillin (100 µg/ml), kanamycin (25 µg/ml) or chloramphenicol (25 µg/ml).The bacterial strains and the plasmids used in this study are listed in Supplementary Table ramA expression, 25.6 mg/ml of a crude ox bile extract purchased under the label ‘sodium choleate’ , or 5 mM sodium cholate hydrate, sodium chenodeoxycholate or sodium deoxycholate monohydrate  was added to the medium. Sodium choleate, whose precise composition was not determined, has previously been used in Salmonella gene expression experiments at concentrations of 3% (w/v), i.e., 30 mg/mL or higher8. We chose a concentration of 25.6 mg/mL, because it was the highest concentration that allowed the normal growth of the Salmonella strains used in this study.In order to investigate the effects of the bile acids on 600 of 0.6) and harvested by centrifugation. Pelleted cells were stabilized with RNAprotect Bacteria Reagent (Qiagen) and stored at −80 °C until needed. Total RNA was extracted using an RNeasy Mini kit (Qiagen). Removal of residual genomic DNA was performed using a Turbo DNA-free kit (Ambion) and checked by negative PCR amplification of the chromosomal sequence. RNA integrity was verified by electrophoresis on a 1% agarose gel. Total RNA (1.5 µg) was reverse-transcribed using random hexamers and the Superscript III First Strand Synthesis System (Applied Biosystems). The expression level of each gene of interest was calculated as the average of the results from three independent cDNA samples. For each cDNA sample and each gene, qRT-PCR runs were performed in duplicated wells and the primers used for qRT-PCR are listed in Supplementary Table gmk, gyrB and rrs). A two-tailed Student’s t test was used to assess significance using a p value < 0.05 as cutoff.Bacteria were grown until mid-log phase , 75 mM NaCl and 2 mM DTT. The crystals grew to optimal size within 1 week and were cryoprotected using a solution containing 20% glycerol. Crystals were picked using LithoLoops (Protein Wave) and subjected to flash cooling in a cold nitrogen gas stream (100 K) from a cryostat (Oxford Cryosystems). All data sets were collected on beamline BL44XU at SPring-8 with a CCD detector MX225-HE (Rayonix) and at a cryogenic temperature of 100 K. The diffraction data were processed and scaled using the HKL200023 package. The crystal structures of bile-bound RamR were determined at 2.00 and 1.78 Å resolution by the molecular replacement method using the program MOLREP24 from the CCP4 software suite. The atomic coordinates of the ligand-free RamR (Protein Data Bank [PDB] code: 3VVX) were used as the search model. The model was refined with the CNS25, REFMAC26 and COOT27 software and stereochemical quality was assessed with RAMPAGE software28. Crystal parameters and refinement statistics are listed in Supplementary Table Purified RamR protein was prepared as described previouslyramA were also analyzed by SPR spectroscopy. A 3′-biotinylated 100-bp DNA fragment of the ramR–ramA intergenic region, as well as a control 100-bp control fragment of the gyrB gene, were each immobilized onto flow cells in a NeutrAvidin (Thermo Scientific)-coated CM5 sensor chip . Analyses were performed at 25 °C and at a flow rate of 30 µl/min. The purified RamR protein was diluted in running buffer , incubated with 1.0–500 µM of cholic, chenodeoxycholic or deoxycholic acids, then injected onto the sensor surface in two replicates for 2 min. Dissociation was also recorded for 2 min. Calculations of affinity constants were performed using BIAevaluation software .The interaction between RamR and each substrate was analyzed by SPR spectroscopy with a Biacore T200 biosensor instrument . RamR was immobilized onto flow cells in a CM5 sensor chip using an amine-coupling method. Binding analyses were carried out at 25 °C and a flow rate of 30 µl/min. Cholic and chenodeoxycholic acids were passed over the RamR at several concentrations, as indicated. An empty flow-cell lacking immobilized protein was used as a reference. The inhibitory effects of cholic, chenodeoxycholic and deoxycholic acids on the interaction between RamR and PProtein Data Bank: coordinates have been deposited under accession codes 6IE8 and 6IE9 for the RamR structures co-crystallized with cholic and chenodeoxycholic acids, respectively.Supplementary Information


Schizoaffective Disorder is a controversial and poorly understood diagnosis. Experts disagree on whether it is a discrete disorder; whether it is on a spectrum between Bipolar Disorder and Schizophrenia or whether it even exists. Lack of individual research attention given to this disorder, changing diagnostic criteria and hence poor diagnostic stability have all contributed to the dearth of knowledge surrounding Schizoaffective Disorder.To describe the profile of mental health care users (MHCUs) diagnosed with Schizoaffective Disorder and determine the degree of agreement between the clinicians’ diagnosis and Operational Criteria (OPCRIT).All MHCUs at Helen Joseph Hospital psychiatric unit with Schizoaffective Disorder between 01 January 2004 and 31 December 2010 were included. The demographic, clinical and treatment profiles as well as data required for OPCRIT were extracted from hospital records and discharge summaries.Most MHCUs with Schizoaffective Disorder were female (68.89%), with a mean age of illness onset of 25 years (SD ± 7.11), had a family history of mood disorders (76.92%) and displayed impaired functioning. Majority (80%) were treated with at least one antipsychotic and one mood stabiliser. No agreement was found between the clinicians’ diagnosis and OPCRIT.While the profile of MHCUs with Schizoaffective Disorder in this study is similar to other studies, the lack of agreement between the clinicians’ and OPCRIT diagnoses calls for further research using larger population samples and a dimensional approach to diagnoses in order to improve understanding and management of Schizoaffective Disorder. The psychosis lasted a few weeks to a few months and was followed by recovery. Kasanin described them as having ‘acute schizoaffective psychoses’ and suggested that they were an intermediate group, with their outcome being better than Schizophrenia and worse than mood disorders.In 1933, Jacob Kasanin reported on nine cases that were diagnosed with Dementia Praecox, but who seemed quite atypical.2 Others believe it to be a psychotic mood disorder and not a discrete entity.3Decades after ‘Schizoaffective Disorder’ was coined by Kasanin, it remains poorly understood. Some consider it to be in the middle of a continuum between Schizophrenia and Bipolar Disorder or a heterogeneous group of the two.4 Furthermore, the inter-rater reliability for the individual items of the DSM-IV Schizoaffective Disorder criteria assessed by two psychiatrists using the Composite International Diagnostic Interview was found to be low (Cohen’s kappa of 0.22).5Schizoaffective Disorder has moreover demonstrated poor diagnostic stability (36%) using DSM IV consensus diagnoses assessed by psychiatrists who were blind to previous research diagnoses.6A re-evaluation of 59 MHCUs’ hospital records of patients diagnosed with Schizoaffective Disorder using OPCRIT as well as agreement by two psychiatrists, found that only 6 users met the ICD-10 criteria and none met the DSM IV criteria for the disorder on a lifetime basis.1 However, Kasanin has provided us with the reality that there is a subset of MHCUs that do not fit into the once popular, neat, dichotomous categories of having either a psychotic disorder or a mood disorder. For decades this subset of users has sparked controversy and hence, has been neglected in psychiatric research. Fortunately, interest in the Schizoaffective Disorder construct has been gaining momentum in recent times and attempts have been made to demystify the illness.7 However, we are not yet at the juncture where we can make firm conclusions or give clear guidelines based on the current available evidence.The current concept of Schizoaffective Disorder is quite disparate from that originally illustrated by Kasanin.Ensuring an accurate diagnosis of Schizoaffective Disorder is imperative and perhaps should be the starting point as this has implications for treatment planning and prognosis. It may also allow for more robust research in the future and hence a greater understanding of the disorder.To date, no studies have examined the agreement of Schizoaffective Disorder between the clinician and OPCRIT in South Africa. The acute psychiatric unit at Helen Joseph Hospital is the point of entry for most MHCUs living in that catchment area and is representative of other acute psychiatric units in Johannesburg. This study sought to provide a local perspective and shed some clarity on the diagnosis and profile of MHCUs with Schizoaffective Disorder.8This study used the DSM IV-TR (text revision) definition of Schizoaffective Disorder as this is what was in use at the study site at the time.N = 28). The profile of these users was described and computer-generated DSM IV diagnoses were extracted from OPCRIT.Data was retrieved from the hospital records of all MHCUs with either a discharge, differential or previous routine clinical diagnosis of Schizoaffective Disorder admitted to Helen Joseph Hospital between 01 January 2004 and 31 December 2010. The rationale for also including patients with a diagnosis of Schizoaffective Disorder both previously or as a differential diagnosis was to prevent poor diagnostic stability and poor reliability of retrospective data from excluding likely candidates. Also, if only a discharge diagnosis was considered, the sample size would have been even smaller (9 OPCRIT has shown good reliability (kappa 0.73)10 and excellent agreement with what some authors deemed the ‘gold standard’ consensus best-estimate lifetime diagnosis.11OPCRIT was developed in 1991 by McGuffin et al utilising a poly-diagnostic approach to psychiatric disorders.OPCRIT for Windows version 4.0 is a 90-item checklist of demographic data, psychopathology, pre-morbid functioning, stressors prior to illness onset, personal history and family history. It has built-in algorithms designed to generate reliable diagnoses from case notes according to operational criteria of 12 major classification systems . It has not incorporated DSM IV TR criteria, but no differences exist between the DSM IV and DSM IV TR criteria for Schizoaffective Disorder. Symptoms are recorded for the time frame being studied . This study used ‘other specified episode’ which recorded the episode that had the most information in the users’ files. Considering Schizoaffective Disorder is a longitudinal illness, OPCRIT has additional questions to account for this such as: total duration of illness .The package is specifically for the needs of the researcher and is intended to be used by clinicians or investigators trained in clinical research. It is not recommended for use by raters without previous experience in psychopathology and psychiatric diagnosis.OPCRIT guidelines state:Also, if one is unfamiliar with OPCRIT, the 90 questions programme should be initially used, where data is entered item by item and an adjacent information button provides additional guidance. Therefore, previous experience in psychopathology and use of the guided questions made OPCRIT easy to use despite being unfamiliar with the tool prior to this study.The data was analysed using Statistica version 8.0. Descriptive analysis used frequencies and percentages for categorical variables, and means (standard deviation) or medians (range) where appropriate. Agreement between the clinicians’ diagnosis and OPCRIT was assessed using the kappa coefficient which measures the agreement between two raters and takes into consideration the agreement occurring by chance. It ranges from 0 (no agreement) to 1 (complete agreement).Ethics approval was obtained from the University of Witwatersrand’s Human Research and Ethics Committee.N = 28), as a differential diagnosis (N = 14) or as noted in the past psychiatric history (N = 3)  .N = 45), 68.89% (N = 31) were female (N = 38) were single. Only 11.11% (N = 5) were employed despite 79.07% (N = 34) having 10 years or more of education. Data regarding disability grants were only available for 34 users of which 64.71% (N = 22) were receiving a disability grant.Of the sample  had 5 or more psychiatric admissions. A Substance Use Disorder was present in 24.44% (N = 11) of users but inadequate information precluded further analysis of this.The mean age of illness onset was 25 years (SD ± 7.11), (range 14–44 years) . The meaN = 25) of the users. Of those for which information was available (52% (N = 13), 76.92% (N = 10) had a family history of a mood disorder: 46.15% (N = 6) with Bipolar Disorder and 30.77% (N = 4) with Depression. A family history of Schizophrenia was present in 7.70% (N = 1) of users, while 15.38% (N = 2) had a family history of both Bipolar Disorder and Schizophrenia.A family history of mental illness was present in 55.56% (N = 28), Schizophrenia 20% (N = 9), Bipolar Disorder 15.56% (N = 7) and Substance-induced Psychotic Disorder 2.22% (N = 1) (N = 21) of users, with 90.48% (N = 19) having the Bipolar and 9.52% (N = 2) having the Depressive subtype.Discharge diagnoses were as follows: Schizoaffective disorder 62.22% ( (N = 1) . The SchN = 20) of the sample and were as follows: Schizoaffective Disorder (70% (N = 14), Bipolar I Disorder , Schizophrenia  and Dementia .Differential diagnoses were given to 44% (N = 34) of users follows: Schizophrenia 47.06% (N = 16), Bipolar I Disorder 38.24% (N = 13), Major Depressive Disorder 5.88% (N = 2) and Schizoaffective Disorder 8.82% (N = 3).Diagnoses on previous admissions available for 76% (N = 35) were treated with 1 antipsychotic while 22.22% (N = 10) were treated with two (N = 36) were on a combination of at least 1 mood stabiliser and 1 antipsychotic. No mood stabiliser was prescribed in 20% (N = 9) of users, while 73.33% (N = 33) received one mood stabiliser and 6.67% (N = 3) received two. Of those on two mood stabilisers (N = 3), two were treated with a combination of Sodium Valproate and Lithium and one with Carbamazepine and Lithium. An antidepressant was prescribed for 11.11% (N = 5) of users.Most MHCUs ; followed by Haloperidol ; Flupenthixol Decanoate depot ; Zuclopenthixol Decanoate Depot ; Clozapine ; Trifluoperazine  and Risperidone Consta .Risperidone was most commonly prescribed  of users received Sodium Valproate while the remainder received Carbamazepine , Lithium  or Lamotrigine .Majority  of MHCUs and Mianserin for 2.22% (N = 1).Citalopram was prescribed for 8.89% (N = 25) were referred to their local psychiatric clinic for follow up and 11.11% (N = 5) were referred to a psychiatric placement facility. The remaining MHCUs were transferred to other psychiatric hospitals for further management.Upon discharge, 55.56% (N = 12) (N = 11) of users. The least common diagnoses generated were Schizophreniform Disorder , Delusional Disorder  and Schizophrenia .The most common diagnosis generated by OPCRIT was Psychosis Not Otherwise Specified  . SchizoaN = 12) of users.Entering data into OPCRIT from hospital records was challenging due to lack of detailed information and may explain why Psychosis Not Otherwise Specified (NOS) was the most common diagnosis generated. Information on ‘relation between mood and psychotic symptoms’ an important criterion used to make the diagnosis of Schizoaffective Disorder, was absent for 27.27% (The Kappa Coefficient was found to be (-0.0014), indicating no agreement between the clinicians’ diagnosis and OPCRIT.7 In this sample, females predominated, in keeping with other studies.7 The majority being single, unemployed and receiving a disability grant suggests that they experienced social and occupational dysfunction and may have a poorer outcome. This is consistent with the Vollmer-Larsen study6 but contrasts Kasanin’s original description.1 Unfortunately, the available literature has been inconsistent in predicting outcomes in these users.14Epidemiological data on Schizoaffective Disorder is lacking and collating data is difficult due to poor diagnostic reliability, stability and changing diagnostic criteria.7The broad range of illness onset (14–44 years) is noted elsewhere, with a third being similar to that of Schizophrenia and a third being similar to that of Bipolar Disorder, which again makes categorising Schizoaffective Disorder difficult.15Despite the results suggesting a higher preponderance of mood disorders in the MHCUs’ families, substantial conclusions cannot be drawn as data was absent for 48% of this sub-sample. The fact that these users had both a family history of Schizophrenia and Bipolar Disorder suggests that Schizoaffective Disorder has a link to both mood and psychotic disorders. This is in line with newer evidence demonstrating abnormalities in the gene DISC1 (Disrupted in Schizophrenia 1) in Schizophrenia, Bipolar Disorder and Schizoaffective Disorder.6 The DSM IV criteria has been criticised for not being specific enough regarding the proportion of the duration of the mood episodes to the total duration of the active and residual phases of the illness. The DSM-5 has addressed some of these concerns.16 However, what constitutes ‘majority’ of total duration of illness is not specified in DSM-5 and making the diagnosis still relies heavily on there being adequate historical information as well as clinical judgment, which requires a degree of experience.This sample received various diagnoses at different admissions. Other studies have also documented this diagnostic instability, citing illness evolution, new information being presented and poor reliability of cross-sectional diagnoses as contributory factors.17 They found the most robust evidence for the use of atypical antipsychotics and highlighted that despite ‘classical’ mood stabilisers (e.g. Lithium and anticonvulsants) being commonly prescribed in clinical practice, there is no good-quality evidence to support this.A systematic review of randomised, double-blind, placebo-controlled trials of treatment specific for Schizoaffective Disorder as well as post-hoc analysis of similar trials for Schizophrenia and Bipolar Disorder noted that research is greatly lacking on specific treatment for Schizoaffective Disorder.18 The findings of this study are somewhat in keeping with these prescribing trends as all users received at least one antipsychotic and 80% received at least one mood stabiliser.A review of clinical practice data for the treatment of Schizoaffective Disorder revealed that 93% of users received an antipsychotic, 48% received a mood stabiliser and 42% received an antidepressant.The finding that OPCRIT most commonly diagnosed Psychosis Not Otherwise Specified (26.67%) while Schizoaffective Disorder was diagnosed in 24.44% of users, is not surprising, given the ambiguities and complexities of diagnosing Schizoaffective Disorder. However, insufficient, retrospective data, with the absence of information regarding relationship between mood and psychotic symptoms for 27% of the sample, is also likely to have contributed to this finding. This information is integral to the diagnosis of Schizoaffective Disorder and is treated by the scoring program as 0 if absent. Certainly, supplementing the OPCRIT with a clinical interview and reaching a consensus best-estimate lifetime diagnosis would have been ideal.6 The advantage of their study is that they supplemented this with a consensus diagnosis by two psychiatrists who had reviewed the files. Being able to do this using retrospective records implies that the records contained adequate and relevant information, which was not possible to do in this study.Vollmer-Larsen et al also found no agreement between the clinicians’ diagnosis and OPCRIT.The lack of agreement between the clinicians’ and OPCRIT diagnoses in this study may seem trivial, but one should also consider the underlying implications of this finding. Schizoaffective Disorder is deemed to be rare, ambiguously defined, inadequately studied and therefore poorly understood. Hence, concurrence on the diagnosis of Schizoaffective Disorder cannot be expected at this point.This retrospective study lends itself to many challenges which could have influenced the results including: the quality of the information in the files, histories and mental state examinations done by doctors with different degrees of experience and accuracy of assessments and the temporal relationship between mood and psychotic features often not being clearly documented. Also, information on possible confounding factors such as the impact of substance use disorders and pre-existing or co-morbid medical conditions were scant.Another important limitation is the small sample size, which precludes any meaningful interpretations or conclusions being made from these results.Of course, having no prior experience or training in the use of OPCRIT is also a valid limitation, but this is not a prerequisite for use of the program. Clinical experience and use of the guided 90-item questions are considered adequate to use OPCRIT.More research is urgently recommended in this area. South Africa has a rich heritage with differing cultures, ethnicities and socio-economic backgrounds. It may therefore prove useful to determine whether the epidemiology, illness pattern, treatment response and outcomes vary in this diverse population. The history of Schizoaffective Disorder, with its poor diagnostic stability and reliability is testament to the inadequacy of a categorical approach to psychopathology. Therefore, further studies on Schizoaffective Disorder alone, done longitudinally and using a more dimensional approach may garner greater understanding of Schizoaffective Disorder. The issue of poor inter-rater reliability and diagnostic stability can only be rectified with a clearer definition and objective measures such as genetic markers for the disorder – all of which require further research.The profile of these users is in many ways akin to descriptions in more contemporary international literature. These include a female preponderance of individuals with impaired functioning who have been given a wide variety of diagnoses over time. These findings are not generalisable however, and require replication using larger sample sizes and improved methodology in future studies. This research may not change our clinical practice, but highlights the need for greater vigilance with careful documentation of these users’ illness trajectory and treatment response.


Postoperative urinary retention (PO‐UR) is an acute and painful inability to void after surgery that can lead to complications and delayed hospital discharge. Standard treatment with a urinary catheter is associated with a risk of infection and can be distressing, undignified and uncomfortable. This systematic review aimed to identify effective interventions for the prevention and treatment of PO‐UR that might be alternatives to urinary catheterization.Electronic databases were searched from inception to September 2017. Randomized trials of interventions for the prevention or treatment of PO‐UR were eligible for inclusion. Studies were assessed for risk of bias using the Cochrane (2.0) tool. Two reviewers were involved at all review stages. Where possible, data were pooled using random‐effects meta‐analysis. The overall quality of the body of evidence was rated using the GRADE approach.Some 48 studies involving 5644 participants were included. Most interventions were pharmacological strategies to prevent PO‐UR. Based on GRADE, there was high‐certainty evidence to support replacing morphine in a regional anaesthetic regimen, using alpha‐blockers (number needed to treat to prevent one case of PO‐UR (NNT) 5, 95 per cent c.i. 5 to 7), the antispasmodic drug drotaverine  and early postoperative mobilization  for prevention, and employing hot packs or gauze soaked in warm water for treatment . Very few studies reported on secondary outcomes of pain, incidence of urinary tract infection or duration of hospital stay.Promising interventions exist for PO‐UR, but they need to be evaluated in randomized trials investigating comparative clinical and cost effectiveness, and acceptability to patients. Postoperative urinary retention (PO‐UR) is frequently regarded as a minor adverse side‐effect of surgery, easily resolved by catheterization. The condition can, however, be distressing. Patients may find catheterization invasive, undignified and uncomfortable. Catheterization also carries risks of urinary tract infectionMultiple perioperative predictors have been proposed for the development of PO‐UR, including age, sex, type of anaesthesia and analgesiaInterventions to prevent or treat PO‐UR include pharmacological , massage, acupuncture and warm compress approaches. Interventions targeting anaesthesia and analgesia represent a potential preventive strategy. A clinical protocol of interventions effective in preventing PO‐UR, and treatment alternatives to catheterization, could lead to improved postoperative morbidity, reduction in associated complications, and improvements for patients with respect to dignity, comfort and psychological wellbeing.The aim of this systematic review was to provide an overall summary of the available trial evidence on interventions to prevent or treat urinary retention after surgery.This review followed Cochrane recommendations for conducting systematic reviewsRCTs investigating any intervention with the specific aim of preventing or treating PO‐UR in an adult surgical population were included. Where the intervention was aimed at preventing PO‐UR, any adult surgical population was included. Where an intervention was aimed at treating PO‐UR, any adult surgical population experiencing PO‐UR according to the authors' definition was included. Populations with known pre‐existing urinary problems, such as benign prostatic obstruction, cancer of the bladder or prostate, or urinary incontinence, were excluded. Studies had to report incidence of PO‐UR, defined as urinary retention with a postoperative onset requiring urgent intervention, such as catheter insertion, recatheterization (where catheter use was part of the treatment protocol), pharmacological or non‐pharmacological treatment. Catheter use was considered as a proxy measure of PO‐UR (if not stated explicitly that the catheter was inserted to treat urinary retention). Outcomes defined as ‘postoperative voiding dysfunction’, ‘voiding difficulties’ or based on a postvoid residual urinary bladder volume alone were not considered PO‐UR.Appendix S1, supporting information). Searches were limited to studies in humans, but were not limited by language, date or publication status.The following electronic databases were searched from inception to September 2017: MEDLINE (OVID) including MEDLINE in process, Embase (OVID), Cochrane Library (Wiley), Cumulative Index to Nursing and Allied Health Literature (CINAHL) (EBSCO), Science Citation Index and Conference proceedings – Science (Web of Science). The search strategies were developed specifically for each database (MEDLINE example included in www.who.int/ictrp/en) and National Institutes of Health Clinical Trials .Supplementary internet searches were undertaken to identify grey literature, including ongoing and completed clinical trials from the WHO International Clinical Trials Registry . Taking on the role of a reviewer, the public contributor was actively involved in all aspects of screening and data extraction. Titles and abstracts were each screened independently by two members of the review team. All references that did not meet the inclusion criteria were excluded. Full‐text papers for each of the remaining references were obtained and examined independently in detail by two reviewers. Discrepancies between reviewers at each stage were resolved by discussion with a third reviewer.® 2010  to include: eligibility criteria, study characteristics, participant characteristics, intervention, comparator, outcome definitions and outcome data for incidence of PO‐UR. Data were also extracted for: rates of urinary tract infection (UTI), duration of hospital stay, patient acceptability, adverse events  and pain scores. Data extraction was performed by one reviewer and checked by a second; disagreements were resolved by discussion with a third reviewer. Where PO‐UR was reported at multiple time points, data for the initial time point were extracted.Data were extracted using a standard form developed in Microsoft AccessIncluded studies were assessed independently for risk of bias by two reviewers, using the Cochrane Risk of Bias tool (ROB 2.0)IComparisons were grouped by aim , intervention type  and intervention category. For dichotomous data, an odds ratio (OR) for each comparison with associated 95 per cent confidence intervals was calculated. Continuous data were analysed as the weighted mean difference (MD) between groups and presented with associated 95 per cent confidence intervals. Where there were two or more studies in an intervention category, data were pooled using random‐effects meta‐analysis. To avoid double‐counting, where multiple different interventions were compared with the same control group, the control group was split into two or more smaller groups of equal size before being entered into the analysis. This approach permitted retention of as many relevant comparisons as possibleThe GRADE approach was used to rate the overall quality of the body of evidence for risk of bias, publication bias, imprecision, inconsistency and indirectness. GRADE ratings of very low, low, moderate or high certainty measure the overall quality of the evidence and reflect confidence in the evidence for each effect estimateFig. TableTables S1–S4 (supporting information).After removal of duplicate records, 6415 reports were identified of which 48 studiesversus 7), and pharmacological interventions were evaluated more commonly than non‐pharmacological measures. All studies used a definition of PO‐UR that involved some form of active treatment, most commonly catheterization. In addition, some included a measure of bladder volume or a palpable bladder.The majority of included studies were conducted in surgical populations with a higher risk of developing PO‐UR. Ten studies included only men and seven included only women. Of the 24 studies that included both men and women, 56·6 per cent were men overall. Seven studies did not report on the sex of included participants. Sample size ranged from 30 to 496 (median 99). More studies targeted prevention than considered treatment of PO‐UR .The main potential limitations of the included studies were methods of randomization/concealment  or blinding of participants/carers (23). Fewer studies were found to have concerns regarding missing data (6), failure to blind outcome assessor (8) and selective reporting (8). Overall and domain‐level assessments of risk of bias for each trial are provided in I2 = 0 per cent; 3 trials) or adding μ‐opioid antagonists to the anaesthetic  reduced the number of patients with PO‐UR.Reducing exposure to morphine was associated with a reduced risk of PO‐UR. There was moderate‐certainty evidence that avoiding morphine as part of a spinal or epidural anaesthetic regimen .There was high‐certainty evidence that the incidence of PO‐UR was lower when the anaesthetic regimen included an alternative to morphine (clonidine or sufentanil) for postoperative analgesia Very few studies reported data on secondary outcomes. Single studies each assessed UTIThere was moderate‐certainty evidence that diclofenac suppositories reduced PO‐UR in patients undergoing haemorrhoidectomy compared with placebo or no suppository There was high‐certainty evidence that the risk of PO‐UR was lower with epidural compared with spinal anaesthesia I2 = 79·5 per cent; 2 trials)There was very low certainty of no difference in the effect of unilateral compared with bilateral spinal anaesthesia  hours)versus local lidocaine plus adrenaline (epinephrine) (MD 2·28 (1·85 to 2·71) hours)Some evidence for increased duration of hospital stay was found when patients received bupivacaine epidurally I2 = 57·6 per cent; 12 trials). Studies compared tamsulosin There was high‐certainty evidence that alpha‐blockers were associated with a lower incidence of PO‐UR , pain (2) or duration of hospital stay (1).I2 = 49·0 per cent; 3 trials)There was low certainty about the effect of cholinergic drugs on PO‐UR There was low certainty regarding the effect of the benzodiazepine lorazepam compared with placebo I2 = 0 per cent; 2 trials)High‐certainty evidence was found for an association between early mobilization and reduced incidence of PO‐UR Low‐certainty evidence suggested a lower incidence of PO‐UR amongst patients restricted to 250 ml or less of oral fluids after surgery compared with patients given free access to fluids There was low‐certainty evidence of reduced PO‐UR in patients assessed with frequent preoperative ultrasound scanning and catheterized when the bladder volume exceeded 400 ml, compared with patients with no preoperative scanning There was no evidence for an effect of bladder stimulatorversus carbachol or alfuzosin versus carbachol and diazepam .There was very low certainty regarding the effect of phenoxybenzamine or alfuzosin compared with placebo There was no evidence of a reduction in PO‐UR among patients given carbachol, with or without diazepam, compared with placebo Interventions for the treatment of urinary retention were evaluated only in single trials. High‐certainty evidence was found for a beneficial effect of applying a hot pack or gauze soaked in warm water to the suprapubic region s for one case of PO‐UR to be prevented ranged from 4 (4 to 6) for morphine avoidance to 11 (9 to 25) for NSAIDs. For patients who developed PO‐UR after surgery, there was high‐certainty evidence that a hot pack or gauze soaked in warm water reduced the need for catheterization, with two patients needing this treatment to resolve one case of urinary retention. There was also evidence that moxibustion, posterior tibial nerve stimulation and caffeinated drinks could help to treat PO‐UR, although the evidence was less certain.This systematic review summarizes the effectiveness of interventions evaluated for the prevention or treatment of PO‐UR. The majority of the 48 RCTs evaluated preventive interventions. Based on GRADE assessments, there was high‐certainty evidence to suggest that the following may help reduce the risk of patients developing PO‐UR after surgery: replacing morphine in the anaesthetic regimen; using alpha‐blockers or the antispasmodic drotaverine; early postoperative mobilization; and epidural anaesthesia or pudendal nerve blocks rather than spinal anaesthesia. The number of patients who would need to receive the intervention for one case of urinary retention to be prevented ranged from 2 (95 per cent c.i. 2 to 3) for morphine replacement to 9 (7 to 30) for drotaverine. There was some evidence that avoiding morphine in the anaesthetic regimen, diclofenac suppositories (NSAID), μ‐opioid antagonists, oral fluid restriction and preoperative ultrasound monitoring were also associated with a lower risk of PO‐UR, but the evidence here was less certain. The number of patients that would need to receive these interventionversus extradural administration), unilateral anaesthesia , general or local anaesthesia  and straight catheterization in the recovery room. For treatment of PO‐UR, there was no evidence to support the use of alpha‐blockers or cholinergic drugs with or without benzodiazepines.There was no evidence that the following could help to reduce the risk of PO‐UR: use of an external bladder stimulator, postoperative anal packing (after haemorrhoidectomy), cholinergic drugs, lorazepam (benzodiazepine) and electroacupuncture, intramuscularly administered morphine (This review had a number of strengths and limitations. Cochrane recommendations for conducting systematic reviewsTable S5 (supporting information).To minimize bias and errors, all stages of the review were performed independently by two reviewers, including a public contributor who had experienced PO‐UR. All included RCTs were assessed using the new Cochrane risk‐of‐bias 2.0 toolThere were a number of limitations in the reviewed studies. A wide variety of definitions for urinary retention were used. Although the majority of trials defined PO‐UR by the need for a urinary catheter, catheterization protocols varied between studies. Catheter use can be considered a strong proxy measure of PO‐UR, but for studies where it was not stated explicitly that catheterization was performed as treatment for urinary retention, it must be noted that there could be other reasons for catheterization, including postoperative fluid balance monitoring or poor mobilityThere are two existing reviews related to PO‐UR. A Cochrane systematic reviewLarge, robust and well designed RCTs are needed to confirm the effects of the interventions studied in this review, and to inform the design of an effective prevention and treatment protocol for surgical patients at risk of developing PO‐UR. Trials should follow CONSORT reporting standardsAppendix S1 Search strategyTable S1 Characteristics of included studies: pharmacological preventionTable S2 Characteristics of included studies: non‐pharmacological preventionTable S3 Characteristics of included studies: pharmacological treatmentTable S4 Characteristics of included studies: non‐pharmacological treatmentTable S5 Summary estimates for the effects of interventions on the incidence of postoperative urinary retention together with GRADE ratings for certainty of the evidenceTable S6 Secondary outcomesAppendix S2 Risk of bias by type of interventionClick here for additional data file.


Cerebrospinal fluid (CSF) neurofilament light (NfL) is a reliable marker of neuro-axonal damage in different neurological disorders that is related to disease severity. To date, all recent studies performed in human CSF have used the same enzyme-linked immunosorbent assay (ELISA). To confirm the large body of evidence for NfL, we developed a new ELISA method and here we present the performance characteristics of this new ELISA for CSF NfL in different neurological disorders.n = 97), including multiple sclerosis , clinically isolated syndrome , and radiologically isolated syndrome ; 2) Alzheimer’s disease , including mild cognitive impairment due to AD  and probable AD dementia ; 3) Parkinson’s disease ; and 4) other neurological noninflammatory and non-neurodegenerative diseases .We produced two monoclonal antibodies (NfL21 and NfL23) directed against the NfL core domain, and developed a novel sandwich ELISA method that we evaluated in patients with: 1) inflammatory demyelinating diseases  between the two methods. CSF NfL levels were significantly higher in MCI-AD/AD-dem and IDD patients as compared with both PD and OND patients. The highest discriminative power was obtained between IDD and OND patients (area under the curve (AUC) 0.87, 95% confidence interval (CI) 0.80–0.95). Within the IDD group, CSF NfL positively correlated with several clinical and radiological disease severity parameters.Our new NfL ELISA showed a good analytical performance (inter-plate coefficient of variation (CV) < 13%), with no cross-reactivity with neurofilament medium and heavy (NfM and NfH). With respect to the other available ELISAs, CSF NfL showed the same range of values with a strong correlation (These results show a good analytical performance of the new ELISA for quantification of NfL concentrations in the CSF. CSF NfL is confirmed to be a reliable marker in AD and MS, and a disease-severity marker in MS patients. In the personalized medicine era, management of diseases requires validated biomarkers to address the issues of making a clinical diagnosis, coming to a prognosis, and monitoring the biochemical effects of pharmacological treatments . CerebroNeurofilaments (Nf) are intermediate filaments of the neuronal cytoskeleton . There aLow amounts of NfL are physiologically secreted into the CSF, but in patients with CNS pathology associated with axonal injury or degeneration, increased amounts of NfL are released from neurons into the interstitial space and into the CSF . BecauseReproducibility of the measurements with different assays is mandatory for the use of a biomarker in a clinical setting . RosengrTo broaden the available choices of NfL measurement methods, we have generated novel monoclonal antibodies (NfL21 and NfL23) specific for NfL and established a new ELISA for the quantification of NfL in CSF. In this paper, we present the development and performance characteristics of this method. Moreover, we have applied this new assay to quantify NfL in the CSF of patients with a variety of neurological disorders, including inflammatory demyelinating diseases (IDD) of the CNS, AD, including both mild cognitive impairment due to AD (MCI-AD) and probable AD dementia (AD-dem), Parkinson’s disease (PD) and other neurological noninflammatory and non-neurodegenerative diseases (OND).Bacillus amyloliquefaciens H restriction enzyme I (BamHI) and 3′ Escherichia coli restriction enzyme I (EcoRI) sites, with full length complementary DNA (cDNA) for NfL  as a template. The PCR fragments were purified and cloned into BamHI/EcoRI digested pGEX2T, a glutathione-S-transferase (GST) expression plasmid . Constructs were sequenced and transformed into E. coli BL21 (DE3). E. coli BL21(DE3) containing the different constructs was incubated overnight in 50 ml of lysogeny broth (LB) medium with ampicillin at a concentration of 100 μg/ml. The overnight culture was used to inoculate 1 liter of LB medium with ampicillin (100 μg/ml), and when the optical density measured at a wavelength of 600 nm (OD600) reached 0.4–0.6, protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at +30 °C. The culture was centrifuged at 6000 rpm for 20 min at +4 °C and the pellet weight was determined. The pellet was stored at –20 °C pending purification. The pellet was re-suspended in 5 ml/mg of lysis buffer  plus complete protease inhibitors  and incubated with rotation for 30 min at room temperature, after which the lysate was centrifuged at 12,000 rpm for 20 min at +4 °C and the supernatant was collected. Protein extract was added to 50% Glutathione-Sepharose 4B  equilibrated with phosphate-buffered saline (PBS) and incubated for 30 min with rotation at room temperature. Sepharose was washed with PBS and the GST-NfL fusion protein was incubated with elution buffer  for 10 min. The unbound fusion protein was eluted and the incubation step was repeated four times. On-bead cleavage of the GST-fusion protein by thrombin was performed in PBS with 50 U thrombin for 2.5 h at room temperature. The cleaved, untagged protein was eluted with PBS containing protease inhibitor.NfL-head + core, amino acid 1–396 of NfL, NfL-core, amino acid 93–396, and NfL-tail, amino acid 397–543, were amplified through a polymerase chain reaction (PCR) using primers containing 5′ Monoclonal antibodies against NfL were generated by immunization of 8-week-old Balb/c mice with the recombinant protein fragments (head + core or core) in complete Freund’s adjuvant (Sigma). After 2–3 dosages with the recombinant protein fragment (approximately 75 μg/mouse), the spleen was removed and B cells were fused with the myeloma cell line SP2/0 following standard procedures. Approximately 10 days after fusion, cell media were screened for NfL antibodies using full-length recombinant NfL, recombinant protein fragment head + core (produced as described above), and purified bovine NfL protein . Clones Anti-mouse antibody  was immobilized according to the kit instructions on a C1 Biacore chip (BR-1005-40) in a Biacore X100 instrument via amine-coupling at 25 °C in running buffer (PBS with 5% DMSO and 0.05% Tween 20). A final response of 1100 RU for the immobilization level was obtained. This chip was then used at 37 °C to bind the NfL capture antibody to be analyzed (3 nM in running buffer with 1 mg/ml CM-Dextran (CMD); Sigma Aldrich 86524) and, subsequently, dilutions of bovine NfL . For regeneration between cycles of this multicycle measuring method, we used injection of 30 mM HCl for 2 min and of 10 mM glycine, pH 1.5, for 30 s , before repeating the cycle for the next NfL concentration. Antibody binding: contact time: 180 s, flow rate 10 μl/min, stabilization period 300 s; NfL antigen binding: contact time 180 s, flow rate 30 μl/min, dissociation time: 900 s. Kinetic analysis was performed using the 1:1 binding model of the Biacore X100 Evaluation software (V2.0.1).For immunoprecipitation and Western blot, the NfL21 and NfL23 monoclonal antibodies were bound to magnetic Dynabeads M-280 Sheep Anti-Mouse IgG, and incubated with CSF samples, head, core and tail recombinant fragments of NfL or full-length NfL recombinant protein (Origene). After elution, bound proteins were electrophoresed on a NuPAGE 4–12% Bis-Tris gel and transferred to Amersham™ Protran™ Nitrocellulose membranes, probed with NfL21 or NfL23 using the ECL Select™ Western Blotting Detection Reagent .We selected 235 CSF samples stored in the CSF Biobank of the Section of Neurology, Department of Medicine, University of Perugia , for the study. CSF was collected over a 10-year period 2006–2016) via lumbar puncture at the same institution using the same standard operating procedures throughout the study following international guidelines [06–2016 vFor the IDD group, we selected CSF samples from patients satisfying the following criteria at the time of CSF collection: 1) a diagnosis of clinically isolated syndrome suggestive of MS (CIS), relapsing-remitting MS (RRMS), primary progressive MS (PPMS) or secondary progressive MS (SPMS) according to the 2010 revision of the McDonald criteria , or a diA senior neurologist expert in inflammatory demyelinating diseases of the CNS examined all the study participants and scored the Kurtzke’s Expanded Disability Status Scale (EDSS) . The preA senior neurologist expert in AD and memory disturbances examined all the study participants. A neuropsychologist performed neuropsychological evaluations, including Mini Mental Status Examination (MMSE) , at the A senior neurologist expert in movement disorders examined all the patients and performed extensive clinical testing, including the scoring of the Unified Parkinson’s Disease Rating Scale (UPDRS) part III  and of t2SO4 and the absorbance was measured at 450 nm (reference wavelength 650 nm) using an ELISA plate reader . A fitted four-parameter logistic model was used to generate the calibration curve and the blank was included as zero concentration of NfL. Validation was performed by evaluating the assay range, the upper and lower limit of quantification , within and inter-plate precision, linear dilution, cross-reactivity, and spike recovery during five separate analyses on different days. Cross-reactivity against NfM and NfH was evaluated by spiking in NfM or NfH into the calibrator curve. Spike recovery was performed by spiking in bovine NfL into five individual CSF samples with endogenous NfL levels ranged between 1208–6649 pg/ml. Recovery was calculated by subtracting the measured concentration of the unspiked samples from the measured concentration of the spiked sample, divided by the spike concentration.NfL21 was used as capturing antibody and was coated on Nunc maxisorp 96-well microtiter plates at a final concentration of 0.5 μg/ml (100 μl/well) in 50 mM bicarbonate buffer, pH 9.6, overnight at +4 °C. All washes were performed at room temperature by adding 4 × 350 μl PBS containing 0.05% Tween20 (PBS-T). After a first wash, the remaining protein binding sites were blocked at room temperature with 1% bovine serum albumin (BSA) in PBS  for 1 h (250 μl/well). Thereafter, plates were washed and 100 μl of calibrator (range 39–5000 pg/ml), blank, quality-control (QC) samples, and CSF were added to the corresponding wells. QC samples and CSF were diluted 1:2 in PBS-T. The plate was shaken for 1 h at 400 rpm and then incubated overnight at +4 °C. After washing, 100 μl of biotinylated NfL23 detection antibody (0.4 μg/ml in PBS-T) was added and incubated for 2.5 h at room temperature. After washing, enhanced streptavidin horseradish peroxidase  diluted 1:20,000 in PBST 0.05% BSA 1% was added (100 μl/well), and incubated for 30 min at room temperature. After a final wash, 100 μl 3,3′,5,5′-tetramethylbenzidine (TMB) substrate  was added to generate the color. After 20-min incubation in the dark at room temperature the reaction was stopped by the addition of 100 μl 2 M Ht test. To control for the confounding effect of age and gender, we carried out an analysis of covariance (ANCOVA) to verify if CSF NfL log values would differ between the diagnostic groups. Whenever appropriate, we considered age and gender as potential confounders in the analyses performed in each diagnostic group. The optimal diagnostic accuracy of NfL was assessed by calculating the area under the curve (AUC) of the receiver operating characteristic (ROC) curve at the point that maximized the Youden index. ROC analysis was performed both adjusting and not adjusting for age. All tests were two-sided and significance was set at p < 0.05. Statistical analyses were performed using R software, version 3.3.1.A likelihood ratio test based on generalized linear models was used to test if the distribution of demographics, clinical, and biochemical features differed between IDD subgroups and between MCI-AD/AD-dem and PD groups. The procedure was carried out to control for the age distribution. Logarithmic transformation was applied to NfL values to reach normality, as verified with the Shapiro-Wilk test. To evaluate potential confounders, we tested the association between CSF NfL logarithmic (log) values and age and gender. The association between CSF NfL log values and gender was tested with Student’s D of NfL21, 0.3 nM; KD of NfL23, 0.5 nM; the association rate constants ka (1/Ms) were similar for both NfL21 and NfL23 (around 4–5 × E05), and the dissociation rate constants (1/s) were also very similar for the two antibodies (around 1–2 × E-04); corresponding half-life of the antibody NfL complex between 58 and 115 min). The residuals for the curve fitting were within the acceptable range; however, NfL showed at the highest used concentration some background binding to the C1 chip even in absence of capture antibody which could not be improved by testing various regeneration conditions. The above given kinetic data must therefore be seen as preliminary estimates only. Immunoprecipitation and Western blot analysis showed that both the NfL21 and NfL23 antibodies recognized a band corresponding to full-length NfL, and reacted with the core domain, but not the head or core domains, of recombinant NfL (data not shown).We used surface plasmon resonance (SPR) measurements to determine the kinetics of binding of bovine NfL to the NfL antibodies captured on a Biacore C1 chip. Both antibodies showed high affinity binding to bovine NfL . In this latter group, 2 patients were diagnosed with SPMS and 6 with PPMS. Patients with PMS had significantly higher mean age, disease duration, EDSS scores, and total number of T2 MRI lesions as compared to the other IDD subgroups. On the contrary, CIS and RRMS patients more frequently had clinical and neuroradiological signs of disease activity (both a recent relapse and a higher number of gadolinium enhancing (Gd+) lesions on MRI). The details of IDD patient characteristics are reported in Table r = 0.19, p = 0.003). Moreover, NfL was slightly increased in males as compared to females . For this reason, the differences in CSF NfL values between the diagnostic groups were tested with ANCOVA adjusted for age and gender. Furthermore, a positive correlation between CSF NfL concentrations and age was also found in the PD group . No such correlation was seen in the IDD or MCI-AD/AD-dem groups. There was no gender difference in CSF NfL concentration within the different diagnostic groups.For the entire cohort we found a positive correlation between CSF NfL concentrations and age (p < 0.001 for both comparisons). Moreover, both IDD and MCI-AD/AD-dem patients had higher CSF NfL concentrations as compared with PD patients . No statistically significant difference was found between the IDD and MCI-AD/AD-dem groups or between the PD and OND groups  and the MCI-AD/AD-dem groups (1003 ± 484 pg/ml) as compared with the OND group  . Furthermore, CSF NfL concentrations were significantly higher in patients with EDSS ≥ 3 than in patients with EDSS < 3   than in patients with no evidence of recent clinical disease activity  and the number of juxtacortical lesions , as well as with Gd+ lesions  at baseline MRI. CSF NfL values were also found to be significantly higher in patients with a higher number of Gd + lesions (p < 0.001). Post-hoc comparison showed that NfL values were increased in those patients with more than 3 Gd + lesions compared to 0 (p = 0.019) or 1–3 Gd + lesions in the baseline MRI (p = 0.025)  Fig. . FinallyNo statistically significant difference in CSF NfL concentrations was observed between patients with MCI-AD and patients with AD-dem. Moreover, no significant correlation was found between CSF NfL values and MMSE at baseline and follow-up. Likewise, the change of MMSE from the baseline to the follow-up did not show any significant correlation with CSF NfL concentrations. In addition, disease duration did not correlate with CSF NfL values.r = 0.48, p = 0.028)  Fig. . HoweverD on Biacore) for NfL with epitopes in the core region of NfL. Given the obtained results, our assay represents a valid alternative for the measurement of NfL concentrations in the CSF.The primary finding of our study was the good analytical performance of the newly established ELISA for NfL. The assay demonstrates robust and accurate measurement of NfL in CSF (inter-plate coefficient of variation (CV) < 13%) with no cross-reactivity towards NfM or NfH. Absolute concentrations were in the same range as the ELISA from UmanDiagnostics and, importantly, there was a clear correlation. Further characterization of the antibodies revealed high affinity (subnanomolar KThe other main results from our study were: 1) a significant increase in CSF NfL in IDD and MCI-AD/AD-dem patients as compared with PD patients and to the control group; 2) no significant differences in CSF NfL in PD patients as compared with controls; 3) a significant association between CSF NfL and several clinical and radiological disease severity measures in IDD patients; and 4) no significant correlations between CSF NfL and clinical variables in MCI-AD/AD-dem and PD patients.n = 8) included in our study. However, we found a nonsignificant increasing trend in CSF NfL values from RIS to CIS, RRMS, and PMS patients, thus suggesting that during the disease course neuro-axonal damage progressively increases.The increase of CSF NfL in IDD patients is in line with the results of several previous studies performed on this type of diseases showing that CSF NfL concentrations are significantly higher in CIS, RRMS, SPMS, and PPMS as compared with controls , 33. MorNevertheless, within the IDD group, we confirmed the positive correlation between CSF NfL values and several clinical and MRI measures of disease severity. Firstly, CSF NfL values are increased in patients with clinical and/or neuroradiological evidence of disease activity. Indeed, in our study CSF NfL values were significantly higher in patients with a recent relapse, similar to what has been previously reported , 33–35. We have additionally found that in IDD patients CSF NfL values correlate with the degree of disability and with the total number of MRI T2 lesions, as previously reported , 38, 40.APOE) ε4 genotype, was 0.88 [In our study, CSF NfL concentration was significantly higher in the MCI-AD/AD-dem group as compared with both PD and control subjects, in line with similar results obtained in previous studies , 44. Morwas 0.88 . NfL lacWithin the MCI-AD/AD-dem group, there was no significant correlation of CSF NfL concentration with MMSE scores at the baseline and at the follow-up or with MMSE score change. This result is in contrast with previous data obtained on larger populations showing a significant correlation between CSF NfL and MMSE performance in AD patients . HoweverFinally, there were no statistically significant differences in CSF NfL concentration between PD and controls, as previously reported in other similar studies performed on CSF NfL . ConcordOur findings showed a good analytical performance of a newly developed ELISA for NfL. CSF NfL is confirmed to be a reliable marker of the severity of neuro-axonal damage processes taking place in different neurological diseases. Our results support the use of CSF NfL as a disease intensity marker in MS and AD. Additional studies are required to confirm these results in different cohorts. This achievement could further confirm the role of CSF NfL in predicting long-term outcomes in MS and AD, as well as to confirm its possible prognostic role in PD patients.


The diagnosis of primary headaches assists health care providers in their decision-making regarding patient treatment, co-management and further evaluation. Chiropractors are popular health care providers for those with primary headaches. The aim of this study is to examine the clinical management factors associated with chiropractors who report the use of primary headache diagnostic criteria.n = 1050) who had reported ‘often’ providing treatment for patients with headache disorders to report on practitioner approaches to headache diagnosis, management, outcome measures and multidisciplinary collaboration. Multiple logistic regression was conducted to assess the factors that are associated with chiropractors who report using International Classification of Headache Disorders (ICHD) primary headache diagnostic criteria.A cross-sectional survey was distributed between August and November 2016 to a random sample of Australian chiropractors who are members of a practice-based research network (n = 381), the majority of chiropractor’s report utilising ICHD primary headache diagnostic criteria (84.6%). The factors associated with chiropractors who use ICHD primary headache diagnostic criteria resulting from the regression analysis include a belief that the use of ICHD primary headache criteria influences the management of patients with primary headaches ; the use of soft tissue therapies to the neck/shoulders for tension headache management ; a belief that primary headache diagnostic criteria are distinct for the diagnosis of primary headaches ; the use of headache diaries ; the use of ICHD criteria improves decision-making regarding primary headache patient referral/co-management ; referral to investigate a headache red-flag  and not referring headache patients to assist headache prevention .With a response rate of 36% (Four out of five chiropractors managing headache are engaged in the use of primary headache diagnostic criteria. This practice is likely to influence practitioner clinical decision-making around headache patient management including their co-management with other health care providers. These findings call for a closer assessment of headache characteristics of chiropractic patient populations and for further enquiry to explore the role of chiropractors within interdisciplinary primary headache management. The global adult prevalence of tension-type headache and migraine is reported to be approximately 40 and 10%, respectively . These hSignificant challenges remain regarding the management of headache patients. Headache patients are often poorly or under diagnosed , under tThe 3rd edition of International Classification of Headache Disorders (ICHD) outlines the current criteria utilised for headache diagnosis . HeadachThe data analysed in this study was drawn from a questionnaire distributed to members (chiropractors) of a national practice-based research network (PBRN) titled the Australian Chiropractic Research Network (ACORN) project . This stn = 1680) of all registered Australian chiropractors joined the ACORN network database. The socio-demographic profile of the ACORN database is representative of the wider chiropractic profession across Australia in terms of gender, age and practice location [Detailed information about the ACORN PBRN recruitment and data base has been previously reported , 32, butParticipants for this PBRN sub-study were randomly selected from the ACORN practitioners who had reported that they ‘often’ provided treatment for patients with headache disorders in the ACORN PBRN invitation pack questionnaire. Participants were asked to complete a 31-item cross-sectional online survey between August and November 2016. An embedded link to the questionnaire was emailed to chiropractors. Three further reminders to complete the survey were sent out during the recruitment period. Participation in the survey was further promoted within routine email newsletters sent out by the Australian Chiropractors Association during that period.The introduction to the questionnaire explained the purpose, contents and approximate duration of the study and that respondent information was anonymous and survey completion voluntary. No incentives were offered to participate, and consent was implied by completing the survey. Questionnaire items specifically developed for this study aimed to examine chiropractic headache management across several clinical themes considered important to frontline headache management practice. With no validated instruments available, the key themes adopted for our study questionnaire were developed after consideration of past surveys examining the management of headache patients in primary care settings , 33, 34.The primary headache management questions included in the questionnaire were based upon primary headaches previously reported as most often treated by chiropractors , 39, 40 Summary statistics were presented by number (percentage), mean (SD) as appropriate. In-order to test the differences in continuous and categorical variables by group, we have used Student’s t-test and chi-square test or Fishers exact test respectively Table . Bivariap < 0.2 included in the regression model. The independent survey variables were dichotomized after consideration of previous research [p < 0.05. Odds ratios were reported with 95% confidence intervals. All statistical analyses were conducted using the statistical software Stata 13.1.Multiple logistic regression modelling was then performed to identify independent predictors associated with those chiropractors who use ICHD primary headache criteria , place of practice (p = 0.916) suggesting survey respondents are generally representative  of the ACORN database participants while our sample was slightly more experienced than the ACORN database members for years in practice (p = 0.003). The majority of questionnaire respondents were male (64%) and the average number of years in practice was 18.1 (SD = 10.9) years. Most participants were educated in Australia including New South Wales (38.6%), Victoria (35.6%), Western Australia (9.7%) and Queensland (0.8%). Place of practice amongst the participants was greatest in New South Wales (35.1%), followed by Victoria (23.2%), Queensland (15.2%), Western Australia (14.7%), South Australia (8.5%), Australian Capital Territory (1.6%), Tasmania (0.9%) and Northern Territory (0.5%). Participant demographic characteristics were consistent with national chiropractic registration records [A total of 1050 chiropractors were invited to participate of which 381 (36.2%) completed the questionnaire. As shown in Table  records .p < 0.001). In addition, those chiropractors who use primary headache diagnostic criteria were also more likely to use a Migraine Disability Assessment Test (MIDAS); the Headache Disability Inventory (HDI); and patient headache diaries .The majority of chiropractors reported utilising ICHD criteria for the diagnosis of primary headaches (84.6%). The clinical management characteristics of chiropractors who use or do not use the ICHD primary headache classification criteria are presented in Table p < 0.001) and dentist (p = 0.002), compared to chiropractors who do not use the ICHD primary headache diagnostic criteria and less likely to receive headache referrals from a physiotherapist (p = 0.684) or osteopath (p = 0.154) although these associations were not statistically significant. Further, chiropractors who use the ICHD primary headache diagnostic criteria were also more likely to refer headache patients for further management to general practitioners (p < 0.001), medical specialists (p < 0.001), psychologist (p = 0.004), dentist (p = 0.001), and CAM practitioners  (p = 0.001), compared to chiropractors do not use the ICHD primary headache diagnostic criteria and were less likely to refer a headache patient to a physiotherapist (p = 0.106) although these associations were not statistically significant. Chiropractors who use ICHD primary headache criteria were more likely to refer patients for reasons of confirming headache diagnosis (p < 0.001), improve coping skills (p < 0.001), investigate headache red-flags (p < 0.001), provide pain relief for acute headache attacks (p = 0.004) and to provide headache prevention (p = 0.004), compared to chiropractors do not use the ICHD primary headache diagnostic criteria.Table p = 0.043), to provide pain relief during headache episode (p = 0.049) and improve headache related coping skills (p = 0.001) and less likely to aim treatment toward headache prevention (p = 0.317) or to improve headache patient overall health and well- being (p = 0.411) although these associations were not statistically significant.The importance of headache treatment outcomes of chiropractors who use or do not use the ICHD primary headache classification criteria are presented in Table p = 0.001); provide massage, myofascial technique, stretching or trigger-points to neck/shoulder area (p < 0.001); use soft tissue or exercise therapy to temporo-mandibular region (p < 0.001); prescribe exercises for the neck and shoulder region (p < 0.001); provide advice on stress management (p = 0.019) and headache triggers (p = 0.005), compared to chiropractors do not use the ICHD primary headache diagnostic criteria. They were less likely to provide manual manipulation (p = 0.751), instrument adjusting (p = 0.407), drop piece adjusting (0.944), electro-physical therapies (p = 0.236) and advice on diet or fitness (p = 0.057) although these associations were not statistically significant. For patients with tension headache, chiropractors who use primary headache diagnostic criteria were more likely to: provide non-thrust spinal mobilisations (p = 0.003); use massage, myofascial technique, stretching or trigger-points to neck/shoulder area (p < 0.001); use soft tissue or exercise therapy to temporomandibular region (p = 0.017); prescribe exercises for the neck/shoulder region (p < 0.001); provide advice on stress management (p = 0.002) and headache triggers (p < 0.001), compared to chiropractors do not use the ICHD primary headache diagnostic criteria. They were less likely to provide manual manipulation (p = 0.291), instrument adjusting (p = 0.810), drop piece adjusting (p = 0.662), electro-physical therapies (p = 0.374), and advice on diet and fitness (p = 0.480) although these associations were not statistically significant.Table The results of the multiple logistic regression modelling used to identify the important independent factors associated with chiropractors who use ICHD primary headache diagnostic criteria compared to those chiropractors who do not use ICHD primary headache diagnostic criteria are presented in Table This is the first study to provide detailed information on the patient management features associated with primary headache diagnosis by chiropractors. The majority of chiropractors in our study report utilising ICHD criteria for the diagnosis of primary headaches, a finding which may suggest that chiropractors are sometimes the first point of provider contact for patients seeking help for the management of primary headache disorders. There are a number of factors that can challenge health care providers delivering an accurate primary headache diagnosis. These include the co-occurrence of migraine with both cervicogenic headache  and tensOur study found several factors that were associated with chiropractors engaged in primary headache diagnosis. These chiropractors include a belief that the use of ICHD primary headache criteria influences their patient management. Previous studies have reported the use of manual therapies, exercise therapies and advice on headache triggers as common to chiropractic headache management , 54. WhiOur analysis found several factors associated with chiropractors engaged in primary headache diagnosis that were related to specific aspects of practitioner decision-making regarding headache patient management. For example, our study found chiropractors engaged in the use of ICHD primary headache diagnostic criteria are more likely to believe doing so improves decision-making related to headache patient referral/co-management. The health care needs of primary headache sufferers can sometimes be multifactorial and multidisciplinary in nature, particularly for those who present with more complex and chronic headache conditions where a greater use of pharmaceutical, behavioural and physical approaches to patient care may be needed , 62. PreAn unexpected finding from our results was that chiropractors engaged in primary headache diagnosis are less likely to undertake patient referral to assist with headache prevention. A recent Australian study showed chiropractors refer headache patients to both complementary health care providers  and general practitioners . For tenOur findings identified chiropractors engaged in primary headache diagnosis are more likely to refer headache patients to investigate a headache red-flag. This finding is not unexpected, since the use of headache diagnostic criteria is more likely to result in the identification of headache features associated with headache red-flag findings. The most important diagnostic consideration for frontline clinicians engaged in headache management is to rule out headaches caused by serious and potentially life-threatening underlying pathology. While rare, the underlying causes associated with headache red-flag symptoms can include stroke, sub-arachnoid haemorrhage, tumour, meningitis and artery dissection  . Since hOur study found that chiropractors engaged in primary headache diagnosis are more likely to use soft tissue therapies such as massage, myofascial technique, stretching or trigger-points to neck/shoulder area for their patients with tension headache. This finding is interesting given a recent systematic review which found manual therapies, including soft tissue therapies, may be more effective than pharmacological care for reducing the short-term frequency, intensity and duration of tension headache . TendernRegression analysis of chiropractors engaged in primary headache diagnosis provides an excellent opportunity to better understand the primary headache management associated with this common headache provider. The self-reported nature of the data collected is a limitation of our study – the data may be subject to recall bias and the use of Likert categories are subject to practitioner interpretation. The headache management characteristics of chiropractors reported in this study may also be influenced by non-respondents to the survey when estimating chiropractors who use of ICHD primary headache criteria and the associations related to their headache management characteristics. Nonetheless, analysis of this cross-sectional survey provides valuable insights into primary headache health management associated with these popular providers and helps to identify key questions for further enquiry into chiropractic headache management.Our research found that most chiropractors managing primary headaches are engaged in primary headache diagnosis and that this practice is likely to influence their clinical-decision making toward key aspects of primary headache patient management and co-management. These findings highlight the need for closer examination of the clinical decision-making that underlies chiropractic primary headache management and the role of these providers toward reducing the burden of this significant public health issue. Gathering this information will help to improve our understanding of the role of chiropractors within multimodal, multidisciplinary headache patient management.