Escherichia coli can be used as indicator organisms to study the resistance in the community. We sought to determine the prevalence of resistance to broad-spectrum antimicrobials with particular focus on the quinolones, which have recently been introduced in parts of Africa, including Ghana.Antimicrobial resistance is under-documented and commensal E. coli isolates evaluated were nalidixic acid-resistant. Thirteen (52%) of 2006 and 2007 isolates and 10 (66.7%) of 2008 isolates were also resistant to ciprofloxacin. All but one of the quinolone-resistant isolates were resistant to three or more other antimicrobial classes. Sequencing the quinolone-resistance determining regions of gyrA and parC, which encode quinolone targets, revealed that 28 quinolone-resistant E. coli harboured a substitution at position 83 of the gyrA gene product and 20 of these isolates had other gyrA and/or parC substitutions. Horizontally-acquired quinolone-resistance genes qnrB1, qnrB2, qnrS1 or qepA were detected in 12 of the isolates. In spite of considerable overall diversity among E. coli from Ghana, as evaluated by multilocus sequence typing, 15 quinolone-resistant E. coli belonged to sequence type complex 10. Five of these isolates carried qnrS1 alleles.Forty (13.7%) of 293 E. coli are commonly present in the faecal flora of Accra residents. The isolates have evolved resistance through multiple mechanisms and belong to very few lineages, suggesting clonal expansion. Containment strategies to limit the spread of quinolone-resistant E. coli need to be deployed to conserve quinolone effectiveness and promote alternatives to their use.Quinolone-resistant  Escherichia coli before 2004 reported no or very low incidences of resistance to nalidixic acid and the fluoroquinolones [st century. More recent anecdotal reports and surveillance studies point to emergence of quinolone resistance among enteric pathogens and faecal enteric bacteria in Ghana and elsewhere in West Africa [E. coli) from nine of the ten regions in Ghana and tested them for antimicrobial susceptibility. They found that over 70% of the isolates were resistant to tetracycline, trimethoprim-sulphamethoxazole, ampicillin and chloramphenicol and reported that 11% of the isolates were ciprofloxacin-resistant [Following emergence of resistance to inexpensive broad-spectrum antimicrobials across much of Africa, quinolone antibacterials have recently been introduced and are widely used. West African studies that sought quinolone resistance in commensal or diarrhoeagenic inolones -4. Thus,t Africa -8. In a gyrA and parC, the two genes that encode DNA gyrase and topoisomerase IV respectively, can lead to conformational changes in these enzymes that cause them to block quinolones from binding to the DNA- substrate complex, yet still preserve their enzymatic function [Escherichia coli and related Gram-negative bacteria, DNA gyrase is the first target for fluoroquinolones. If gyrA has resistance-conferring mutations, the primary target of fluoroquinolone switches from DNA gyrase to topoisomerase IV [gyrA first, and then parC.Quinolones inhibit the activity of bacterial DNA gyrase and DNA topoisomerase enzymes, which are essential for replication. Single nucleotide polymorphisms (SNPs) in the quinolone resistance determining regions (QRDR) of function . In Escherase IV ,11. StudgyrA and parC are the most commonly documented resistance mechanisms, resistance has also been known to be conferred by mutations in the second topoisomerase gene, parE. Another mechanism of quinolone resistance relies on upregulation of efflux pumps, which export quinolones and other antimicrobials out of the bacterial cell. For example, mutations in the gene encoding a repressor of the acrAB pump genes, acrR, are associated with quinolone resistance [qnr) or other DNA. The Qnr gene product inhibits quinolones binding to target proteins [aac(6')-Ib , encoding a fluroquinolone acetylating enzyme, as well as qepA and oqxAB, which encode horizontally transmitted efflux pumps [Although mutations in the QRDR of sistance . Quinoloproteins . Other hux pumps -16.Resistance to the quinolones often emerges at low-levels by acquisition of an initial resistance-conferring mutation or gene. Acquisition of subsequent mutations leads to higher levels of resistance to the first-generation quinolone, nalidixic acid and a broadening of the resistance spectrum to include second-generation quinolones (first-generation fluoroquinolones) such as ciprofloxacin, followed by newer second- and third-generation fluoroquinolones .E. coli isolates from Accra, Ghana in 2006, 2007 and 2008. We identified isolates that were resistant to nalidixic acid and screened these strains for mutations in the QRDR of gyrA and parC as well as horizontally-acquired quinolone-resistance genes. In order to gain some insight into resistance dissemination, we also studied inter-strain relatedness among quinolone-resistant E. coli isolates by multilocus sequence typing.Although multiple mechanisms of quinolone resistance have been reported from other continents, there are few data from sub-Saharan Africa on the molecular basis for quinolone resistance. We performed antimicrobial susceptibility testing on fecal Escherichia coli isolates were recovered from culture of the 335 stool specimens. Consistent with the results of recent studies from West African countries, including Ghana [E. coli isolates were resistant to the broad-spectrum antimicrobials ampicillin, streptomycin, sulphonamides, tetracycline and trimethoprim  of the quinolone-resistant E. coli were resistant to ciprofloxacin  were resistant to at least one other antimicrobial and all but three of the QREC isolates were resistant to four or more non-quinolone antibacterials. Most QREC demonstrated high-level resistance to nalidixic acid with 21 of 40 of the QREC isolates showing a nalidixic acid MIC that exceeded 1024 mg/L. Among 2006 isolates, low-level resistance was more common, with the MIC50 in that year being 128 mg/L. In both 2007 and 2008, the MIC50 was >1024 mg/L.Quinolone resistance was almost always seen in multiply-resistant gyrA and parC in the 40 QREC isolates. As shown in Table gyrA and 18 of these isolates also had one or more non-synonymous mutations in parC. Twenty-seven of the 28 isolates with at least one mutation in gyrA had a serine to leucine substitution at position 83, one of the most commonly documented resistance conferring mutations [Increasing nalidixic acid MICs, accompanied by resistance to fluoroquinolones is often due to the acquisition of multiple mutations in quinolone targets. We sequenced the quinolone-resistance determining regions (QRDRs) of utations . Twenty parC with a serine to isoleucine substitution at position 80, present in 16 strains, being the most common substitution parC withqnrA, qnrB, qnrS and qepA genes and confirmed all amplicons by sequencing. Of the 40 strains evaluated twelve carried one horizontally acquired quinolone resistance gene. These were qnrB1 (2 isolates), qnrB2 (1 isolate), qnrS1 (7 isolates) and qepA (2 isolates). In two isolates, without mutations in gyrA and parC QRDRs, horizontally-acquired resistance genes could account for the resistance seen. However, in the vast majority of cases, horizontally acquired resistance was seen in combination with QRDR mutations.We used PCR to screen for http://www.mlst.net. We identified 30 Sequence Types (STs) among 40 QREC isolates from Ghana (0.75 STs per strain). As shown in Figure qnrS1 alleles were in all but two cases detected among strains belonging to the ST10 complex.We hypothesized that clonal expansion might account, at least in part, for the rise in resistance seen in the course of the study. To test this hypothesis, we subjected all the 40 QREC isolates to multi-locus sequence typing by the scheme of Wirth et al  and depoE. coli from Ghana in the database . Moreover six other QREC isolates were single- or double- locus variants of ST10. Thus ST10 appeared to be over-represented among QREC isolates from Ghana. Because most of the isolates from Ghana were deposited in the database two to four years in advance of our own study, we sequence typed eight non-QREC isolates selected at random from our 2008 isolates. All eight belonged to different sequence types , five of the eight sequence types were novel, and only one 2008 non-QREC strain was an ST10 isolate. Therefore our data suggest that ST10-complex QREC may represent a successful quinolone-resistant lineage.Nine of the 40 QREC isolates obtained in this study belonged to ST10, in contrast to 10 of 125 other E. coli in many parts of the world [E. coli isolates are resistant to ampicillin, sulphonamides, tetracycline and trimethoprim, as well as streptomycin, which have been used to treat actual and supposed bacterial infections in Ghana for over four decades, and that resistance to these agents is increasing with time. We also found that about a third of isolates were resistant to chloramphenicol. Fluoroquinolone antimicrobials have been recently introduced as an effective alternative to older antibacterials that have been compromised by resistance. However, although resistance rates were markedly lower for this class of drugs, we also found that quinolone resistance was increasingly common among fecal E. coli in this study.Evolution of reduced susceptibility to the quinolones is causing concern following rapidly rising rates of fluoroquinolone-resistant he world . In AfriE. coli isolated from healthy individuals in Accra in 2006, 2007 and 2008 are quinolone resistant. Twenty-three of the 40 QREC isolated were resistant to the fluoroquinolone ciprofloxacin. Ciprofloxacin-resistant QREC, showing high-level nalidixic acid resistance, were more commonly isolated in 2008 than in 2006 and 2007. Strains with one or no mutations in gyrA were typically ciprofloxacin sensitive. However most isolates had accumulated a second gyrA mutation and/or mutations in parC and were fluoroquinolone resistant. The QRDR polymorphisms most commonly detected in this study are those most frequently reported in the literature [parC substitutions in strains also harbouring substitutions in gyrA [gyrA and parC mutations did not absolutely correlate with nalidixic acid MICs, partly due to horizontally-acquired quinolone-resistance genes. We sought qnrA, qnrB, qnrS and qepA genes by PCR and confirmed all amplicons by sequencing. We found that two isolates without mutations in the QRDRs of gyrA and parC, as well as ten isolates with QRDR mutations carried a qnrS1, a qnrB or a qepA allele. The presence of horizontally-acquired genes accounted in part for elevated nalidixic acid MICs in strains that harboured these genes, but not completely. It is therefore possible that other resistance mechanisms, such as ParE polymorphisms, other horizontally acquired resistance genes (such as oqxAB and aac(6')-Ib for example), over-active efflux, or even novel mechanisms are present in some of the isolates.We determined that 12-18% of fecal terature . As has  in gyrA . HoweverVibrio cholerae isolates recovered in the same time frame as the E. coli strains presented in this report [E. coli phylogenetic clades: ECOR A, B1, B2 and D. Few studies have looked at the geographical variance in the distribution of these groups but overall, QREC from Ghana were predominantly drawn from ECOR group A. Of the STs identified in this study that are classified into ECOR clades at the E. coli MLST database, ST10 complex (14 isolates) belong to ECOR group A, ST131 (1 isolate) to ECOR B2, STs101 and 410 (3 isolates) to ECOR B1 and STs 156, 206 and 210 (4 isolates) are hybrids of ECOR A and B1, that is AxB1. Available data appear to suggest that ECOR A strains are highly prevalent in Africa, compared to some other world regions [qnrS1 is associated with ST10 complex.Resistance patterns in pathogens often mirror those in commensals. This is borne out by our recent documentation of quinolone resistance in s report . Fifteen regions . However regions -28. For  regions -28. HoweE. coli and fluoroquinolone consumption at population levels [E. coli were multiply resistant, selective pressure from other, even more commonly applied, antimicrobials will help to maintain the quinolone-resistant clonal groups we identified in this study.A recent paper by Davidson et al suggests that the antimalarial chloroquine may select for fluoroquinolone-resistant fecal bacteria in malaria endemic areas and proposes that chloroquine-mediated selection accounts for high levels of QREC in fecal flora in villages in South America . Howevern levels ,31. Our Fluoroquinolones, largely ciprofloxacin, were introduced very recently to Ghana with high expectations. This study demonstrates that resistance to these drugs is already common and occurs through multiple mechanisms, suggesting that heavy use of these valuable drugs may rapidly obliterate their usefulness. In addition to the impact that the emergence and dissemination of quinolone resistant bacteria may have on the use of fluoroquinolone antibacterials, we found that QREC were almost invariably resistant to multiple antimicrobials. This is worrisome because it means that, if the commensal flora is reflective of resistance profiles in pathogens, there may be few low-cost alternatives for managing infections due to Gram-negative enteric organisms. Additionally, horizontally-acquired resistance to the quinolones, and presumably other agents may be present on mobile elements that could be transmitted to pathogens. Recent calls for antimicrobial development have spotlighted hospital pathogens and Gram-positive community-acquired pathogens such as Staphylococci . Our datE. coli isolates were recovered from stool specimens collected from consenting, apparently healthy individuals who presented for medical check-ups at the Korle-Bu Teaching Hospital and the Microbiology Department of the University of Ghana Medical School. Colonies with a typical E. coli morphology on MacConkey agar were subjected to biochemical testing, and where this was inconclusive, by 16 S amplification and sequencing [This study was approved by the Institutional Review Board of the University of Ghana Medical School. quencing . ColonieE. coli ATCC 35218 were obtained from Remel. The antimicrobial discs used contained ampicillin (10 μg), streptomycin (10 μg), trimethoprim (5 μg), tetracycline (30 μg), nalidixic acid (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg) and sulphonamide (300 μg). Inhibition zone diameters were interpreted in accordance with CLSI guidelines with WHONET software version 5.3 [E. coli ATCC 35218 as control [Each isolate was tested for susceptibility to eight antimicrobials using the Clinical and Laboratory Standards Institute  disc diffusion method . Antimicsion 5.3 . Minimum control .gyrA and parC genes were amplified from DNA templates by PCR using Platinum PCR supermix (Invitrogen) and the primer pairs listed in Table gyrA amplifications were annealed at 58°C and parC reactions were annealed at 52°C. E. coli K-12 MG1655 [DNA was extracted from each quinolone-resistant isolate, using the Promega Wizard genomic extraction kit. The QRDR of the 2 MG1655  was used2 MG1655  by pair-qepA gene and qnrA, qnrB, and qnrS were identified with PCR using the primer pairs published by Wu et al [Horizontally-acquired quinolone-resistance genes were identified by PCR. The primers of Liu et al  were useWu et al  (Table 2adk, fumC, gyrB, icd, mdh, purA and recA were amplified using primers listed in Table E. coli MLST database at http://www.mlst.net. Phylogenetic inferences about ancestral allelic profiles and strain interrelatedness were made using eBURSTv3 at http://eburst.mlst.net/ defining clonal complexes based on groups sharing five identical alleles and bootstrapping with 1000 samplings.Gene fragments from the 2 or Fisher's exact test with p-values less than 0.05 being considered significant.Proportions were compared using the χSSN performed molecular experiments, analysed and interpreted data, and contributed to writing the paper. JAO collected isolates and performed microbiology experiments. RSL designed and performed molecular experiments. MJN co-conceived the study and collected isolates. INO co-conceived the study, performed microbiology and molecular experiments, analysed and interpreted data and wrote the manuscript. All authors read and approved the final manuscript.This work was supported by a Branco Weiss Fellowship from the Society in Science, ETHZ, Zürich to INO. SSN and RSL were HHMI-supported undergraduate researchers, and RSL was also an Arnold and Mabel Beckman Scholar, at Haverford College.


Gram-negative bacterial bloodstream infection (BSI) is a serious condition with estimated 30% mortality. Clinical outcomes for patients with severe infections improve when antibiotics are appropriately chosen and given early. The objective of this study was to estimate the association of prior healthcare exposure on time to appropriate antibiotic therapy in patients with gram-negative BSI.invitro activity against the infecting organism. Healthcare exposure status was categorized as community-acquired, healthcare-associated, or hospital-acquired. Time to appropriate therapy among groups of patients with differing healthcare exposure status was assessed using Kaplan-Meier analyses and multivariate Cox proportional hazards models.We performed a multicenter cohort study of adult, hospitalized patients with gram-negative BSI using time to event analysis in nine community hospitals from 2003-2006. Event time was defined as the first administration of an antibiotic with The cohort included 578 patients with gram-negative BSI, including 320 (55%) healthcare-associated, 217 (38%) community-acquired, and 41 (7%) hospital-acquired infections. 529 (92%) patients received an appropriate antibiotic during their hospitalization. Time to appropriate therapy was significantly different among the groups of healthcare exposure status (log-rank p=0.02). Time to first antibiotic administration regardless of drug appropriateness was not different between groups (p=0.3). The unadjusted hazard ratios (HR)  were 0.80 (0.65-0.98) for healthcare-associated and 0.72 (0.63-0.82) for hospital-acquired, relative to patients with community-acquired BSI. In multivariable analysis, interaction was found between the main effect and baseline Charlson comorbidity index. When Charlson index was 3, adjusted HRs were 0.66 (0.48-0.92) for healthcare-associated and 0.57 (0.44-0.75) for hospital-acquired, relative to patients with community-acquired infections.Patients with healthcare-associated or hospital-acquired BSI experienced delays in receipt of appropriate antibiotics for gram-negative BSI compared to patients with community-acquired BSI. This difference was not due to delayed initiation of antibiotic therapy, but due to the inappropriate choice of antibiotic. Timely and appropriate antimicrobial therapy is critically important for treatment of patients with severe infections. Inadequate timing or choice of antibiotics increases the risk that patients will die; even a few hours of delay increases mortality risk in patients with severe infections -7. MultiAlthough the impact of inappropriate empiric antibiotic therapy has been well-established, unanswered questions remain. Specifically, what types of patients are at risk for delayed appropriate therapy? Antibiotic management of Gram-negative infections is increasingly difficult due to drug resistance and lagging antibiotic development . Gram-nePrior studies have identified patient groups at increased risk for inappropriate empiric antibiotic therapy, which include patients with Gram-negative infections or nosocomial acquisition ,4,14-18.Time to appropriate antibiotic therapy has recently been proposed as a quality measure and needs further study . This prThe objective of this multicenter cohort study was to estimate the effect of healthcare exposure status on the time to receive appropriate antibiotic therapy in patients with Gram-negative BSI from the community hospital setting.This study was reviewed and approved by the Institutional Review Board (IRB) of Duke University Health System. Participating community hospitals deferred to the Duke IRB (n=5), or reviewed and approved the study via their local IRB (n=4). Written patient consent was waived by all sites.This retrospective cohort study included patients at nine community, non-academic hospitals in North Carolina and Virginia affiliated with the Duke Infection Control Outreach Network (DICON) ,21. The BSI events were defined using Centers for Disease Control and Prevention (CDC) criteria for laboratory-confirmed BSI: ≥ 1 positive blood culture for all bacterial pathogens except common skin contaminants, which required ≥ 2 positive blood cultures within a 48 hour period ,23. The Time of BSI event was defined as collection date and time of first positive blood culture growing the Gram-negative pathogen. The main outcome was time to receipt of appropriate antibiotic therapy.in vitro, and 2) antibiotic and route of administration would provide adequate bioavailability for treatment of BSI. For example, a pathogen may show in vitro susceptibility to nitrofurantoin; however this was deemed inappropriate for BSI due to poor antimicrobial activity in plasma.Antibiotic therapy was appropriate if two criteria were met: 1) the pathogen was susceptible to the antibiotic Time to appropriate therapy was defined as starting 24 hours prior to collection of first positive blood culture (“time 0”) and ending with receipt of first dose of appropriate antibiotic (the event). Ideally, time 0 starts with the first opportunity for a clinician to recognize the onset of bacteremia; however, this was impossible to measure retrospectively. Therefore, we included appropriate antibiotic choices made during the 24 hours leading up to collection of blood cultures. Although blood cultures should be collected prior to initiation of antibiotic therapy, empiric antibiotic therapy before obtaining blood cultures is a common practice. To be clear, time 0 was defined as the time of blood culture collection minus 24 hours for the purposes of this analysis. Thus, the time interval analyzed in this study included time in the emergency room for patients presenting from the community. Patients who were receiving an appropriate antibiotic for >24 hours at the time of blood culture collection were defined as “breakthrough” BSIs [Polymicrobial infection was defined as presence of >1 pathogen during any single BSI event. If multiple Gram-negative pathogens were present during the same BSI event, appropriate therapy did not occur until at least one agent active against each Gram-negative pathogen was administered. Patients who never received an appropriate antibiotic were administratively censored at noon on day of discharge or 240 hours (10 days), whichever came first. Patients who received an appropriate antibiotic >10 days from time 0 were censored at 240 hours and considered not to have experienced the outcome. Administrative censoring was utilized to dampen the skewing effect of the few patients who had a prolonged admission without appropriate therapy.in vitro activity or bioavailability. Administrative censoring was the same as described above.Time to any antibiotic therapy, regardless of appropriateness, was explored as a secondary outcome and defined similarly. Time 0 started 24 hours prior to collection of first positive blood culture. Event was receipt of first dose of any antibiotic without consideration of Healthcare exposure status was the main exposure of interest, categorized into three groups using a definition modified from Friedman et al.  HealthcaMultidrug-resistant (MDR) phenotype was defined using published consensus definitions . InfectiCharlson comorbidity index measured at hospital admission and APACHE II score measured on day of BSI were calculated as previously described ,28.Descriptive statistics summarized the patient cohort. Kaplan-Meier curves were constructed for time to appropriate therapy and time to any antibiotic therapy. Log-rank tests were used to compare among groups of healthcare exposure status. Alpha level of 0.05 and two-sided tests were used with any exceptions noted below.Cox proportional hazard models were fit to measure the hazard ratios (HR) and 95% confidence intervals (CI) for the association between healthcare exposure and time to appropriate therapy. Proportional hazards assumptions were tested using time-dependent variables. Clustering due to hospital-specific factors were accounted for using the methods of Lee et al. and the robust covariance estimator . Patienta priori to be important and were included in modeling regardless of statistical significance.Covariates were chosen from factors theorized to impact the association between healthcare exposure and time to appropriate therapy. Covariates directly used in definitions of healthcare exposure  were excluded. Charlson index and age were determined a priori alpha<0.1). Each covariate deemed not to be a potential modifier was evaluated individually for association with the main exposure  among all patients, and then the main outcome among patients in the referent category  to determine potential confounding.Selected covariates were evaluated for effect measure modification using hierarchical models with interaction terms and likelihood ratio tests (LRT) for model fit .The study cohort included 578 patients with Gram-negative BSIs after removal of 19 patients with breakthrough BSI. A total of 320 (55%) patients had healthcare-associated, 217 (38%) had community-acquired, and 41 (7%) had hospital-acquired BSI . Most paEnterobacteriaceae. E. coli was observed more commonly in community-acquired BSI, whereas Enterobacter was predominately found in hospital-acquired BSI.A total of 598 Gram-negative pathogens were isolated from 578 patients . Forty-eMDR phenotype was observed in 84 (15%) patients in the entire cohort. MDR was more common in healthcare-associated  and hospital-acquired  BSIs, than in community-acquired BSIs  . SeventyA total of 1,648 courses of antibiotics were administered to study patients during their admission . The most common antibiotics administered were ceftriaxone , levofloxacin , ciprofloxacin , and piperacillin-tazobactam .We observed significant differences in secondary outcomes, including mortality, discharge to skilled nursing facility, intubation, and readmission in 90 days among the three groups of healthcare exposure status .Five hundred twenty-nine (92%) of 578 patients received appropriate therapy during the 10-day risk period. Forty-nine (8%) patients were administratively censored at discharge or day 10. Time to appropriate therapy was significantly different for the three groups  .Unadjusted Cox analysis suggested that patients with healthcare-associated  or hospital-acquired  BSI experienced delays in receiving appropriate therapy when compared to patients with community-acquired BSI. Tests for violation of the proportional hazards assumption were not significant.Charlson score, body mass index, and active malignancy were found to be potential effect measure modifiers. We pursued a single interaction only due to lack of power to evaluate multivariable interactions. Therefore, only interaction terms between Charlson and the main exposure were included in subsequent modeling.a priori criteria reduced the covariate pool to Charlson, age, body mass index, male sex, Medicare/Medicaid, active malignancy, non-white race, and functional status. Following backward elimination, the final adjusted Model 1 included the main exposure, Charlson plus interaction terms, age, active malignancy, Medicare/Medicaid, and dependence in ≥ 3 activities of daily living. Final adjusted HR (aHR) estimates among strata of Charlson revealed that healthcare-associated and hospital-onset infections were associated with delay in receipt of appropriate antibiotic therapy compared with community-acquired infections  that make clinical evaluation more challenging. These healthcare-related events were not captured in our study.Time of initial clinical evaluation may also impact the degree of clinical uncertainty. Community-acquired patients who present for care several days after infection onset may have clear manifestations of their primary source. In contrast, healthcare-exposed patients may be evaluated early in their infection course and lack symptoms of a localized source. Early access to care could affect disease latency at the time of first clinical evaluation and thus impact the ease of diagnosis and antibiotic choice.We found evidence of interaction with baseline comorbidity and the association between healthcare exposure and time to appropriate therapy. The hazard ratio appeared strongest when Charlson index was low, which may be related to lack of statistical power when Charlson was high. Alternatively, more appropriate (or aggressive) antibiotic therapy may have occurred in patients with high baseline comorbidity. We cannot, however, make this conclusion with our data.Definition of the time component for appropriate antibiotic therapy is not standardized. Most prior studies have examined inappropriate empiric therapy, defined as the lack of receipt of an effective antibiotic within 24 hours from blood culture collection, or in the 48 hour period surrounding blood culture collection (e.g. 1 day before to 1 day after) ,18,32. IThis study is limited by its retrospective design, which may have resulted in measurement error or uncontrolled confounding. Limited data could be gathered regarding a primary source of BSI, and the true timing of blood culture collection as compared to the documented date and time could not be validated. Also, unmeasured or unknown confounding factors could have affected the multivariate analysis. Second, the study period 2003-2006 may not reflect current antibiotic prescribing practices or prevalence of multidrug resistance. Third, we did not examine antibiotic dose, which is important for optimal antibiotic therapy. We believe, however, that this study has relevance for today’s prescribers. It highlights the need to assess healthcare-associated risk factors and provides greater understanding of effects of prior healthcare exposure on treatment decisions.Early, effective antibiotic therapy is important for providing high quality care for patients with Gram-negative BSI. We demonstrated that healthcare-exposed patients are at risk for delayed receipt of appropriate antibiotics.


Excessive reliance on wheelchairs in individuals with tetraplegia or paraplegia due to spinal cord injury (SCI) leads to many medical co-morbidities, such as cardiovascular disease, metabolic derangements, osteoporosis, and pressure ulcers. Treatment of these conditions contributes to the majority of SCI health care costs. Restoring able-body-like ambulation in this patient population can potentially reduce the incidence of these medical co-morbidities, in addition to increasing independence and quality of life. However, no biomedical solution exists that can reverse this loss of neurological function, and hence novel methods are needed. Brain-computer interface (BCI) controlled lower extremity prostheses may constitute one such novel approach.One able-bodied subject and one subject with paraplegia due to SCI underwent electroencephalogram (EEG) recordings while engaged in alternating epochs of idling and walking kinesthetic motor imagery (KMI). These data were analyzed to generate an EEG prediction model for online BCI operation. A commercial robotic gait orthosis (RoGO) system (suspended over a treadmill) was interfaced with the BCI computer to allow for computerized control. The subjects were then tasked to perform five, 5-min-long online sessions where they ambulated using the BCI-RoGO system as prompted by computerized cues. The performance of this system was assessed with cross-correlation analysis, and omission and false alarm rates.−4). Also, there were on average 0.8 false alarms per session and no omissions.The offline accuracy of the EEG prediction model averaged 86.30% across both subjects (chance: 50%). The cross-correlation between instructional cues and the BCI-RoGO walking epochs averaged across all subjects and all sessions was 0.812±0.048 (p-value <10These results provide preliminary evidence that restoring brain-controlled ambulation after SCI is feasible. Future work will test the function of this system in a population of subjects with SCI. If successful, this may justify the future development of BCI-controlled lower extremity prostheses for free overground walking for those with complete motor SCI. Finally, this system can also be applied to incomplete motor SCI, where it could lead to improved neurological outcomes beyond those of standard physiotherapy. Individuals with tetraplegia or paraplegia due to spinal cord injury (SCI) are unable to walk and most are wheelchair bound. Decreased physical activity associated with prolonged wheelchair use leads to a wide range of co-morbidities such as metabolic derangements, heart disease, osteoporosis, and pressure ulcers . UnfortuA brain-computer interface (BCI) controlled lower extremity prosthesis may be one such novel approach. It can be envisioned that a combination of an invasive brain signal acquisition system and implantable functional electrical stimulation (FES) electrodes can potentially act as a permanent BCI prosthesis. However, for safety reasons, the feasibility of brain-controlled ambulation must first be established using noninvasive systems.This concept was explored in the authors’ prior work -4 in whiTo facilitate the development of a BCI-controlled RoGO, EEG data were recorded from subjects as they engaged in alternating epochs of idling and walking KMI. These data were then analyzed offline to generate an EEG prediction model for online BCI operation. A commercial RoGO system (suspended over a treadmill), was interfaced with the BCI computer to allow for computerized control. In a series of five, 5-min-long online tests, the subjects were tasked to ambulate using the BCI-RoGO system when prompted by computerized cues. The performance of this system was assessed by calculating the cross-correlation and latency between the computerized cues and BCI-RoGO response, as well as the omission and false alarm rates.Ethical approval was obtained from the Institutional Review Board at the Long Beach Veterans Affairs Medical Center (LBVA) and the University of California, Irvine (UCI). Subjects were recruited from a population of able-bodied individuals, or those with chronic, complete motor paraplegia due to SCI (> 12 months post-injury). The exclusion criteria for subjects with SCI were severe spasticity, contractures, restricted range of motion, or fractures in the lower extremities, pressure ulcers, severe osteoporosis, or orthostatic hypotension. These criteria were ruled out in a safety screening evaluation consisting of an interview, a physical exam, lower extremity dualenergy x-ray absorptiometry (DEXA) scan and x-rays, and a tilt-table exam.Ω. EEG signals were acquired using 2 linked NeXus-32 bioamplifiers  at a sampling rate of 256 Hz. The subjects were suspended into a treadmill-equipped RoGO  using partial weight unloading ; cooperative walking ; and passive walking . Three pairs of surface EMG electrodes were placed over the left quadriceps, tibialis anterior, and gastrocnemius  was measured to rule out BCI control by voluntary leg movements in able-bodied subjects. To this end, baseline lower extremity EMG were measured under 3 conditions: s Figure , and sigs Figure  5]. App. Appactiet al. was found by increasing the lower and upper frequency bounds and repeating the above procedure until the classifier performance stopped improving , the CPCA-AIDA transformation, and the classifier parameters, were then saved for real-time EEG analysis during online BCI-RoGO operation. The above signal processing and pattern recognition algorithms were implemented in the BCI software and were optimized for real-time operation . Let Pk} is wide-sense stationary with mean μ and variance σ2, the coefficients α and β can be determined from: where ρ is the correlation coefficient between Pk+1 and Pk. Using these coefficients, 10,000 Monte Carlo trials were performed for each online session. Each sequence of simulated posteriors, {Yk}, was then processed as in Section “Calibration” above, and the cross-correlation between the cues and simulated BCI-RoGO state sequence was calculated. An empirical p-value was defined as a fraction of Monte Carlo trials whose maximum correlation was higher than that of the online session.where TI and TW were set at 0.04 and 0.65 for Subject 1, and 0.50 and 0.90 for Subject 2.Two subjects (one able-bodied and one with paraplegia due to SCI) were recruited for this study, provided their informed consent to participate, and consented to the publication of the biomedical data and media (photographs and videos) presented in this report. Their demographic data are described in Table −4. Also, there were no omissions for either subject. The false alarm rate averaged 0.8 across all sessions and both subjects. While the duration of these false alarm epochs averaged 7.42±2.85 sec, much of this time can be attributed to the RoGO’s locked-in startup sequence (∼5 sec). In addition, each subject managed to achieve 2 sessions with no false alarms. Videos of a representative online session for the able-bodied subject (Subject 1) and for the subject with SCI (Subject 2) are provided as downloadable supplemental media. Alternatively, online versions can be found at: http://www.youtube.com/watch?v=W97Z8fEAQ7g, and http://www.youtube.com/watch?v=HXNCwonhjG8.The performances from the 5 online sessions for both subjects are summarized in Table active or cooperative walking conditions (p < 10 −13), and were not different from those of passive walking (p = 0.37). These results confirm that the BCI-RoGO system was wholly BCI controlled. Note that passive walking is known to generate EMG activity [For Subject 1, the EMG and leg movement data from online sessions were analyzed as described in Section “Online evaluation”. EMG and gyroscope measurements indicated that no movement occurred prior to the initiation of BCI decoded “walking” states  was able to accomplish this with minimal prior BCI experience and after only a brief 10 min training data acquisition session. To the best of the authors’ knowledge, this represents the first-ever demonstration of a person with paraplegia due to SCI re-gaining brain-driven basic ambulation and completing a goal-oriented walking task.The EEG prediction models for both subjects in this study had a high offline classification accuracy. In the case of Subject 1, the performance was higher than his performances in prior BCI walking avatar studies  was higher than those achieved with lower (0.67) and upper (0.78) extremity BCI-prostheses ,12, desp was highWith no more than ∼5 hr of relevant BCI experience , bothBased on the above observations, this data-driven BCI approach may be necessary for future intuitive and practical BCI-controlled lower extremity prostheses for people with SCI. This approach would enable subjects with SCI to use intuitive BCI control strategies such as KMI of walking or attempted  walking. Similar to Subject 2 in this study, this can potentially be accomplished with minimal user training and supervision from the experiment operator. Finally, this approach may enhance the appeal and practicality of future BCI-controlled lower extremity prostheses for ambulation by reducing the time burden and associated costs.In conclusion, these results provide convincing evidence that BCI control of ambulation after SCI is possible, which warrants future studies to test the function of this system in a population of subjects with SCI. Since participants with SCI were able to operate the BCI-walking simulator ,4, it isSCI: Spinal cord injury; BCI: Brain-computer interface; EEG: Electroencephalogram; KMI: Kinesthetic motor imagery; RoGO: Robotic gait orthosis; DEXA: Dual-energy x-ray absorptiometry; PSD: Power spectral densities; CPCA: Classwise principal component analysis; EMG: Electromyogram.CEK has received salary from HRL Laboratories, LLC . The remaining authors declare no competing interests.AHD conceived the study, recruited and evaluated subjects, implemented the interface between the computer and RoGO, carried out the experiments, and wrote the paper. CEK and PTW carried out the experiments, collected the data, analyzed the data, and co-wrote the paper. PTW also implemented the BCI software. SNC contributed to conception of the study and co-wrote the paper. ZN conceived the study, designed the signal processing algorithm, carried out the experiments, and co-wrote the paper. All authors read and approved the final manuscript.Video of representative BCI-RoGO session in able-bodied subject. This video is a recording of a representative BCI-RoGO session for Subject 1. The Idle/Walk cues are presented to the subject on the monitor. The subject responds by generating idle/walking KMI to control the BCI-RoGO system.Click here for fileVideo of representative BCI-RoGO session in subject with paraplegia due to SCI. This video demonstrates a representative BCI-RoGO session for Subject 2 (paraplegia due to T6 ASIA B SCI). The video first demonstrates the mounting procedure specifically adapted to SCI. Similar to Subject 1, the Idle/Walk cues are presented on the monitor, and the subject responds by generating idle/walking KMI to control the BCI-RoGO system. This is followed by a dismounting procedure that is specific to SCI. Due to some mild discomfort with breathing while wearing the restrictive body-weight unloading harness, Subject 2 was allowed to rest his arms on the hand rails. Note that the video has been time-compressed by a factor of 4.Click here for file


Protein and antibody arrays have emerged as a promising technology to study protein expression and protein function in a high-throughput manner. These arrays also represent a new opportunity to profile protein expression levels in cancer patients’ samples and to identify useful biosignatures for clinical diagnosis, disease classification, prediction, drug development and patient care. We applied antibody arrays to discover a panel of proteins which may serve as biomarkers to distinguish between patients with ovarian cancer and normal controls.Using a case-control study design of 34 ovarian cancer patients and 53 age-matched healthy controls, we profiled the expression levels of 174 proteins using antibody array technology and determined the CA125 level using ELISA. The expression levels of those proteins were analyzed using 3 discriminant methods, including artificial neural network, classification tree and split-point score analysis. A panel of 5 serum protein markers  was identified, which could effectively detect ovarian cancer with high specificity (95%) and high sensitivity (100%), with AUC =0.98, while CA125 alone had an AUC of 0.87.Our pilot study has shown the promising set of 5 serum markers for ovarian cancer detection. Ovarian cancer represents the third most frequent cancer and is one of the leading causes of cancer death among females in the United States and Europe -3. Most Currently, CA-125 and imaging are the 2 most common approaches for ovarian cancer screening tests. However, these 2 markers, either used alone or in combination, are not useful screening or diagnostic purposes due to low specificity and/or sensitivity. For example, serum CA-125 has been shown to have a sensitivity of >98% but a specificity of only 50-60% for early-stage disease -6.P<0.05). By classification analysis and split-point score analysis of these 2 groups, a 6-marker panel of proteins, which consisted of interleukin-2 receptor alpha (IL2Rα), endothelin, osteoprotegerin (OPG), vascular endothelial growth factor D (VEGF-D) and betacellulin (BTC), can be used to distinguish ovarian cancer patients from normal subjects [Multiple studies have been reported to identify serum ovarian cancer biomarkers using multiplex antibody array technology -9. Dr. LIn this study, we used our 174-marker, sandwich ELISA-based antibody array panels to screen serum samples from 34 ovarian cancer patients and 53 normal healthy subjects in order to identify a serum protein marker panel for detection of ovarian cancer.In this study, we applied antibody array technology to determine the expression profiles of 174 cytokines in the serum from ovarian cancer patients and age-matched healthy normal controls. Cytokines in this study included anti-inflammatory cytokines, proinflammatory cytokines, growth factors, angiogenic factors or chemotactic cytokines, among others. Some of these cytokines reportedly are altered in ovarian cancer patients from our own studies and literature, but our broad screen of 174 proteins also included many other types of markers as part of an “unbiased” approach of using high-content, high-throughput cytokine antibody arrays to profile the cytokine levels from ovarian cancer patients’ serum with the goal of identifying potential diagnostic biomarkers.P<0.001) and 0.899 (P<0.001) respectively, suggesting high reproducibility of the assay. In contrast, the Pearson correlation coefficient for cancer vs. normal samples were 0.226 (P<0.005), suggesting that the cancer samples and normal samples are from two different populations.First, we further determined the reproducibility of the assay in the analysis of human serum using scatter-plot analysis. Intra-slide reproducibility for the glass-slide–based arrays was assessed by testing replicate aliquots of the same samples with two sub-arrays printed on the same slide and assayed at the same time. The inter-slide reproducibility was determined using two different slides printed with the same arrays were assayed using duplicate aliquots of the same samples on two different days. The Pearson correlation coefficients for intra-slide and inter-slide reproducibility were 0.923  analysis. The signal intensity data for individual patients were randomly divided into the training set (N= 51) or prediction set (N=36). In prediction discovery phase, the training set was analyzed using leave-one cross-validation approach. Through this analysis, a total of 8 predictors were identified. These 8 predictors were then used to predict the disease status in prediction set. The correct agreement of predicted disease status using the 8-marker panel with clinical diagnosis in the training set and prediction set was 82% and 80% respectively.Next, of these 8 markers, we chose 4, macrophage stimulating protein alpha , tissue inhibitor of metalloproteinases-4 (TIMP-4), platelet derived growth factor receptor alpha , and osteoprotegerin (OPG), for hierarchal cluster analysis using SPSS software. Using the 4-marker panel above, 83% of samples were correctly identified .Finally, all 87 samples were analyzed by the above identified 4 serum markers plus CA125 using split-point score analysis. Using the cutoff score of 3, 100% ovarian cancer and 95% healthy control samples were correctly identified, giving the total correct agreement of 96.6%.Since CA125 is the most widely used marker for ovarian cancer, we compared the AUC between CA125 alone to that of our 5-marker panel, as determined by ROC curves. CA125 alone has an AUC of 0.87. On the other hand, our newly identified 5-marker panel has an AUC of 0.98. Thus, our pilot study has identified a promising set of 5 serum markers for early detection of ovarian cancer.To confirm the multiplex detection of the array data, we performed single-target ELISA assays to quantitatively measure the expression levels of these cytokines individually, and these results were compared with the array data. The relative expression levels for proteins measured by the array and ELISA were similar see . All 4 mCA125 is one of the most important biomarkers for ovarian cancer. It is often used effectively for monitoring treatment response and detecting recurrence of ovarian cancer. However, CA125 alone is not a useful diagnostic marker for clinical application due to its low specificity; with a reference cutoff value of 35 IU/ml, CA125 showed limited specificity of 50–60% with the sensitivity of >98% for early-stage disease -6. ElevaOwing to the complexity and heterogeneity of ovarian cancer, it is unlikely that a single biomarker will be able to detect all subtypes and stages of the disease with a high specificity and sensitivity. By searching the literature and other source, Drs. Polanski and Anderson have compile a list of 1261 proteins believed to be differentially expressed in human cancer [Cytokines are a diverse group of proteins comprised of cytokines, chemokines, growth factors, interferons, adipokines and lymphokines and play many critical roles of physiological and pathological processes. It is also well known that cytokines, chemokines, growth factors, angiogenesis factors, proteases, apoptotic factors, receptors, adhesion molecules and adipokines play important roles in cancer development, progression and metastasis. Growing evidence suggests that a complex cytokine network is involved in ovarian cancer. A number of autocrine and paracrine cytokine loops have been identified in ovarian cancer and influence the biology of this tumor. Detection of expression patterns of multiple cytokines can provide new insights on cancer biology, identify new molecular targets for cancer treatment and discover new biomarkers for diagnosis and prognosis of disease ,14.In this study, we have demonstrated the effectiveness of screening a semi-quantitative, sandwich-based antibody array detecting a panel of 174 markers in the serum of 34 ovarian cancer patients and 53 age-matched healthy controls to identify a panel of 5 serum protein markers, including CA125, that can effectively detect ovarian cancer with high specificity (95%) and high sensitivity (100%) with AUC of 0.98. These markers were validated with ELISA assay.We observed that CA125 alone has an AUC of 0.87, on the other hand, our newly identified 5-marker panel has an AUC of 0.98, indicating improved efficiency when detection of CA125 is combined with other 4 putative protein biomarkers for detection of ovarian cancer .TIMP-4 belongs to the matrix metalloproteinase (MMP) superfamily. MMPs are essential elements in extraceullular matrix (ECM) degradation, including regulating the release of ECM-bound cytokines and growth factors, which leads to angiogenesis, cellular invasion and, eventually in many cancers, metastasis. These MMPs are tightly controlled and regulated by several TIMPs, several of which appear to play a critical role in tumorigenesis. Chegini’s Lab has reported elevated expression of TIMP-4 in ovarian cancer tissues by IHC analysis, indicating its potential role in tumorigenesis of ovarian cancer .OPG belongs to TNF superfamily and can be linked to the nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) signaling pathways. OPG was first identified by its ability to regulate the homeostasis of bone remodeling. However, Piche’s Lab reported that OPG can serve as such survival factor by protecting TRAIL-induced apoptosis in ovarian cancer cells, indicating its potential role in the development and progression of ovarian cancer .PDGF-R alpha is a receptor in the PDGF superfamily. Serving as angiogenic growth factors, PDGFs play important role in cell growth, chemotaxis, angiogenesis and, in the context of cancer, reconstruction of tumor stromal microenvironments. Jakobsen’s Lab reported that PDGF-R alpha showed higher expression in ovarian cancer tissues in comparison with adjacent normal tissues . It was MSP is a growth factor involved in activating macrophage stimulating receptor-1 (MSTR1). The alpha chain of MSP  is secreted by cleavage of pro-MSP. There are reports showing that the MSP pathway plays an important role in tumor metastasis .In summary, using 174-marker cytokine antibody array technology, we identified a panel of 5 serum protein markers which can detect ovarian cancer with both high specificity and high sensitivity, indicating its promising application in personalized medicine for ovarian cancer detection. Additionally, considering that a relatively small sample size (N<100) used in this investigation achieved an extremely high sensitivity (100%) and relatively high specificity (95%), we offer some hope that validation of multi-biomarker panel may someday be useful to screen for a deadly cancer that rarely gets diagnosed in its early stages.Protein and antibody arrays have emerged as a promising technology to study protein expression and protein function in a high throughput manner. These protein and antibody arrays present a new opportunity to profile protein expression levels in cancer patients’ samples and identify useful biosignatures for drug development and patient care. Our 5-marker panel could effectively distinguish ovarian cancers from healthy controls These 5 individual markers are not unique to ovarian cancers, as shown by their expression in other cancer types, including breast cancers -22, lungThis protocol has been approved by sterling institutional review board. IRB ID: 3303. The review board is sterling independent service, Inc located in 6300 powers ferry road, suite 600-351, Atlanta, GA 30339. Written consent was obtained when collecting samples from both patients and healthy controls.Written consent was obtained when collecting samples from both patients and healthy controls.The serum samples from 34 patients diagnosed with early-stage (I and II) or late-stage (III and IV) ovarian cancers and 53 age-matched healthy controls included in the study were collected from the affiliated hospital, Sun Yat-sen University. Briefly, about 2 ml of venous blood was drawn from patients. Serum was collected and stored at -80°C until needed. Information about ovarian cancer diagnosis, staging, histology, grade and age was available to us, but the unique patient identifiers, such as name, address, day of birth, was not provided.® Human Cytokine Array G-Series 2000) were developed as 3 distinct arrays , each representing a unique set of 54 to 60 antigen-specific antibodies to detect a total of 174 serum markers on a glass slide matrix. A pair of antibodies is required to detect each analyte. Glass slides were printed as 4 or 8 identical sub-arrays consisting of spots of each antigen-specific apture antibody for that array. The corresponding detection antibodies were biotin-labeled and combined as a single cocktail reagent for later use. Printed slides were placed in chamber assemblies to allow for incubation of each sub-array with a different sample. After blocking each sub-array with a blocking buffer, sub arrays were incubated with serum samples. Following extensive washing to remove non-specific binding, the cocktail of biotinylated detection antibodies were added to the arrays. After extensive washing, the array slides were incubated with a streptavidin-conjugated fluor . The fluorescent signals were then visualized using laser-based scanner system  using the green channel. To increase the accuracy, two replicates per antibody were spotted, and the averages of the median signal intensities for both spots  were used for all calculations. Through these improvements, we can get a coefficient of variation (CV) of about 10% using our glass slide platform.Semi-quantitative sandwich-based antibody arrays . In brief, pre-coated 96-well ELISA plates with captured antibodies were first blocked using a blocking buffer. Duplicate aliquots (100 microliter per well) of diluted sera and multiple dilutions  of standard protein were loaded onto the ELISA plate. The plates were then incubated for 2 h at room temperature (RT). Unbound materials were washed out, and biotinylated anti-cytokine detection antibody was added to each well. The plates were incubated for 1 h at RT. After washing, 100 microliter of streptavidin-conjugated HRP reagent was added to the wells, and the plate was incubated for 30 minutes at RT. After extensive washing, color development was performed by incubation with HRP substrate. After adding stop solution, the optical density (O.D.) at 450 nm was determined for each well using a microplate reader, and the concentrations of the samples were determined by comparison to the standard concentration curves.P values less than 0.05 were considered to be statistically significant.An adjusted t-test was used to test the significance between protein expression levels in ovarian cancer and healthy control samples. To determine the signal threshold, signals from the arrays were measured in the absence of samples (using blocking buffer as a blank) and repeated 10 times. The signals generated using blanks were averaged and the standard deviation (SD) was calculated. Signals with values lower than the average blank signal +2xSD were considered as background.The data was also analyzed using neural network. This powerful tool allows us the find common protein expression profiles to predict cancer. In phase one study, 80% of samples were randomly assigned to training set and the remaining 20% of samples were used as test set. The advantage of this approach is the success of prediction will become more accurate over time, as more data become available.N different markers. Therefore, the range of such score was between 0 to N. A given threshold (T) was chosen to optimally separate ovarian cancer from healthy controls, i.e. a given individual with a total score <T is predicted to have normal status, whereas an individual with a total score >T was diagnosed as ovarian cancer.The data were also analyzed by split-point score analysis. The split point divides the sample space into two intervals, one for ovarian cancer and one for normal controls. The best split-point score of each marker was chosen to ensure the minimization of misclassified samples. For each marker, a score of 0 was assigned to a sample if it fell in the normal control interval for that marker; a score of 1 was assigned to a sample if it fell in the ovarian cancer interval. Overall, an individual was assigned a score as the sum of these assigned scores for From the above data, we calculated the specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV). The ROC was also determined.


Severe alcohol misuse as measured by the Alcohol Use Disorders Identification Test–Consumption (AUDIT-C) is associated with increased risk of future fractures and trauma-related hospitalizations. This study examined the association between AUDIT-C scores and two-year risk of any type of trauma among US Veterans Health Administration (VHA) patients and assessed whether risk varied by age or gender.Outpatients  who returned mailed AUDIT-C questionnaires were followed for 24 months in the medical record for any International Statistical Classification of Diseases and Related Health Problems (ICD-9) code related to trauma. The two-year prevalence of trauma was examined as a function of AUDIT-C scores, with low-level drinking (AUDIT-C 1–4) as the reference group. Men and women were examined separately, and age-stratified analyses were performed.Having an AUDIT-C score of 9–12  was associated with increased risk for trauma. Mean (SD) ages for men and women were 68.2 (11.5) and 57.2 (15.8), respectively. Age-stratified analyses showed that, for men ≤50 years, those with AUDIT-C scores ≥9 had an increased risk for trauma compared with those with AUDIT-C scores in the 1–4 range . For men ≥65 years with average comorbidity and education, those with AUDIT-C scores of 5–8  and 9–12  were at significantly increased risk for trauma compared with men ≥65 years in the reference group. Higher AUDIT-C scores were not associated with increased risk of trauma among women.Men with severe alcohol misuse (AUDIT-C 9–12) demonstrate an increased risk of trauma. Men ≥65 showed an increased risk for trauma at all levels of alcohol misuse (AUDIT-C 5–8 and 9–12). These findings may be used as part of an evidence-based brief intervention for alcohol use disorders. More research is needed to understand the relationship between AUDIT-C scores and risk of trauma in women. Trauma is the leading cause of death in the United States in persons aged ≤44 years, and alcohol misuse is linked to higher risk of traumatic injury . AlcoholRisk of future trauma may differ between individuals based not only on the amount of alcohol consumed but also on age and gender. Klatsky and Armstrong  found thThe Alcohol Use Disorders Identification Test–Consumption (AUDIT-C) is becoming an increasingly relied upon screening tool for alcohol use problems in modern medical practice . It consScores on the AUDIT-C have been associated with risk of future trauma in two prior studies of US veterans. Harris et al.  found thThis study builds on existing research on the relationship between AUDIT-C scores and risk of future trauma. Unlike prior work, which evaluated the risk of fracture and trauma-related hospitalization, this study examines AUDIT-C scores in relation to two-year risk of any type of trauma across all inpatient, residential, and outpatient settings in a large integrated health-care system. Also, the present study is an effort to verify the results of previous studies in a much larger sample with a specific focus on the differential risks associated with age and gender. The overarching goal is to provide more comprehensive sex- and age-tailored data for health-related discussions of alcohol misuse and risk of future trauma. Giving patients specific and tailored feedback about alcohol consumption has been shown to reduce hazardous alcohol use -21 and rAUDIT-C and covariable data were collected by the US Veterans Health Administration (VHA) as part of the population-based Survey of Healthcare Experiences of Patients (SHEP). The sampling strategy and logistics of the SHEP are described elsewhere . Institua priori. Patients with AUDIT-C scores of 0 were past-year nondrinkers. Patients with AUDIT-C scores of 1–4 were considered low-level drinkers in this study. Patients with AUDIT-C scores 5–8 were considered positive for moderate alcohol misuse. Patients with AUDIT-C scores of ≥9 were considered positive for severe alcohol misuse . One standard drink per the US National Institute on Alcohol Abuse and Alcoholism (NIAAA) is 12 ounces of beer, five ounces of wine, or 1.5 ounces of hard liquor [The AUDIT-C consists of the following three questions: (1) How often did you have a drink containing alcohol in the past year? (2) How many drinks containing alcohol did you have on a typical day when you were drinking in the past year? (3) How often did you have six or more drinks on one occasion in the past year? Each response is scored 0–4, with total AUDIT-C scores ranging from 0–12 . Based od liquor , and patPatients who received ambulatory care in Veterans Affairs (VA) health-care facilities in 2004 and 2005, and who had not been surveyed in the past 12 months, were eligible for the SHEP in the month after an outpatient visit. Respondents’ International Statistical Classification of Diseases and Related Health Problems (ICD-9) diagnoses were obtained for VA health services received in the two years following the survey. Respondents had to have used VA health services at least once during the two-year follow-up period to be eligible. Utilization was assessed across diverse treatment settings in a large integrated health care system, including inpatient , residential , outpatient , and emergency departments.Figure Outcomes included all clinically recorded ICD-9 diagnoses for trauma in the two years after completing the AUDIT-C. Trauma was defined as any wounds, amputations, musculoskeletal injuries, fractures (osteoporotic or nonosteoporotic), spinal cord injuries, burns, complications of trauma, poisoning, external causes of injury , concussions and brain injuries, internal injuries, and abuse . (A list ICD-9 codes used to define trauma is available from the authors.) The primary outcome was an indicator (0 or 1) signifying whether the patient experienced any form of trauma, as defined above, during the two-year observation period. Analyses were conducted on each subtype of trauma  to determine if the overall pattern for all traumas obscured underlying differences.The SHEP included questions about race, education , married status (no/yes), and cigarette smoking status . Age was obtained from the VA National Patient Care Database (NPCD). The Deyo comorbidity index, adapted from the Charleston Index for use with ICD-9 administrative data, was constructed from the NPCD based on inpatient and outpatient ICD-9 diagnostic codes assigned to participants in the year prior to taking the SHEP .A priori AUDIT-C group by gender and age interaction effects were evaluated. The interaction of gender and AUDIT-C group was marginally significant (p = 0.06). Given this interaction and the lack of data evaluating the association between alcohol screening scores and health outcomes in women, we stratified all subsequent analyses by gender. Because the recommended alcohol consumption limits for women are lower than those for men, and their recommended threshold for a positive AUDIT-C score is ≥2 compared with ≥3 for men, we examined different AUDIT-C groupings  for women in sensitivity analyses. We also performed age-stratified analyses in men because significant interactions were found, and because prior studies of AUDIT-C and health outcomes suggested age-moderated risk [Initial analyses described the sample and calculated the unadjusted prevalence of the outcomes in each AUDIT-C group. Then, covariable-adjusted logistic regression models were used to assess the risk of each outcome based on AUDIT-C risk group. ted risk ,14.Tables Table For men aged <50 years, those with AUDIT-C scores of ≥9 (indicating severe misuse) had an increased risk for trauma compared with those with AUDIT-C scores in the 1–4 range . Covariables associated with increased risk of trauma were Caucasian race/ethnicity, being single, having more comorbid conditions, and being a current or past smoker. No significant interactions between AUDIT-C group and covariables were found in this subgroup.For men aged 50–65 years, only those with AUDIT-C scores of 0 (past-year nondrinker) had an increased risk for trauma compared with those with AUDIT-C scores in the 1–4 range . In these middle-aged men, covariables associated with increased risk of trauma were non-Caucasian race/ethnicity, being single, having more comorbid conditions, and being a current or past smoker. No significant interactions between AUDIT-C group and covariables were found in this subgroup.For men aged >65 years with average comorbidity and education, both those with AUDIT-C scores of 5–8  and ≥9  had significantly increased risk of trauma compared with the reference group. Nondrinkers aged ≥65 years were also at increased risk of trauma compared with those in the AUDIT-C 1–4 group . In all men ≥65 years, significant interactions between AUDIT-C group, Deyo comorbidity index, and education were found: in general, compared with patients in the AUDIT-C 1–4 category, patients in the other categories were at greater risk for trauma if they had fewer comorbidities or more or less than a high-school education.Age-stratified analyses of specific types of trauma in men revealed similar patterns across age groups for fractures as for all other types of trauma combined Table . Men ageNo association between AUDIT-C scores and trauma outcomes was found among women Table . SensitiTo our knowledge, this is the first study to examine the relationship between alcohol screening scores (AUDIT-C) and risk of inpatient, residential, and outpatient medical treatment for any type of trauma. This is also one of few studies to look at women’s trauma risk separate from men’s. In male VHA patients, severe alcohol misuse (AUDIT-C 9–12) was associated with a significantly increased risk for trauma, particularly fractures. Men in the oldest age group (>65 years) showed increased risk of medical care for trauma even at lower levels of alcohol misuse (AUDIT C 5–8). These findings augment prior research, which found an association between severe alcohol misuse and specific risk of fracture , and betThe AUDIT-C was not a predictor of two-year trauma risk in female VHA patients. The absence of an association is counterintuitive, since women have been shown to be more vulnerable to the toxic effects of alcohol ,31. OtheSeveral factors may be affecting the results on for women in the present study. First, increased stigma for heavy drinking among women typically leads to underreporting drinking levels compared with men ,32. AlsoFractures comprised the third leading cause of trauma for men 2.28%), exceeded only by wounds/amputations and musculoskeletal injuries. The association between alcohol misuse and fractures may be related to the increased risk of falls due to intoxication, with potential contribution from neurologic complications of alcohol misuse such as cerebellar degeneration, Wernicke-Korsakoff syndrome, peripheral neuropathy , and inc.28%, excOn average, older men (>65) had a heightened risk of trauma with lower levels of misuse (AUDIT-C 5–8) as well as severe misuse (AUDIT-C 9–12). This finding is consistent with two prior studies of AUDIT-C and future trauma risk ,18, bothNondrinkers aged >50 years were at increased risk for injuries and accidents over the two-year follow-up. Previous studies found that nondrinkers were at increased risk for adverse health outcomes, including fractures and primary or secondary trauma-related hospitalization ,18 compaAlthough our data do not distinguish between former at-risk drinkers and lifetime nondrinkers, nondrinking men aged <50 years had no increased trauma risk. It is likely that, with age, some formerly heavy-drinking men migrate to the nondrinking group and potentially increase the trauma risk of nondrinkers. Consistent with this hypothesis, older nondrinking men had more comorbid illnesses than younger nondrinking men and older men with alcohol misuse. In other words, the increased risk of trauma among abstainers might be due, in part, to alcohol-related harm from former heavy drinking . Given tThese results need to be understood in light of several limitations. The sample consisted exclusively of patients of the VHA, who are predominantly male and older than the general population. Only 69% of selected patients responded to the SHEP, and only 83% of those provided complete AUDIT-C data. Although we have no reason to expect that the relationship between AUDIT-C scores and subsequent trauma is different in the observed and unobserved samples, this possibility must be considered. Also, with samples this large, some  of the statistically significant differences in risk were small in absolute and relative terms.Also, misclassification and under-ascertainment of outcomes threaten the internal validity of this study. Outpatient ICD-9 codes are not as valid as inpatient codes . Many paIn summary, the AUDIT-C is a useful scaled marker of two-year risk of trauma across different treatment settings in men. Severe alcohol misuse is most strongly associated with risk of subsequent fracture in older men who are otherwise relatively healthy. Older men with moderate alcohol misuse are also at increased risk. We did not find an association between AUDIT-C scores and risk of trauma diagnoses in women, but we hypothesize that this had more to do with under-ascertainment bias than lack of an association between alcohol use and trauma in women. Our data add to the growing body of evidence in support of the AUDIT-C as a marker for future health risk among men, including trauma, medication nonadherence , gastroiThe authors declare that they have no competing interests.AH conceived of the study, supervised the acquisition and analysis of data, and helped draft the manuscript. AL drafted the manuscript and assisted in the interpretation of the data. PH and SG led the statistical analyses. RM and KB helped conceive the study, helped with interpretation of the data, and helped draft the manuscript. All authors read and approved the final manuscript.


It is unclear whether there is a clinical benefit to adding hydroxyapatite (HA) coatings to total knee implants, especially with the tibial component, where failure of the implant more often occurs. A systematic review of the literature was undertaken to identify all prospective randomized trials for determining whether the overall clinical results  favored HA-coated tibial components.A comprehensive literature search was performed for the years 1990 to September 16, 2010. We restricted our search to randomized controlled trials involving participants receiving either an HA-coated tibia or other forms of tibial fixation. The primary outcome measures evaluated were durability, function, and acute adverse events.2 = 39%, M-H random effects model). There was no significant difference in durability, as measured from revision and evaluated at 2 and 8–10 years, between groups. Also, functional status using different validated measures showed no significant difference at 2 and 5 years, no matter what measure was used. Lastly, there was no significant difference in adverse events. Limitations included small numbers of evaluable patients (≤ 50) in 7 of the 14 trials identified, and a lack of “hard” evidence of durability with need for replacement .Data from 926 evaluable primary total knee implants in 14 studies were analyzed. Using an RSA definition for durability, HA-coated tibial components (porous or press-fit) without screw fixation were less likely to be unstable at 2 years than porous and cemented metal-backed tibial components (RR = 0.58, 95% CI: 0.34–0.98; p = 0.04, IIn patients > 65 years of age, an HA-coated tibial implant may provide better durability than other forms of tibial fixation. Larger trials should be undertaken comparing the long-term durability, function, and adverse events of HA-coated implants with those of other porous-coated tibial implants in younger, more active OA patients. Experts have recommended that uncemented TKA is preferable in patients < 60 years of age , for a nThe price of implants remains a major cost consideration for these types of procedures in the US. Hydroxyapatite (HA) coating on implants is usually an added cost  for providers, insurers, and patients. HA coatings may be applied to non-porous or porous surfaces. HA coatings are osteoconductive, meaning that they facilitate the formation of bone structure and may aid in more stable fixation over the years. Preclinical studies have shown that HA coatings can enhance the stability of implants by promoting early bone ingrowth even in the presence of interfacial gaps or initial interfacial instability . HA-coatWe examined all the published prospective, randomized clinical trials (level-1 evidence), comparing patients with OA who received HA-coated tibial components or other tibial designs including uncemented porous coated metal components, uncemented press-fit metal components, and cemented metal components (porous and non-porous coated) to determine whether the clinical results of durability, function, and adverse events (both short and longer term) seen with HA-coated tibial components were superior.The following hypotheses were tested: 1. HA-coated tibial components as part of a total knee implant system have better durability than metal-backed (MB) tibial components  irrespective of the manufacturer/brand and patient age at time of implant over the short term (2 years), and longer term (≥ 8 years). 2. HA-coated tibial components as part of a total knee implant system have better functionality than MB tibial components  irrespective of the manufacturer/brand and patient age at time of implant over the short term (2 years) and longer term (≥ 8 years). 3. HA-coated tibial components as part of a total knee implant system have a lower incidence of adverse events/complications than MB tibial components irrespective of the manufacturer/brand or method of fixation .The Cochrane Review Manager software version 5.0.2 was used in assembling and reviewing the data .A study protocol was not published prior to this analysis. However, the above hypotheses were developed a priori, i.e. before the results were available. These hypotheses were developed based on a previous current concept review  and on sTypes of studies. Randomized controlled trials (RCTs) were eligible for inclusion if they compared the use of an HA-coated tibial component and any other metal-backed tibial component in the short and longer term, specifically examining the durability (length of time the implant survived in the patient without having to perform a repeat procedure for loosening or pain or loss of function—i.e. revision surgery), functionality and pain , LEFS), and adverse events.Durability and/or functionality (as defined below) must have been one of the primary outcome measures evaluated in the trial.Definitions used for eligibility criteria. The following definitions were used to determine clinical outcomes: durability, function, and adverse events. The definitions below are consistent with other technology assessments in total knee replacement  in everyday living, as measured by a number of validated scoring systems including: the Knee Society score, Hospital for Special Surgery score (HSS) (re (HSS) , the WesAdverse events were defined as complications resulting from the surgical procedure and/or subsequent need for surgical intervention and included: DVT, instability due to late varus or valgus with need for surgical intervention, patellofemoral problems with need for surgical intervention, knee flexion problems (including stiffness) with need for surgical manipulation, hematoma requiring evacuation, allergic reactions to medications and infections (over the life of the implant).Types of participants. Any person undergoing a first-time total knee arthroplasty.Interventions: Patient of any age undergoing a total knee implant that contained an HA-coated tibial component vs. other total knee implant with a different mode of fixation of the tibial component. Comparison groups in prospective randomized trials included:Total knee implant with HA-coated tibial component  compared to a total knee implant with porous coated or press-fit stemmed metal tibial tray with polyethylene bearing surface.Total knee implant with HA-coated tibial component  compared to a total knee implant with porous coated modular (for polyethylene insert) metal tibial tray with polyethylene bearing surface; with or without screw fixation.Total knee implant with HA-coated tibial component  compared to a total knee implant with a cemented metal tibial tray with polyethylene insert bearing surface.Types of outcome measuresDurability of implant with at least 2-year follow-up (including availability of MTPM or radiographs at 2 years).Functionality of implant with at least 2-year follow-up.Adverse events occurring during procedure or afterwards, such as deep vein thrombosis, allergic reactions, infections, decreased range of motion.Secondary outcomesOsteoarthritis vs. rheumatoid arthritis patients  and comparing above primary outcomes.Cost-effectiveness analysis.Electronic and other searches  from included studies. One review author (JDV) independently extracted the data, and a second review author (MM) validated the extracted data. If more than one publication arose from the same study, all versions were considered to maximize data extraction and the primary publication was identified along with the secondary references.Studies were assessed using the Cochrane Collaboration tool for assessing risk of bias—at both the study level and the outcome level . This toAn assessment of risk of bias using a “risk of bias summary figure”, which presents all of the judgments in a cross-tabulation of studies by entry was evaluated. This display of internal validity indicates the weight the reader may give the results of each study.Studies other than RCTs  were assessed using the same criteria. We incorporated the results of the risk-of-bias assessment into the review through systematic narrative description and commentary about each of the domains, leading to an overall assessment of the risk of bias of included studies and a judgment about the internal validity of the results.Each study is reported separately. The results of binary outcomes (e.g. revision or not) are presented as risk ratios (RRs) with corresponding 95% confidence intervals (CIs). For continuous data, we used the mean difference if outcomes were measured in the same way between trials. Furthermore, if pooling of data was not possible, we used the statistics used in the study for analysis of treatment effect; in most cases the Mann-Whitney U test was used (a nonparametric test).In such cases, we attempted to contact authors where data was missing and requested it. We also addressed the effect of missing data in the discussion section. In the case of abstracts, we attempted to contact authors to determine whether a paper had been published in a peer-reviewed journal. If a paper had been written from an abstract but was unpublished, we attempted to obtain it from the authors.2 statistic, in order to determine appropriateness for meta-analysis. If the I2 statistic was at or below 60%, the heterogeneity was considered moderate and meta-analysis was considered appropriate. If the value was greater than 60%, sensitivity analysis was undertaken in an attempt to identify which studies were most likely to be causing the problem. If there were only a few such studies, and they could be identified, the reasons for their difference were explored and the appropriateness of removing these studies was determined. When appropriate, meta-analysis excluding any such studies was performed.Assessment of statistical heterogeneity was done using the IWe used a funnel plot to assess reporting bias. Each primary outcome was reported separately. Furthermore, an assessment was made of publication bias (including a review of unpublished studies), location bias , and language bias.2 = 0%) or in the presence of low heterogeneity (I2 statistic less than 40%), a fixed-effects model was used. If heterogeneity was moderate , a random-effects model was used.Where possible, we grouped studies together that were similar. In the absence of heterogeneity ; and porous coated metal-backed tray with screw fixation and polyethylene insert bearing surface (non-cemented). No studies were identified that compared an HA-coated tibial component to a cemented all-polyethylene tibial component. This included a total of 926 evaluable total knee implants .Based on the available data, the comparisons evaluated were as follows:HA-coated  vs. all other implants (cemented MB and porous-coated MB with or without screw fixation). Evaluation of performance against the RSA (MPTM) endpoint was done at 2 years HA-coated  vs. other implants (cemented MB and porous coated MB without screw fixation). Evaluation of performance against the RSA (MPTM) endpoint was done at 2 years HA-coated  vs. porous coated MB without screw fixation). Evaluation of performance against the RSA (MPTM) endpoint was done at 2 years HA-coated  vs. cemented MB. Evaluation of performance against the RSA (MPTM) endpoint was done at 2 years HA-coated  vs. porous coated MB with screw fixation). Evaluation of performance against the RSA (MPTM) endpoint was done at 2 years .HA-coated vs. cemented MB tibia – durability (failure requiring a revision) at 5 years HA-coated vs. cemented MB tibia – durability (failure requiring a revision) at 8–10 years Functional assessment:– KSS clinical score at 2 years —comparin– HSS score at 2 years .RAND-36 and WOMAC at 5 yearsHA-coated vs. cemented implants—adverse events .See Two trials were excluded, as they were earlier trials  Önsten eAllocation. Sequence generation (randomization scheme used) and allocation to treatment arm were not well defined in the majority of the studies.Blinding. Physicians performing the procedure could not be blinded regarding the treatment arm. In a number of the studies, it was unclear whether patients had also been blinded to the treatment arm. It was also unclear in approximately 50% of the studies whether clinicians evaluating radiographs over time  were blinded to the treatment arm. In addition, it was unclear approximately 50% of the time whether clinicians evaluating for functionality, pain, and quality of life were blinded to the treatment arm.Incomplete outcome data: A substantial proportion of the missing data, especially in the longer-term outcome studies, were related to survival of the patient. According to the appendix “risk of bias assessment”, this represents a low risk of bias.Durability as measured by RSA with MPTM of > 0.2 mm/2 years constituting instability: Funnel plot analysis of this outcome for the 7 studies included  showed sDurability as defined by need for revision at 5 years: Funnel plot analysis of this outcome for the 3 studies included  showed sDurability as defined by need for revision at 8–10 years: Funnel plot analysis of this outcome for the 2 studies included  showed sAdverse events: Funnel plot analysis of this outcome for the 6 studies included showed symmetry, indicating that bias was minimal  and porous coated tibial implants (n = 8), Peterson et al. (2005) found no significant effect of HA coating on bone remodeling in the proximal tibia.Durability as defined above by Durability as defined by Function was evaluated using validated scoring systems as outlined above. Functional scores were reported on below from data at 2 years and 5 years .The functional status (as assessed using the KSS) of 2 = 0%; Mantel-Haenszel (M-H) fixed-effects model)  .All studies identified had been published in English. However, as noted above in the funnel plot analyses for overall assessment of reporting bias, symmetry was noted, indicating low expectation of reporting bias. Furthermore, in half of the studies identified there were low numbers of patients (≤ 50).All of these studies were conducted outside the US, with 7 of the 14 from Sweden, 1 from Denmark, 3 from the Netherlands, 2 from the UK, and 1 from Canada.The majority of patients enrolled in the trials were 67–70 years of age. Only 3 trials evaluated patients < 65 years of age . These s2 = 27%; M-H random-effects model)  between the first and second year with HA-coated tibial implants as opposed to all other types of tibial implants when used in patients requiring primary total knee replacement. It should be noted that in the analysis of MPTM, a strict definition of > 0.2 mm in 2 years was used, according to s model) . HA coats model) , 2005.As mentioned above, when those HA implants with screw fixation were excluded from the analysis , a statiAs The trial by As it relates physician- and patient-assessed functioning, there appeared to be no clinically meaningful difference in mean KSS, HSS, WOMAC, and RAND-36 between HA-coated and other tibial implants. One of the issues with the data available was that a pre- and postoperative (2- and 5-year) analysis could not be performed. The only analysis that could be undertaken was to examine a difference between groups regarding function at 2 and 5 years, which again demonstrated no difference.2 > 0), they compare the fixed- and random-effects estimates of the intervention effect. If the estimates are similar, then any small-study effects have little effect on the intervention effect estimate. If the random-effects estimate is more beneficial, review authors should consider whether it is reasonable to conclude that the intervention was more effective in the smaller studies.” This analysis was performed, and demonstrated similarity in the intervention effect shown in 2 = 39%; vs. fixed-effects model: RR = 0.53, 955, CI: 0.36–0.77; p < 0.001; I2 = 39%). Thus, it does appear that HA coating  may promote early fixation/stability compared to other tibial components (cemented and porous coated) in younger patients, as assessed by MPTM at 2 years. The main concern is that there is limited long-term evidence regarding “hard” durability (need for revision/replacement). The functioning of patients with HA coatings appears to be comparable to that with other implant types at 2 and 5 years, regardless of the measure. Adverse events were similar between HA coatings and other implant types.Due to the limited numbers of patients, recommendations on the applicability of the evidence cannot be made with complete confidence. In a prior analysis on small study effects in meta-analysis of osteoarthristis trials , it was determined that on average, treatment effects were more beneficial in small trials (< 100) than in large trials (> 100)—leading to a distortion of the results in meta-analyses . It furtThe trials evaluated in this meta-analysis were performed mainly on patients with osteoarthritis, with a very small minority in patients with rheumatoid arthritis. There were no studies that compared the cost effectiveness of HA-coated tibial components with that of any other method of tibial fixation. In the trials that examined patients < 65 years of age, the endpoints varied between studies and further analysis could not be undertaken by combining studies.The overall quality of the evidence can be considered “average”. It was not possible to blind the clinician performing the procedure. Blinding of patients, of clinicians assessing RSA, and of clinicians evaluating function was not clear in most studies. Also, the randomization method used in allocating patients to treatment groups was not clear in the majority of the studies. Most results (including missing data) were accounted for. Most clinicians performing the trials were funded from outside sources, either industry, medical associations, or foundations.  later on in life.Larger-sized studies (i.e. with > 100 patients) should be undertaken to compare the longer-term durability (as measured by need of revision), function, and adverse events with HA-coated implants vs. other porous coated tibial implants in much younger, more active patients (< 65 years of age); durability in patients aged < 65 years (as measured by RSA); and outcome of HA-coated implants in patients with RA.


Breastfeeding self-efficacy (BFSE) supports breastfeeding initiation and duration. Challenges to breastfeeding may undermine BFSE, but second-line strategies including nipple shields, syringe, cup, supply line and bottle feeding may support breastfeeding until challenges are resolved. The primary aim of this study was to examine BFSE in a sample of women using second-line strategies for feeding healthy term infants in the first week postpartum.A retrospective self-report study was conducted using the Breastfeeding Self-Efficacy Scale - Short Form (BSES-SF), demographic and infant feeding questionnaires. Breastfeeding women who gave birth to a singleton healthy term infant at one private metropolitan birthing facility in Australia from November 2008 to February 2009 returned anonymous questionnaires by mail.A total of 128  women participated in the study. The mean BSES-SF score was 51.18 . The median BSES-SF score was 53. Of women using a second-line strategy, 16 exceeded the median, and 42 were below. Analyses using Kruskal-Wallis tests confirmed this difference was statistically significant . The rate of second-line strategy use was 48%. The four most commonly used second-line strategies were: bottles with regular teats (77%); syringe feeding (44%); bottles with wide teats (34%); and nipple shields (27%). Seven key challenges were identified that contributed to the decision to use second-line strategies, including: nipple pain (40%); unsettled infant (40%); insufficient milk supply (37%); fatigue (37%); night nursery care (25%); infant weight loss > 10% (24%); and maternal birth associated pain (20%). Skin-to-skin contact at birth was commonly reported (93%). At seven days postpartum 124 women (97%) were continuing to breastfeed.The high rate of use of second-line strategies identified in this study and high rate of breastfeeding at day seven despite lower BFSE indicate that such practices should not be overlooked by health professionals. The design of this study does not enable determination of cause-effect relationships to identify factors which contribute to use of second-line strategies. Nevertheless, the significantly lower BSES-SF score of women using a second-line strategy highlights this group of women have particular needs that require attention. During the first week postpartum many breastfeeding challenges can occur. Maternal challenges include nipple shape, pain, or damage, perceived insufficient milk supply and engorgement. FurtherSecond-line strategies include a range of devices and techniques that compensate for challenges whilst women continue to pursue their breastfeeding goal, and proBreastfeeding self efficacy (BFSE) is an important focus of interventions designed to support breastfeeding women, enable success of first and second-line strategies, and achieve successful breastfeeding outcomes. Numerous studies have identified that BFSE is an important factor in breastfeeding outcomes including duration-14. One Little is known about the relationship between BFSE and use of second-line strategies during the first week postpartum. The primary purpose of this research was to measure BFSE retrospectively across the first seven days postpartum, and to examine the relationship between use of second-line strategies in the first week postpartum and BFSE.The study design was a quantitative, observational retrospective self-report survey. The research was conducted at one Australian metropolitan UnitingCare Health private birthing facility, located in Brisbane.Participants were identified through systematic chart review of all current inpatients, from 1 November 2008 to 27 February 2009. Following the birth of their healthy term singleton infant, and initiating any breastfeeding, participants were invited to complete a questionnaire mailed to their home by day seven. Length of postnatal inpatient stay at this facility was four/five days. Figure Ethical approval was granted from UnitingCare Health Human Research Ethics Committee (200837), and Queensland University of Technology Human Research Ethics Committee (0800000784). No identifying information was contained on the questionnaires and consent was implied by return of the questionnaire.Women were asked to respond to a range of questions regarding demographic, birthing, feeding variables, and the 14 item Breastfeeding Self Efficacy Scale - Short Form (BSES-SF), yielding nominal and interval data. Permission to use the scale was granted by the owner. International studies have measured BFSE, with repeated reliability and validity, including one Australian study-26. The All questionnaires were checked for missing data. One respondent omitted a page, and two others had other missing data but this was minimal so their available data were included in the analysis. Seven respondents omitted an item in the BSES-SF, and these were not included in analysis. Data were analysed using SPSS version 16.0. Descriptive statistics were used to describe the demographic profile of the respondents, present the rates of use of second-line strategies and the reasons for their use. Due to large variances, the non-parametric Kruskal-Wallis Analysis of Variance,28 was uOf 225 women identified for inclusion Figure , 128 retThe majority of infants weighed between 3000 g and 4000 g, and just over half the women were multiparous, with a history of breastfeeding a previous infant for more than six months Table . Almost The frequency of second-line strategy use and parity is shown at Table Knowledge and preference was noted for all respondents. For each second-line strategy women indicated whether or not they previously knew of the strategy, and if not, to indicate if they would prefer to have known. As shown in Table The seven key challenges cited by women for using second-line strategies are shown in Table The overall BSES-SF mean was 51.18 SD = 12.48). The mean BSES-SF score of women using a second-line strategy was 43.43 (SD = 12.19), and of women not using a second-line strategy it was 58.32 (SD = 7.40). Mean BSES-SF scores, parity, and Kruskal-Wallis analysis of median scores are reported at Table 8. The meThe first important finding in this study is that mean BFSE levels in the group of women not using any second-line strategies was relatively high 58.32) (43% primiparas). Other studies measuring BFSE at 7 days range from 44.7 (48.5% primiparas) to 55.8 2  Ten steps to successful breastfeeding step nine is to “give no artificial teats or pacifiers to breastfeeding infants”. Almost Woman centred care revolves around the guiding principles of control, choice, and continuity. In manyThe sample in this study described high rates of positive obstetric practice, such as skin-to-skin contact at birth and positive breastfeeding experiences. A notable proportion of women also reported use of second-line strategies. Importantly, the women using second-line strategies had significantly lower BFSE than those who did not use these strategies. The complex matrix of factors contributing to BFSE requires the implementation of woman-centred principles to breastfeeding and postpartum care which include the informed choice and control of second-line strategies, and continuity of care. Further research investigating woman-centred care for breastfeeding women would be valuable.BFSE: Breastfeeding self-efficacy; BFHI: Baby friendly health initiative; BSES: Breastfeeding self-efficacy scale; BSES-SF: Breastfeeding self-efficacy scale – short form; SPSS: Statistical package for social sciences.The author declares that she has no competing interests.