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Magdolna Barra,1 Edit Remák,1 Dong Dong Liu,2 Li Xie,2 Lucy Abraham,3 Alesia B Sadosky4 1Evidera, Budapest, Hungary; 2Health Economics & Outcomes Research, Pfizer Investment Co., Ltd., Beijing, China; 3Health Economics & Outcomes Research, Pfizer Ltd, Tadworth, UK; 4Health Economics & Outcomes Research, Pfizer Inc, New York, NY, USA Purpose: The use of parecoxib plus opioids for postoperative analgesia in noncardiac surgical patients seems to be cost-saving in Europe due to a reduction in opioid use and opioid-related adverse events. Given the lack of information on postoperative analgesic use in Asia, this study assessed the economic consequences of the addition of parecoxib to opioids vs opioids alone to treat postsurgical pain in China.Methods: A cost-consequence economic evaluation assessed direct medical costs related to opioid-related clinically meaningful events (CMEs) utilizing dosing information and reported frequency of events from a Phase III, randomized, double-blind, global clinical trial (PARA-0505-069) of parecoxib plus opioids vs opioids alone for 3 days following major orthopedic, abdominal, gynecologic, or noncardiac thoracic surgery requiring general or regional anesthesia. The cost of CMEs was calculated using information on resource utilization and unit costs provided by a panel of clinical experts in China. Sensitivity analyses were performed to test the robustness of the results.Results: Patients treated with parecoxib plus opioids reported fewer CMEs (mean 0.62 vs 1.04 events per patient [P

Alesia B Sadosky,1 Marco DiBonaventura,2 Joseph C Cappelleri,3 Nozomi Ebata,4 Koichi Fujii4 1Pfizer Inc, New York, NY, USA; 2Kantar Health, New York, NY, USA; 3Pfizer Inc, Groton, CT, USA; 4Pfizer Japan Inc, Tokyo, Japan Introduction: This study investigated the effect of pain severity on health status, work productivity, health care resource use, and costs among respondents with lower back pain (LBP), in Japan. Materials and methods: Data from the 2013 Japan National Health and Wellness Survey, a survey of Japanese adults, were analyzed (N=30,000). All respondents provided informed consent, and the protocol was institutional review board-approved. Respondents who reported experiencing LBP were propensity score–matched to those without LBP, based on demographics and health history. Using regression modeling, patients with mild, moderate, and severe pain were compared against matched controls, with respect to health status (Mental and Physical Component Summary scores, and health utilities from the Short Form®-36 Health Survey version 2), work productivity (Work Productivity and Activity Impairment – General Health version), health care resource use, and annual per-patient costs (estimated using published annual wages and resource use event costs). Results: A total 1,897 patients reported experiencing LBP in the past month (6.32%); 52.45% reported their pain as mild, 32.79% as moderate, and 14.76% as severe. Increasing pain severity was associated with significantly lower levels of mental component scores (46.99 [mild], 42.93 [moderate], and 40.58 [severe] vs 48.10 [matched controls]), physical component scores (50.29 [mild], 46.74 [moderate], and 43.94 [severe] vs 52.93 [matched controls]), and health utilities (0.72 [mild], 0.66 [moderate], and 0.62 [severe] vs 0.76 [matched controls]) (all P

Jeffrey J Ellis,1 Alesia B Sadosky,2 Laura L Ten Eyck,1 Joseph C Cappelleri,2 Courtney R Brown,3 Brandon T Suehs,1 Bruce Parsons2 1Comprehensive Health Insights Inc., Louisville, KY, USA; 2Pfizer Inc., New York, NY, USA; 3Humana Inc., Louisville, KY, USA Purpose: To examine the impact of newly initiated pregabalin or duloxetine treatment on fibromyalgia (FM) patients' encounters with potential drug–drug interactions (DDIs), the health care cost and utilization consequences of those interactions, and the impact of treatment on opioid utilization.Patients and methods: Subjects included those with an FM diagnosis, a pregabalin or duloxetine prescription claim (index event), ≥1 inpatient or ≥2 outpatient medical claims, and ≥12 months preindex and ≥6 postindex enrollment. Propensity score matching was used to help balance the pregabalin and duloxetine cohorts on baseline demographics and comorbidities. Potential DDIs were defined based on Micromedex 2.0 software and were identified by prescription claims.Results: No significant differences in baseline characteristics were found between matched pregabalin (n=794) and duloxetine cohorts (n=794). Potential DDI prevalence was significantly greater (P

We report results from a phase 2, randomized, double-blind, 2-period trial (48 weeks each) of domagrozumab and its open-label extension in patients with Duchenne muscular dystrophy (DMD). Of 120 ambulatory boys (aged 6 to <16 years) with DMD, 80 were treated with multiple ascending doses (5, 20, and 40 mg/kg) of domagrozumab and 40 treated with placebo. The primary endpoints were safety and mean change in 4-stair climb (4SC) time at week 49. Secondary endpoints included other functional tests, pharmacokinetics, and pharmacodynamics. Mean (SD) age was 8.4 (1.7) and 9.3 (2.3) years in domagrozumab- and placebo-treated patients, respectively. Difference in mean (95% CI) change from baseline in 4SC at week 49 for domagrozumab vs placebo was 0.27 (-7.4 to 7.9) seconds (p = 0.94). There were no significant between-group differences in any secondary clinical endpoints. Most patients had ≥1 adverse event in the first 48 weeks; most were mild and not treatment-related. Median serum concentrations of domagrozumab increased with administered dose within each dose level. Non-significant increases in muscle volume were observed in domagrozumab- vs placebo-treated patients. Domagrozumab was generally safe and well tolerated in patients with DMD. Efficacy measures did not support a significant treatment effect. Clinicaltrials.gov identifiers: NCT02310763 and NCT02907619., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose The purpose of the study was to determine the impact of educational text messages on diabetes self-management activities and outcomes in patients with painful diabetic peripheral neuropathy (pDPN). Methods Patients with pDPN identified from a large integrated health system who agreed to participate were randomized to 6 months of usual care (UC) or UC plus twice-daily diabetes self-management text messages (UC+TxtM). Outcomes included the Pain Numerical Rating Scale, Summary of Diabetes Self-Care Activities (SDSCA), questions on diabetes health beliefs, and glycated hemoglobin (A1C). Changes from baseline were evaluated at 6 months and compared between groups. Results Demographic characteristics were balanced between groups (N = 62; 53% female, mean age = 63 years, 94% type 2 diabetes), as were baseline measures. After 6 months, pain decreased with UC+TxtM from 6.3 to 5.5 and with UC from 6.5 to 6.0, with no difference between groups. UC+TxtM but not UC was associated with significant improvements from baseline on all SDSCA subscales. On diabetes health beliefs, UC+TxtM patients reported significantly increased benefits and reduced barriers and susceptibility relative to UC at 6 months. A1C declined in both groups, but neither change was significant relative to baseline. Conclusions Patients with pDPN who receive twice-daily text messages regarding diabetes management reported reduced pain relative to baseline, although this change was not significant compared with usual care. In addition, text messaging was associated with increased self-management activities and improved diabetes health beliefs and total self-care. These results warrant further investigation.

Objectives: This analysis compared the therapeutic response of pregabalin in patients with neuropathic pain (NeP) who had been previously treated with gabapentin to the therapeutic response in patients who had not received gabapentin previously., Methods: Data were pooled from 18 randomized, double-blind, placebo-controlled trials of pregabalin in patients with NeP. Pregabalin-mediated changes in pain and pain-related sleep interference scores, patient global impression of change scores at endpoint, and the occurrence of adverse events were compared between patients who had received gabapentin previously (+GBN) and patients who had not received gabapentin previously (-GBN)., Results: There were no significant differences between the -GBN and +GBN cohorts with regard to the extent of pain relief and relief of pain-related sleep interference for any dose of pregabalin (150, 300, 600, or 150 to 600 mg/day) at any time point (6, 8, or 12 weeks). Additionally, there was no significant difference in the distribution of patient global impression of change scores at study endpoint, or the occurrence of adverse events, between the -GBN and +GBN cohorts., Discussion: The findings presented here support the idea that pregabalin may be used successfully to treat patients with NeP who may be refractory, respond inadequately, or are intolerant to gabapentin. These findings highlight the importance of tailoring treatment of NeP based on individual patient response to different treatments, including the trial of multiple agents within the same mechanistic class., (© 2016 World Institute of Pain.)

Background: Anticipating and controlling drug-drug interactions (DDIs) in older patients with painful diabetic peripheral neuropaty (pDPN) presents a significant challenge to providers. The purpose of this study was to examine the impact of newly initiated pregabalin or duloxetine treatment on Medicare Advantage Prescription Drug (MAPD) plan pDPN patients' encounters with potential drug-drug interactions, the healthcare cost and utilization consequences of those interactions, and opioid utilization., Methods: Study subjects required a pregabalin or duloxetine pharmacy claim between 07/01/2008-06/30/2012 (index event), ≥1 inpatient or ≥2 outpatient medical claims with pDPN diagnosis between 01/01/2008-12/31/2012, and ≥12 months pre- and ≥6 post-index enrollment. Propensity score matching was used to balance the pregabalin and duloxetine cohorts on pre-index demographics and comorbidities. Potential DDIs were defined by Micromedex 2.0 and identified by prescription claims. Six-month post-index healthcare utilization (HCU) and costs were calculated using pharmacy and medical claims., Results: No significant differences in pre-index demographics or comorbidities were found between pregabalin subjects (n = 446) and duloxetine subjects (n = 446). Potential DDI prevalence was significantly greater (p < 0.0001) among duoxetine subjects (56.7%) than among pregabalin subjects (2.9%). There were no significant differences in HCU or costs between pregablin subjects with and without a potential DDI. By contrast, duloxetine subjects with a potential DDI had higher mean all-cause costs ($13,908 vs. $9,830; p = 0.001), more subjects with ≥1 inpatient visits (35.6% vs 25.4%; p = 0.02), and more subjects with ≥1 emergency room visits (32.8% vs. 20.7%; p = 0.005) in comparison to duloxetine subjects without a potential DDI. There was a trend toward a difference between pregabalin and duloxetine subjects in their respective pre-versus-post differences in milligrams (mg) of morphine equivalents/30 days used (60.2 mg and 176.9 mg, respectively; p = 0.058)., Conclusion: The significantly higher prevalence of potential DDIs and potential cost impact found in pDPN duloxetine users, relative to pregabalin users, underscore the importance of considering DDIs when selecting a treatment.

Study Design: A cross-sectional observational study of physicians and patients with chronic low-back pain (CLBP) in the United States., Objective: To evaluate the association of patient-reported CLBP severity with other patient-reported outcomes., Summary of Background Data: Appropriate management of patients with CLBP can be enhanced by understanding how patients perceive the severity of their CLBP., Methods: Data were from the 2009 Adelphi Disease Specific Programme. Patients reported the severity of their CLBP condition by answering the question Please rate how your chronic lower back pain condition is today with responses of mild, moderate, and severe. Patient-reported severity was evaluated with respect to scores on standard patient self-report measures of pain, pain interference, health status, functional disability, work productivity, and questions addressing satisfaction with medications for treating CLBP., Results: Of 1860 subjects in the CLBP database, 1363 (73.3%) agreed to complete the survey. This sample was similarly distributed between males (49%) and females (51%), with mean age 54.8 years: 52% were employed at least part-time. CLBP severity was rated as mild, moderate and severe by 28.6%, 53.3%, and 18.2% of patients, respectively. With increasing CLBP severity, significant differences were observed in increased pain (P<0.0001), pain interference with function (P<0.0001), and impairment while working due to CLBP (P<0.01), mainly due to presenteeism. Increased work impairment resulted in higher costs related to lost work productivity; annual lost productivity costs were estimated at $7080, $16,616, and $25,032 per patient for mild, moderate, and severe CLBP, respectively (P<0.0001 for pairwise comparisons). Patient satisfaction with pain-related medication was inversely associated with CLBP severity., Conclusions: The association between patient-reported CLBP severity and other patient-reported outcomes demonstrates that in the clinical setting, patient-reported CLBP severity provides an accurate and suitable indicator of patient-reported health status. This indicator may be useful for guiding management strategies for CLBP patients.

Background: Recommended pain treatments for osteoarthritis (OA) and chronic low back pain (CLBP) are suboptimal, and limited information is available regarding patterns of pharmacotherapy among patients with these conditions., Aims: Evaluate patterns of therapy switching, augmentation, and discontinuation after treatment initiation with select pain medications in patients with OA and CLBP., Methods: Using the U.K. The Health Improvement Network (THIN) database, OA and CLBP patients newly prescribed (index-event) nonselective nonsteroidal anti-inflammatory drugs (NS-NSAIDs), cyclooxygenase-2 inhibitors (Cox-2s), acetaminophen, tramadol and weak or strong opioids were selected. Descriptive statistics, Kaplan-Meier analyses, and COX proportional hazards models were used to evaluate patterns of changes in pharmacotherapy during the 12-month postindex period., Results: Rates of therapy switching, augmentation, and discontinuation, respectively, were significantly different (all P values<0.0001) across the evaluated medication cohorts for both OA and CLBP patients. Discontinuation rates in OA patients were 91.9% (NS-NSAIDs), 86.9% (Cox-2s), 91.4% (acetaminophen), 89.7% (tramadol), 93.2% (weak opioids), and 84.3% (strong opioids); and in CLBP patients were 97.2% (NS-NSAIDs), 94.0% (Cox-2s), 95.0% (acetaminophen), 92.8% (tramadol), 97.0% (weak opioids), and 86.8% (strong opioids). The rates of switching (range 30.0% to 59.6%) and augmentation (range 7.5% to 15.2%) were lower. Estimated probability evaluations suggested that two-thirds of patients who switched, augmented, or discontinued therapy did so within the first couple of months, and a majority did so within 6-months of treatment initiation., Conclusions: This study demonstrates that therapy switching and discontinuation of select pain medications were common among OA and CLBP patients in the U.K. and may result from inadequate pain relief or undesirable side effects., (© 2011 The Authors. Pain Practice © 2011 World Institute of Pain.)

Background: Characterization of chronic low back pain (CLBP) severity from a patient's perspective can provide a context within which management strategies may be determined and therapeutic outcomes evaluated., Objective: The aim of our study was to evaluate the association between patient-rated CLBP severity and medication resources., Methods: Data were drawn from the Adelphi CLPB Disease Specific Programmme, a cross-sectional study of patients undertaken between September and November 2009. Patients reported the severity of their CLBP by answering the statement "Please rate how your chronic lower back pain condition is today" with responses of "mild," "moderate," or "severe." Severity was evaluated relative to physician-reported use of medications for the relief of CLBP and patient-reported satisfaction with pain relief and medications., Results: Data from 170 physicians and 1363 patients (mean age 55 years; 52.3% female) were analyzed. CLBP severity was rated as mild, moderate, and severe by 28.3%, 52.8%, and 18.0% of patients, respectively. Physician-reported analgesia requirements increased with CLBP severity (P < 0.05). Opioids, nonsteroidal antiinflammatory drugs, and muscle relaxants were the most commonly prescribed medications for CLBP. Opioid prescriptions increased with increasing severity (P < 0.05), and nonsteroidal antiinflammatory drug prescriptions declined. Purchase of over-the-counter medications was similar across severity categories (23%-26% of patients), but the monthly amount spent on over-the-counter drugs was more than twice as high in patients with severe CLBP ($29.90) than in other severity categories. Patient and physician satisfaction with pain-related medication was inversely associated with CLBP severity; inadequate response was the primary reason for physician dissatisfaction. Factors limiting generalizability include potential differences between participants and those who refused to participate; potential misdiagnosis of CLBP in a proportion of patients; and an inability for cause-and-effect imputation due to the cross-sectional nature of the study., Conclusions: The relationship between patient-reported CLBP severity and medication prescribing patterns suggests that this rapid assessment may be of value for informing decisions regarding treatment options. The data also suggest that despite greater use of medications at greater CLBP severity, current options remain less than optimal in providing analgesic efficacy., (Copyright © 2011 Elsevier HS Journals, Inc. All rights reserved.)

Introduction: Understanding the relationship between patient-reported osteoarthritis (OA) severity and other patient-reported outcomes in the real-world clinical setting can provide a basis for appropriate patient management. The objective of this study was to determine how patient-reported OA severity correlates with patient-reported outcomes including pain, function and productivity., Methods: We used the Adelphi Disease Specific Programme (DSP) for OA, a database aggregated from large, multinational, observational studies for specific chronic diseases. Data were obtained based on a 0 to 100 mm pain visual analogue scale (VAS) and a series of questions including functioning (that is, activities of daily living) and work productivity. OA severity was rated by the patients based on the question "How bad would you say your arthritis is now?" with potential responses of "mild," "moderate," and "severe." Regression models and chi-square analyses were used to evaluate the relationships between self-reported OA severity and other outcomes., Results: Of 998 subjects in the OA DSP U.S. database, 714 (72.5%) agreed to participate. This sample was predominantly female (61.7%) with a mean age of 63.8 ± 12.9 years. Increased OA severity was associated with an older population (P < 0.05). With increasing OA severity (mild, moderate, severe), statistically significant differences (P < 0.05) were observed in increased pain VAS scores (23.5, 50.2, 70.8, respectively), lower functioning outcomes, and a higher percent of overall work impairment due to OA (17%, 37%, 48%, respectively). The increased work impairment at greater severity levels also resulted in higher costs related to lost work productivity, with annual costs due to lost productivity estimated at $6,096, $13,2510, and $17,214 per patient for self-reported mild, moderate, and severe OA, respectively (P < 0.05 for pairwise comparisons)., Conclusions: In the clinical practice setting, patient-reported OA severity was associated with other key patient-reported outcomes and thus may provide an accurate and tangible assessment of patients' perceptions of their disease. Identifying OA patients by their perceived severity level may be of benefit to patients and health-care providers when choosing treatment options aimed at reducing pain, and improving function and productivity.

Objective: To characterize comorbidities, pain-related pharmacotherapy, and healthcare resource use among patients with fibromyalgia (FM) newly prescribed pregabalin or gabapentin in clinical practice., Methods and Design: Using the PharMetrics Database, FM patients (International Classification of Diseases, Ninth Revision, Clinical Modification code 729.1X) newly prescribed pregabalin (n = 1,606; mean age 49.9 +/- 9.6 years; 87.9% female) and gabapentin (n = 930; mean age 49.5 +/- 9.6 years; 86.6% female) on/after July 1, 2007 were identified. Prevalence of comorbidities, pharmacotherapy, and healthcare resource use/costs (pharmacy, outpatient, inpatient, total) were examined during the 6 months preceding (preindex) and following (postindex) the date of their first pregabalin or gabapentin (index) prescription., Results: Patients in both cohorts had a variety of comorbidities and used multiple medications. There were significant decreases (P values < 0.05) in the use of nonsteroidal anti-inflammatory drugs (32.1% vs. 29.5%), anticonvulsants (27.0% vs. 22.0%), and combination therapies in the pregabalin cohort in the postindex period. There were significant increases (all P values < 0.05) in use of short-acting opioids (58.8% vs. 63.7%), any opioids (61.5% vs. 65.6%), serotonin-norepinephrine reuptake inhibitors (22.5% vs. 24.5%), anticonvulsants (16.3% vs. 26.2%), benzodiazepines (33.2% vs. 36.6%), topical agents (6.6% vs. 9.0%), and combination therapies in the gabapentin cohort. Although there were no changes in units of healthcare resources used, there were increases in the postindex period in hospitalization, medications, and total costs for pregabalin, and office visits and medication costs for gabapentin (all P values < 0.05)., Conclusions: Results suggest a high comorbidity and medication use burden in FM patients in this study. Further evaluation is warranted to clarify differences in resource utilization/costs observed with these two anticonvulsants.

Background: Sleep disturbances are a common and bothersome symptom of fibromyalgia (FM). This study reports psychometric properties of a single-item scale to assess sleep quality among individuals with FM., Methods: Analyses were based on data from two randomized, double-blind, placebo-controlled trials of pregabalin (studies 1056 and 1077). In a daily diary, patients reported the quality of their sleep on a numeric rating scale ranging from 0 ("best possible sleep") to 10 ("worst possible sleep"). Test re-test reliability of the Sleep Quality Scale was evaluated by computing intraclass correlation coefficients. Pearson correlation coefficients were computed between baseline Sleep Quality scores and baseline pain diary and Medical Outcomes Study (MOS) Sleep scores. Responsiveness to treatment was evaluated by standardized effect sizes computed as the difference between least squares mean changes in Sleep Quality scores in the pregabalin and placebo groups divided by the standard deviation of Sleep Quality scores across all patients at baseline., Results: Studies 1056 and 1077 included 748 and 745 patients, respectively. Most patients were female (study 1056: 94.4%; study 1077: 94.5%) and white (study 1056: 90.2%; study 1077: 91.0%). Mean ages were 48.8 years (study 1056) and 50.1 years (study 1077). Test re-test reliability coefficients of the Sleep Quality Scale were 0.91 and 0.90 in the 1056 and 1077 studies, respectively. Pearson correlation coefficients between baseline Sleep Quality scores and baseline pain diary scores were 0.64 (p < 0.001) and 0.58 (p < 0.001) in the 1056 and 1077 studies, respectively. Correlations between the Sleep Quality Scale and the MOS Sleep subscales were statistically significant (p < 0.01), except for the MOS Snoring subscale. Across both studies, standardized effect sizes were generally moderate (0.46 to 0.52) for the 300 mg group and moderate (0.59) or moderate-to-large (0.70) for the 450 mg group. In study 1056, the effect size for the 600 mg group was moderate-to-large (0.73). In study 1077, the effect size for the 600 mg group was large (0.82)., Conclusion: These results provide evidence of the reproducibility, convergent validity, and responsiveness to treatment of the Sleep Quality Scale and provide a foundation for its further use and evaluation in FM patients.

Objective: The Fibromyalgia Impact Questionnaire (FIQ) is a disease-specific composite instrument that measures the effect of problems experienced by patients with fibromyalgia (FM). Utilization of the FIQ in measuring changes due to interventions in FM requires derivation of a clinically meaningful change for that instrument. Analyses were conducted to estimate the minimal clinically important difference (MCID), and to propose FIQ severity categories., Methods: Data from 3 similarly designed, 3-month placebo-controlled, clinical treatment trials of pregabalin 300, 450, and 600 mg/day in patients with FM were modeled to estimate the change in the mean FIQ total and stiffness items corresponding to each category on the Patient Global Impression of Change. FIQ severity categories were modeled and determined using established pain severity cutpoints as an anchor., Results: A total of 2228 patients, mean age 49 years, 93% women, with a mean baseline FIQ total score of 62 were treated in the 3 studies. Estimated MCID on a given measure were similar across the studies. In a pooled analysis the estimated MCID (95% confidence interval) was 14% (13; 15) and for FIQ stiffness it was 13% (12; 14). In the severity analysis a FIQ total score from 0 to <39 was found to represent a mild effect, >or= 39 to <59 a moderate effect, and >or=59 to 100 a severe effect., Conclusion: The analysis indicates that a 14% change in the FIQ total score is clinically relevant, and results of these analyses should enhance the clinical utility of the FIQ in research and practice.

Aims: To determine the patient burden of painful diabetic peripheral neuropathy (DPN) with respect to pain intensity and impact on patient functioning and to characterize relevant DPN treatment patterns., Methods: Patients (n=140) with painful DPN identified during an observational survey of neuropathic pain syndromes in six European countries were included in the current analysis. Patients primarily recruited from community-based general practices answered a questionnaire that included pain severity and interference items from the modified Short-Form Brief Pain Inventory, the EuroQol survey, and questions related to productivity and health resource utilization. Physicians provided information on disease duration and current medications prescribed for painful DPN and pain-related comorbidities., Results: The mean patient age was 65.6+/-11.2 years; 58% of the patients were >or=65 years. Duration of painful DPN was >1 year in 74% of patients. The mean Pain Severity Index was 5.0+/-2.0; 57% of patients reported moderate pain and 25% reported severe pain. Patients reported moderate interference with functioning despite 91% of patients reporting use of prescription medications for painful DPN including antiepileptics (56%), standard analgesics (63%), and amitriptyline (26%). Use of prescription medication for concomitant anxiety, depression, or sleep disturbance was reported for 43% of the patients. Disruption in employment was reported by 35% of the patients. Pain severity was significantly associated with reduced health state valuation (P<.001), greater pain interference scores (P<.001), greater employment disruption (P<.05), and more physician visits (P<.05)., Conclusions: Painful DPN is associated with substantial patient burden resulting from interference with daily functioning, especially in patients with suboptimal pain management.

The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection. The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection. This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element. A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements. The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation. Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups. In the course of this analysis, two new Rhs elements absent from E. coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily.

Legionella pneumophila, the causative agent of Legionnaires' disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular life-style is the ability of the organism to replicate within a specialized phagosome which does not fuse with lysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome-lysosome fusion. In a previous study, a 12 kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-lysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-lysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1-2.4 kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.

Legionella pneumophila, the causative agent of Legionnaires' disease, contains two superoxide dismutases (SODs), a cytoplasmic iron enzyme (FeSOD) and a periplasmic copper-zinc SOD. To study the role of the FeSOD in L. pneumophila, the cloned FeSOD gene (sodB) was inactivated with Tn903dIIlacZ, forming a sodB::lacZ gene fusion. By using this fusion, expression of sodB was shown to be unaffected by a variety of conditions, including several that influence sod expression in Escherichia coli: aeration, oxidants, the redox cycling compound paraquat, manipulation of iron levels in the medium, and the stage of growth. A reproducible twofold decrease in sodB expression was found during growth on agar medium containing charcoal, a potential scavenger of oxyradicals, in comparison with growth on the same medium without charcoal. No induction was seen during growth in human macrophages. Additional copies of sodB+ in trans increased resistance to paraquat. Construction of a sodB mutant was attempted by allelic exchange of the sodB::lacZ fusion with the chromosomal copy of sodB. The mutant could not be isolated, and the allelic exchange was possible only if wild-type sodB was present in trans. These results indicate that the periplasmic copper-zinc SOD cannot replace the FeSOD. The data strongly suggest that sodB is an essential gene and that FeSOD is required for the viability of L. pneumophila. In contrast, Sod- mutants of E. coli and Streptococcus mutans grow aerobically and SOD is not required for viability in these species.

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Tn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5' truncated 'lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cis-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig-) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposon-linked mutation into a wild-type background results in a Pig- phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and beta-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.

Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.

The Escherichia coli K-12 chromosome contains a family of five large, unlinked sequences known as the Rhs elements. They share several complex homologies, the most prominent being a 3.7 kb Rhs core. The elements are divided into two subfamilies, RhsA-B-C and RhsD-E, according to the sequence similarities of the cores. The RhsD core is 3747 bp long compared to 3714 bp for RhsA. Despite a 22% sequence divergence, the RhsD core conserves features previously noted for RhsA. Similar to RhsA, the RhsD core maintains a single ORF, the start codon coinciding with the first nucleotide of the homology. The RhsD core-ORF continues 177 codons beyond the homology, resulting in a carboxy terminal extension unrelated to that of RhsA. The RhsD core retains all 28 copies of the repeated motif GxxxRYxYDxxGRL(I/T) seen in RhsA. The other member of the RhsD-E subfamily, RhsE, has been mapped to minute 32 of the E. coli map. It appears defective in that it contains only the last 1550 bp of the 3.7 kb core. Its sequence is more closely related to that of RhsD than RhsA. In addition, RhsE and RhsB share a 1.3 kb homology, known as the H-repeat. The H-repeats from RhsE and RhsB are more closely related than their cores, showing only 1% nucleotide divergence.

The complete nucleotide sequence of the rhsA locus and selected portions of other members of the rhs multigene family of Escherichia coli K-12 have been determined. A definition of the limits of the rhsA and rhsC loci was established by comparing sequences from E. coli K-12 with sequences from an independent E. coli isolate whose DNA contains no homology to the rhs core. This comparison showed that rhsA comprises 8,249 base pairs (bp) in strain K-12 and that the Rhs0 strain, instead, contains an unrelated 32-bp sequence. Similarly, the K-12 rhsC locus is 9.6 kilobases in length and a 10-bp sequence resides at its location in the Rhs0 strain. The rhsA core, the highly conserved portion shared by all rhs loci, comprises a single open reading frame (ORF) 3,714 bp in length. The nucleotide sequence of the core ORF predicts an extremely hydrophilic 141-kilodalton peptide containing 28 repeats of a motif whose consensus is GxxxRYxYDxxGRL(I or T). One of the most novel aspects of the rhs family is the extension of the core ORF into the divergent adjacent region. Core extensions of rhsA, rhsB, rhsC, and rhsD add 139, 173, 159, and 177 codons to the carboxy termini of the respective core ORFs. For rhsA, the extended core protein would have a molecular mass of 156 kilodaltons. Core extensions of rhsB and rhsD are related, exhibiting 50.3% conservation of the predicted amino acid sequence. However, comparison of the core extensions of rhsA and rhsC at both the nucleotide and the predicted amino acid level reveals that each is highly divergent from the other three rhs loci. The highly divergent portion of the core extension is joined to the highly conserved core by a nine-codon segment of intermediate conservation. The rhsA and rhsC loci both contain partial repetitions of the core downstream from their primary cores. The question of whether the rhs loci should be considered accessory genetic elements is discussed but not resolved.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.