scholarly journals AmpC β-Lactamase Variable Expression in Common Multidrug-Resistant Nosocomial Bacterial Pathogens from a Tertiary Hospital in Cairo, Egypt

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Aliaa ELshamy ◽  
Zainab Zakaria ◽  
Mahmoud M. Tolba ◽  
Nermeen Salah Eldin ◽  
Al-Taher Rabea ◽  
...  
Keyword(s):  
Tract Infection ◽  
Bacterial Species ◽  
Expression Patterns ◽  
Tertiary Hospital ◽  
Clinical Samples ◽  
Resistant Bacteria ◽  
Variable Expression ◽  
Wound Swab ◽  
Significant Difference ◽  
Lactamase Gene

The emergence of AmpC (pAmpC) β-lactamases conferring resistance to the third-generation cephalosporins has become a major clinical concern worldwide. In this study, we aimed to evaluate the expression of AmpC β-lactamase encoding gene among the pathogenic Gram-positive and Gram-negative resistant bacteria screened from clinical samples of Egyptian patients enrolled into El-Qasr El-Ainy Tertiary Hospital in Cairo, Egypt. A total of 153 bacterial isolates of the species Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecium were isolated from patients diagnosed with urinary tract infection (UTI), respiratory tract infection (RTI), and wound infections. The total number of E. faecium isolates was 53, comprising 29 urine isolates, 5 sputum isolates, and 19 wound swab isolates, whereas the total number of P. aeruginosa isolates was 49, comprising 27 urine isolates, 7 sputum isolates, and 15 wound swab isolates, and that of the K. pneumoniae isolates was 51, comprising 20 urine isolates, 25 sputum isolates, and 6 wound swab isolates. Our results indicated that there is no significant difference in the expression of AmpC β-lactamase gene among the tested bacterial species with respect to the type of infection and/or clinical specimen. However, the expression patterns of AmpC β-lactamase gene markedly differed according to the antibacterial resistance characteristics of the tested isolates.

scholarly journals Rapid Detection of New Delhi Metallo-β-Lactamase Gene Using Recombinase-Aided Amplification Directly on Clinical Samples From Children

2021 ◽  
Vol 12 ◽  
Author(s):  
Yanling Feng ◽  
Guanhua Xue ◽  
Junxia Feng ◽  
Chao Yan ◽  
Jinghua Cui ◽  
...  
Keyword(s):  
Klebsiella Oxytoca ◽  
Multi Drug Resistance ◽  
Clinical Samples ◽  
Resistant Bacteria ◽  
New Delhi ◽  
Analytical Sensitivity ◽  
Acinetobacter Junii ◽  
Carbapenem Resistant ◽  
Three Samples ◽  
Lactamase Gene

New Delhi metallo-β-lactamase, a metallo-β-lactamase carbapenemase type, mediates resistance to most β-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect blaNDM genes in children’s clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5−15 min, was established to target blaNDM genes in children’s clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without blaNDM genes did not amplify. This method was used to detect blaNDM genes in 112 children’s stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying blaNDM in children, 15 carbapenem-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca, Acinetobacter junii, and Proteus mirabilis) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. blaNDM–1 and blaNDM–5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid blaNDM gene screening test for clinical samples. The common existence of blaNDM and multi-drug resistance genes presents major challenges for pediatric treatment.

scholarly journals Antibacterial Activities of Five Medicinal Plants in Ethiopia against Some Human and Animal Pathogens

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Gebremedhin Romha ◽  
Birhanu Admasu ◽  
Tsegaye Hiwot Gebrekidan ◽  
Hailelule Aleme ◽  
Gebreyohans Gebru
Keyword(s):  
Medicinal Plants ◽  
Bacterial Species ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Positive Control ◽  
Resistant Bacteria ◽  
Traditional Medicines ◽  
Animal Pathogens ◽  
Significant Difference

Objective. To evaluate thein vitroantibacterial activities of five plant extracts which have been used as traditional medicines by local healers against three multidrug resistant bacteria, namely,Staphylococcus aureus,Escherichia coli, andPseudomonas aeruginosa.Results. The highest mean zone of inhibition (4.66 mm) was recorded from methanol extract ofCalpurnia aurea(Ait.) Benth. at a concentration of 200 mg/ml againstS. aureus, followed byCroton macrostachyusDel. (4.43 mm) at the same dose and solvent for the same bacterial species, while methanol and chloroform extracts ofE. bruceiSchwein. did not inhibit growth of any bacterial species. The lowest value (100 μg/ml) of minimum inhibitory concentration (MIC) was observed from both methanol and chloroform extracts ofC. aurea(Ait.) Benth. against all the three bacteria. The results of the positive control had no statistically significant difference (P>0.05) when compared with crude extracts ofC. aurea(Ait.) Benth. at concentration of 200 mg/ml againstS. aureus.Conclusion. The results of the present study support the traditional uses of these medicinal plants by the local healers. ExceptErythrina bruceiSchwein., all the plants investigated in this study exhibited antibacterial activities against the test bacterial species. Further researches are needed to be conducted to evaluate efficacy of these medicinal plant species on other microbes in different agroecological settings and their safety levels as well as their phytochemical compositions.

scholarly journals Phenotypic Distribution of Serine- and Zinc-Type Carbapenemases Among Clinical Bacterial Isolates in a Tertiary Hospital in Benin, Nigeria

2020 ◽  
Vol 8 (1) ◽  
pp. 3-7
Author(s):  
Ephraim E. Ibadin ◽  
Angela Eghiomon ◽  
Nosakhare L. Idemudia ◽  
Nana A. Anogie ◽  
Richard E. Eriamiatoe ◽  
...  
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Tertiary Hospital ◽  
Test Methods ◽  
Clinical Samples ◽  
Bacterial Isolates ◽  
Gram Negative ◽  
Clinical Specimens ◽  
Significant Difference ◽  
Acinetobacter Spp ◽  
Synergy Test

Background: Serine and zinc type carbapenemases are distributed in many genera of bacteria and are typically associated with specific regions or countries. Objectives: This study phenotypically determined the prevalence of serine and zinc-type carbapenemases among Gram-negative bacilli recovered from clinical specimens in Benin, Nigeria. Materials and Methods: Totally, 158 consecutive non-duplicate bacterial isolates (gram-negative bacilli) recovered from clinical samples were screened for serine and zinc-type carbapenemases using the simplified carbapenemase inactivation (sCIM) and ethylenediaminetetraacetic acid -double-disc synergy test methods. Results: The isolates recovered from clinical specimens included 126 Enterobacteriaceae (79.7%), 7 Acinetobacter spp (3.7%), and 28oxidase positive gram negative bacilli (17.7%). Twenty-eight isolates (17.7%) out of the 158 tested samples were carbapenemase positive. There was no significant difference in the prevalence of serine- and zinc-type carbapenemases (P=0.0748). However, the prevalence of zinc-type carbapenemase was significantly higher in Pseudomonas aeruginosa compared with other isolates (P=0.0028) while that of serinetype carbapenemase was not affected by the type of clinical isolates (P=0.7216). Finally, the prevalence of both serine- and zinc-type carbapenemases were not affected (P>0.05) by clinical specimens and the source of isolates (in-patient vs. out-patient) respectively. Conclusion: In general, the prevalence of zinc-type (12%) carbapenemases was insignificantly higher than that of serine-type (5.7%) carbapenemases. The measures to reduce infections caused by carbapenemase-producing organisms (CPOs) are advocated accordingly.

Evaluation of Culture Top transport systems for assessing the bacterial diversity of microbiota by culturomics as compared to a routine transport system

2021 ◽  
Vol 70 (10) ◽  
Author(s):  
Nadim Cassir ◽  
Souad Belkacemi ◽  
Mehdi Ballouche ◽  
Saber Khelaifia ◽  
Bernard La Scola
Keyword(s):  
Bacterial Diversity ◽  
Transport System ◽  
Bacterial Species ◽  
Storage Conditions ◽  
Rapid Identification ◽  
Culture Conditions ◽  
Transport Systems ◽  
Clinical Samples ◽  
Icu Patients ◽  
Significant Difference

In recent years, metagenomics and then culturomics, which consists of the multiplication of media and culture conditions and the rapid identification of all bacterial colonies, have generated renewed interest in the human microbiota, and diseases associated with modifications in its composition in particular. The sample transport media included in diverse swab transport systems and the storage conditions are among the factors that influence the results of the culturomics. In this study, we compared the results of culturomics from paired skin, oral and rectal swabs from intensive care unit (ICU) patients using Culture Top sample transport medium as compared to our routine one. From 152 clinical samples, we were able to isolate and identify 45 600 colonies, belonging to 338 different bacterial species. The transport system Culture Top identified 282 different bacterial species, while 244 were identified by our routine system. Of these, 188 different bacterial species were commonly identified using both transport systems, while 94 (27.8 %) and 56 (16.5 %) were only identified using Culture Top and our routine system, respectively (P<0.001), but there was no significant difference in bacterial diversity at the genus or phylum level, or in terms of their type of respiration and cell wall. In conclusion, the Culture Top transport system appears to be complementary to our routine system, although it seems slightly superior in terms of isolated bacterial species.

scholarly journals Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Associated With Klebsiella pneumoniae Carbapenemase-Producing Enterobacterales During a Hospital Outbreak in Barcelona, Spain

2021 ◽  
Vol 12 ◽  
Author(s):  
Marta Marí-Almirall ◽  
Núria Ferrando ◽  
Mariana José Fernández ◽  
Clara Cosgaya ◽  
Joaquim Viñes ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Tertiary Hospital ◽  
Clinical Samples ◽  
Resistant Bacteria ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Flight Mass Spectrometry ◽  
Hospital Outbreak ◽  
Resistance Plasmids ◽  
Long Read ◽  
Clonal Spread

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain.Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids.Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae, Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the blaKPC–2 allelic variant. The blaKPC–2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved.Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the blaKPC–2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones.

scholarly journals Tracking of chloramphenicol, erythromycin, and sulfamethoxazole antibiotic-resistant bacteria from untreated wastewater effluents to receiving river

2019 ◽  
Vol 6 (2) ◽  
pp. 89-96 ◽  
Author(s):  
Rahim Aali ◽  
Sepideh Baragh ◽  
Esrafil Asgari ◽  
Reza Fouladi Fard ◽  
Hassan Izanloo ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Antibiotic Resistance Genes ◽  
Resistant Bacteria ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Untreated Wastewater ◽  
Significant Difference ◽  
Receiving River ◽  
Comparison Of The Results ◽  
Wastewater Effluents

Background: The aim of this study was to investigate the frequency and type of bacteria resistant to chloramphenicol (CHL), erythromycin (E), and sulfamethoxazole (SXT) antibiotics from untreated wastewater effluents to receiving river. Methods: In total, 32 samples were taken from eight sites located in the raw wastewater to the downstream of the receiving Ghotor river in Khoy city. Resistant microorganisms were studied through modified HPC method and CLSI standards. Different and specific colonies were selected and re-cultured in R2A culture medium and enough colonies were used for DNA extraction. Bacterial 16sr RNA target gene was amplified and sequenced. Pseudomonas sp., Comamonas sp., and Thiobacillus sp. were predominant bacterial species identified in water samples. Also, antibiotic-resistant genes (ARGs) including sul1, ermB, and cmlA1 were tracked by PCR. Results: The average total number of antibiotic-resistant bacteria (ARB) increased in the downstream (2.4×102 to 2.6×104 CFU/100 mL). The comparison of the results related to the river’s upstream and downstream also indicated a significant difference between ARB and ARGs contents (P<0.05). The average number of bacteria resistant to SXT, E, and CHL antibiotics was obtained to be 2.3×104 , 2.3×104 , and 3.4×104 CFU/mL, respectively. Finally, the evaluation of water revealed that only the aeration lagoon was able to decline the number of CHL-resistant bacteria (88.9%) while this process increased the number of SXT- (51%) and E- (16%) resistant bacteria. The lowest and highest percentages of the identified genes were related to ermB (12.5%) and sul1 (81.25%), respectively. Conclusion: According to the results, wastewaters play an important role in releasing ARB and their antibiotic resistance genes to downstream of Ghotor rivers in Khoy city.

Risk Factors in Diabetes and Pregnant Women with Urinary Tract Infections Compared to Younger Aged Female.

2018 ◽  
Vol 1 (3) ◽  
pp. 26-38
Author(s):  
Abdulghani Mohamed Alsamarai ◽  
Shler Ali Khorshed
Keyword(s):  
Risk Factors ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Pregnant Women ◽  
Female Student ◽  
Mean Value ◽  
Infected Female ◽  
Significant Difference ◽  
Tract Infections

Background: Urinary tract infection is common with health impact in women and characterised by failure to treatment and recurrent episodes. Aim: This study was conducted to determine the risk factors for the development of urinary tract infection in diabetic and pregnant women in comparison to student female. Materials and methods: A prospective cross-sectional study conducted during the period from 1st of June 2015 to the end of January 2016. The population included in the study are 563 women, of them 425 were outpatients, and 138 were inpatients. Their age range between 18 and 80 years, with a mean age of 33.59±15.29 years. Urine samples collected and cultured on blood agar and MacConkey agar by spread plate technique. Bacterial colonies with different morphology were selected, purified and identified according to their biochemical characteristics using conventional standard methods. Results: In diabetic women, there were no significant difference in mean age and BMI values between culture positive and culture negative groups. However, pus cell mean scale was significantly higher [P=0.000] in women with urinary tract infection [1.76±1.25] than in those with negative culture [0.69±1.00]. In pregnant women, BMI mean value was significantly [P=0.013] lower in pregnant women with UTI [26.14] as compared to those without infection [26.99]. Pus cell scale mean value was significantly [P=0.000] higher in pregnant women with UTI [1.55] than women with negative UTI [0.85]. While there was no significant difference in mean age between UTI positive and negative pregnant women. In female student, there was a significant difference between UTI infected and non-infected in mean age [P=0.041] and pus cell scale [P=0.000]. However, BMI was not significantly different between infected and non-infected female student. Other risk factors association are variables in the 3 groups when analysed using X2, while AUC and OR show different trends of association between risk factors and UTI. Conclusion: BMI, pus cell scale, child number, delivery method, operation history and hospital setting were significantly associated with culture positivity in the 3 studied groups as determined by AUC. While OR confirmed association with pus sale scale in the 3 groups.

Assessment of Bacterial Contamination of Orthodontic Arch wire

2019 ◽  
Vol 31 (1) ◽  
pp. 48-51
Author(s):  
Suha S Hassan ◽  
Nidhal H. Ghaib ◽  
Batool H Al-Ghurabi
Keyword(s):  
Bacterial Contamination ◽  
Microbial Contamination ◽  
Ethylenediaminetetraacetic Acid ◽  
Bacterial Species ◽  
Brain Heart Infusion ◽  
Control Groups ◽  
Dental Practitioners ◽  
Significant Difference ◽  
Bacillus Spp ◽  
Droplet Transmission

Background: The microorganisms can impend the life of health care professional and particularly the dental practitioners. They can be transmitted by different ways like airborne and droplet transmission. The current study was carried out to identify whether the arch wires that received from the manufactures are free from microbial contamination and to determine the bacterial species attached to the arch wires. Materials and Methods: This study involved eighty samples, consisted of two types of arch wires (nitinol and stainless-steel) from four companies (3M, G&H, Jiscop, OrthoTechnology). These wires inserted in a plane tube that contains 10 -ml of (Tris [tris(hydroxymethyl)aminomethane] and EDTA (ethylenediaminetetraacetic acid) tris-EDTA and brain heart infusion (BHI) broth. A 0.1 ml was withdrawn from the tube and spread on agar plates. The control groups consist of 16 plane tube (8 tubes with tris-EDTA and other 8 tubes with (BHI). Results: Microbial sampling yielded growth from 5 of the 80 arch wires. The predominant bacteria that isolated were Bacillus spp. No growth was recovered from 75 of the samples and from controls. The bacteria were isolated by BHI reagent and no growth was observed by tris-EDTA reagent with statistically significant difference (P<0.05). The Bacillus spp. found only in the G&H and Jiscop companies, however, no statistically significant difference was found among them (P>0.05). With regard to the presence and distribution of bacteria according to the types of wires, the present results clarified that cases of contamination with Bacillus spp. were found in the nitinol arch wires with statistically significant difference (P<0.05). Conclusions: The results of the current study revealed low count of bacterial contamination in the two types of companies (G&H and Jiscop). Not all materials that received from the manufactures are free from contamination and an effective sterilization regimen is needed to avoid cross-contamination.

scholarly journals Effect of Surgical Setting on Hospital-Reported Outcomes for Elective Lumbar Spinal Procedures: Tertiary Versus Community Hospitals

2019 ◽  
Vol 10 (4) ◽  
pp. 375-383 ◽  
Author(s):  
Tristan B. Weir ◽  
Neil Sardesai ◽  
Julio J. Jauregui ◽  
Ehsan Jazini ◽  
Michael J. Sokolow ◽  
...  
Keyword(s):  
Pay For Performance ◽  
Linear Regression Analysis ◽  
Degenerative Spondylolisthesis ◽  
Hospital Costs ◽  
Patient Characteristics ◽  
Tertiary Hospital ◽  
Community Hospitals ◽  
Single Level ◽  
Significant Difference ◽  
Reported Data

Study Design: Retrospective cohort study. Objective: As hospital compensation becomes increasingly dependent on pay-for-performance and bundled payment compensation models, hospitals seek to reduce costs and increase quality. To our knowledge, no reported data compare these measures between hospital settings for elective lumbar procedures. The study compares hospital-reported outcomes and costs for elective lumbar procedures performed at a tertiary hospital (TH) versus community hospitals (CH) within a single health care system. Methods: Retrospective review of a physician-maintained, prospectively collected database consisting of 1 TH and 4 CH for 3 common lumbar surgeries from 2015 to 2016. Patients undergoing primary elective microdiscectomy for disc herniation, laminectomy for spinal stenosis, and laminectomy with fusion for degenerative spondylolisthesis were included. Patients were excluded for traumatic, infectious, or malignant pathology. Comparing hospital settings, outcomes included length of stay (LOS), rates of 30-day readmissions, potentially preventable complications (PPC), and discharge to rehabilitation facility, and hospital costs. Results: A total of 892 patients (n = 217 microdiscectomies, n = 302 laminectomies, and n = 373 laminectomy fusions) were included. The TH served a younger patient population with fewer comorbid conditions and a higher proportion of African Americans. The TH performed more decompressions ( P < .001) per level fused; the CH performed more interbody fusions ( P = .007). Cost of performing microdiscectomy ( P < .001) and laminectomy ( P = .014) was significantly higher at the TH, but there was no significant difference for laminectomy with fusion. In a multivariable stepwise linear regression analysis, the TH was significantly more expensive for single-level microdiscectomy ( P < .001) and laminectomy with single-level fusion ( P < .001), but trended toward significance for laminectomy without fusion ( P = .052). No difference existed for PPC or readmissions rate. Patients undergoing laminectomy without fusion were discharged to a facility more often at the TH ( P = .019). Conclusions: We provide hospital-reported outcomes between a TH and CH. Significant differences in patient characteristics and surgical practices exist between surgical settings. Despite minimal differences in hospital-reported outcomes, the TH was significantly more expensive.

scholarly journals COPD-Lower Respiratory Tract Infection Visual Analogue Score (c-LRTI-VAS) validation in stable and exacerbated patients with COPD

2021 ◽  
Vol 8 (1) ◽  
pp. e000761
Author(s):  
Hendrik Johannes Prins ◽  
Ruud Duijkers ◽  
Johannes M A Daniels ◽  
Thys van der Molen ◽  
Tjip S van der Werf ◽  
...  
Keyword(s):  
Tract Infection ◽  
Respiratory Tract ◽  
Respiratory Tract Infection ◽  
Lower Respiratory Tract Infection ◽  
Lower Respiratory Tract ◽  
Visual Analogue Score ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Significant Difference ◽  
Related Quality

BackgroundWe developed the chronic obstructive pulmonary disease (COPD)-Lower Respiratory Tract Infection-Visual Analogue Score (c-LRTI-VAS) in order to easily quantify symptoms during exacerbations in patients with COPD. This study aimed to validate this score.MethodsIn our study, patients with stable COPD as well as those with an acute exacerbations of COPD (AECOPD) were included. The results of c-LRTI-VAS were compared with other markers of disease activity (lung function parameters, oxygen saturation and two health related quality of life questionnaires (St Georges Respiratory Questionnaire (SGRQ) and Clinical COPD Questionnaire (CCQ)) and validity, reliability and responsiveness were assessed.ResultsEighty-eight patients with clinically stable COPD and 102 patients who had an AECOPD completed the c-LRTI-VAS questionnaire. When testing on two separate occasions for repeatability, no statistically significant difference between total scores was found 0.143 (SD 5.42) (p=0.826). Internal consistency was high across items (Cronbach’s apha 0.755). Correlation with SGRQ and CCQ total scores was moderate to high. After treatment for hospitalised AECOPD, the mean c-LRTI-VAS total score improved 8.14 points (SD 9.13; p≤0.001).Conclusionsc-LRTI-VAS showed proper validity, responsiveness to change and moderate to high correlation with other questionnaires. It, therefore, appears a reliable tool for symptom measurement during AECOPD.Trial registration numberNCT01232140.

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