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Microbial diagnosis of infection and colonization of cardiac implantable electronic devices by use of sonication

Open AccessPublished:July 24, 2015DOI:https://doi.org/10.1016/j.ijid.2015.07.018

      Highlights

      • Infected cardiac devices were more associated with previous local complications.
      • Gram-positive cocci (CoNS, Streptococcus, and S. aureus) were frequently isolated.
      • Compared to traditional cultures, sonication was more sensitive and less specific.
      • Sonication identified a higher number of organisms in colonized cardiac devices.
      • Previous use of antibiotic reduces microbial identification, mainly tissue cultures.

      Summary

      Objectives

      The clinical utility of sonication as an adjunctive diagnostic tool for the microbial diagnosis of cardiac implantable device-associated infections (CIDAIs) was investigated.

      Methods

      The implants of 83 subjects were investigated, 15 with a CIDAI and 68 without a clinical infection. Clinical data were analyzed prospectively and sonication fluid cultures (83 patients, 100%) and traditional cultures (31 patients, 37.4%) were performed

      Results

      Generator pocket infection and device-related endocarditis were found in 13 (86.7%) and four (26.7%) subjects, respectively. The mean numbers of previous technical complications and infections were higher in the infected patients compared to the non-infected patients (8 vs. 1, p < 0.001; 2 vs. 0, p < 0.031, respectively). The sensitivity and specificity for detecting CIDAI was 73.3% (11/15) and 48.5% (33/68) for sonication fluid culture, and 26.7% (4/15) and 100% (16/16) for traditional culture (p < 0.001), respectively. A higher number of organisms were identified by sonication fluid than by tissue culture (58 vs. 4 specimens; p < 0.001). The most frequent organisms cultured were Gram-positive cocci (66.1%), mainly coagulase-negative staphylococci (35.5%). Thirty-five (51.5%) non-infected subjects were considered colonized due to the positive identification of organisms exclusively through sonication fluid culture.

      Conclusions

      Sonication fluid culture from the removed cardiac implants has the potential to improve the microbiological diagnosis of CIDAIs.

      Keywords

      1. Introduction

      The surgical implantation of cardiovascular electronic devices, including permanent pacemakers (PPMs), implantable cardioverter defibrillators (ICDs), and cardiac resynchronization devices (CRTDs), has been indicated increasingly worldwide in the last 30 years for the treatment of many different medical conditions, such as bradycardia, ventricular arrhythmia, and heart failure, and for the prevention of sudden cardiac death.
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      • Patanè S.
      Cardiovascular implantable electronic device infective endocarditis.
      Indeed, data collected in 2009 from 61 different countries have shown a continuous rise in the number of cardiac implantable electronic devices (CIEDs) being inserted worldwide, with the highest number of implanted PPMs in the USA (225 567) and Germany accounting for 927 new implants per million population.
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      The 11th world survey of cardiac pacing and implantable cardioverter-defibrillators: calendar year 2009—A World Society of Arrhythmias Project.
      Although infection following CIED use remains relatively uncommon, it may affect exclusively the generator pocket, intra-vascular electrode components, or endocardial structures, or may even present in different combinations, making the clinical suspicion often delayed and not considered.
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      Moreover, accurate and evidence-based diagnostic tools for CIED-associated infections (CIDAIs) are lacking, as the clinical presentation is highly variable, echocardiography may show low accuracy, and blood and conventional cultures of peri-implant fluid (swab) or tissue samples may show low sensitivity.
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      False-negative microbiological results varying from 12% to 49% have been associated with previous antibiotic use and to the nature of a biofilm-associated infection in which organisms are enclosed in a polymeric matrix substance exhibiting altered phenotype and gene expression.
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      • et al.
      Guidelines for the diagnosis, prevention and management of implantable cardiac electronic device infection. Report of a joint working party project on behalf of the British Society for Antimicrobial Chemotherapy (BSAC, host organization), British Heart Rhythm Society (BHRS), British Cardiovascular Society (BCS), British Heart Valve Society (BHVS) and British Society for Echocardiography (BSE).
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      Antibiotic resistance of bacteria in biofilms.
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      • et al.
      Bacteriology of infected extracted pacemaker and ICD leads.
      Furthermore, local positive cultures in subjects showing no signs or symptoms of active infection have been associated with microbial colonization of the generator pocket.
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      • et al.
      Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.
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      • et al.
      Prevalence of bacterial colonization of generator pockets in implantable cardioverter defibrillator patients without signs of infection undergoing generator replacement or lead revision.
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      • Bara C.
      • et al.
      High prevalence of asymptomatic bacterial colonization of rhythm management devices.
      A few studies have recently suggested that by using techniques that dislodge bacterial cells from the biofilm, such as vortexing and sonication, microbial diagnosis is increased among CIDAI subjects.
      • Oliva A.
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      • Mascellino M.T.
      • D’Abramo A.
      • Iannetta M.
      • Ciccaglioni A.
      • et al.
      Sonication of explanted cardiac implants device infections improves microbial detection in cardiac devices infections.
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      • Frei R.
      • et al.
      Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.
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      • Bilchick K.C.
      • et al.
      Sonication of explanted cardiac rhythm management devices for the diagnosis of pocket infections and asymptomatic bacterial colonization.
      Compared to conventional cultures, sonication has shown higher sensitivity and specificity for microbial diagnosis for a variety of implant-associated infections.
      • Trampuz A.
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      • Jacobson M.J.
      • Hanssen A.D.
      • Unni K.K.
      • Osmon D.R.
      • et al.
      Sonication of removed hip and knee prostheses for diagnosis of infection.
      • Piper K.E.
      • Jacobson M.J.
      • Cofield R.H.
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      • Osmon D.R.
      • et al.
      Microbiologic diagnosis of prosthetic shoulder infection by use of implant sonication.
      • Yano M.H.
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      • da Silva C.B.
      • Nigro S.
      • Avanzi O.
      • Mercadante M.T.
      • et al.
      Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.
      In the present study, the clinical utility of sonication as an adjunctive diagnostic tool for CIDAIs was investigated, by comparing it with traditional cultures (blood cultures and intraoperative peri-implant tissue cultures). Furthermore, the utility of sonication in identifying microbial colonization of the generator pocket was also assessed among subjects with no signs of infection.

      2. Materials and methods

      2.1 Study population

      Eighty-three subjects who underwent complete or partial surgical removal of a CIED (including PPMs and ICDs) for any reason, between September 2010 and October 2013, at the Cardiac Surgery Unit of Santa Casa de Sao Paulo School of Medical Sciences, São Paulo (Brazil), were included prospectively in this study. Subjects were excluded if clinical data were unavailable for analysis, when the retrieved CIED was not submitted to the sonication technique, or when contamination occurred during implant removal, transportation, or processing in the microbiology laboratory. Subject demographics, the type of CIED, comorbidities, previous CIDAIs and surgeries, the length of time between implantation and implant retrieval, clinical signs and symptoms of infection, and the presence of endocarditis were recorded. The study protocol was reviewed and approved by the institutional review board.

      2.2 Diagnosis of CIED-associated infection

      Clinical features of CIDAI were considered to be present when the generator pocket showed localized cellulitis, swelling, discharge, wound dehiscence, or local pain; intraoperative tissue showed visible purulence as determined by the surgeon; a draining fistula communicating with the internal implant was evident; and when signs and symptoms of systemic infection (fever, chills, night sweats, malaise) were present. The Duke criteria were also used for the diagnosis of CIED infective endocarditis.
      • Durack D.T.
      • Lukes A.S.
      • Bright D.K.
      New criteria for diagnosis of infective endocarditis: utilization of specific echocardiographic findings. Duke Endocarditis Service.
      • Li J.S.
      • Sexton D.J.
      • Mick N.
      • Nettles R.
      • Fowler V.G.
      • Ryan T.
      • et al.
      Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis.

      2.3 Specimen collection and microbiological methods

      In the surgical ward, blood cultures, sterile cotton swabs of the prosthetic (generator and leads), peri-prosthetic fluids, and tissue samples were collected and processed for microbiology and histopathology. Tissue was homogenized in 3 ml of brain–heart infusion (BHI) broth for 1 min. Homogenized tissue and wood shaft cotton swabs were inoculated onto aerobic sheep blood agar, chocolate agar, and anaerobic blood agar and into thioglycolate broth (BD Diagnostic Systems, Sparks, MD, USA). The time limit for processing samples was 6 h. Aerobic and anaerobic plates were incubated aerobically at 35–37 °C in 5–7% CO2 for 7 days and anaerobically at 37 °C for 14 days, respectively. Additionally, 0.5 ml of tissue homogenate was inoculated in thioglycolate broth, incubated for 14 days, and the turbid thioglycolate broth was sub-cultured on blood agar plates when cloudy. Colonies of microorganisms growing on plates were identified, and their susceptibility to antibiotics was tested by standard microbiological techniques. Bacterial identification was performed with routines established in the laboratory, assessing the morphology and tinctorial properties displayed on Gram staining. Catalase tests were applied on Gram-positive colonies to identify Staphylococcus spp and Streptococcus spp. DNase tests differentiated Staphylococcus aureus from coagulase-negative staphylococci (CoNS), while groups of Streptococcus spp and Enterococcus spp were identified by their hemolysis (alpha, beta, or gamma), ability to hydrolyze esculin in the presence of bile, and growth on 6.5% NaCl associated with susceptibility testing to optochin and bacitracin or the CAMP test, when necessary. Gram-negative colonies were identified by biochemical methods to determine their genus and species, including glucose non-fermenting bacteria. Low-virulence microorganisms (CoNS, Corynebacterium sp, Chryseobacterium sp, Bacillus sp) were considered pathogens when the same organism was identified in at least two different tissue samples, or when at least one additional (culture-independent) criterion for CIDAI was also fulfilled.

      2.4 CIED sonication

      In the operating room, the explanted CIEDs were removed aseptically and placed in sterilized solid polyethylene containers, to which 250 ml of Ringer solution was added; these were sealed with an air-tight cover. In the microbiology laboratory, containers with the retrieved implants were vortexed for 30 s using a Vortex-Genie 2 (Scientific Industries Inc., Bohemia, NY, USA) and then sonicated (ultrasound bath BactoSonic; Bandelin GmbH, Berlin, Germany) for 5 min at a frequency of 40 ± 2 kHz and power density 0.22 ± 0.04 W/cm2, followed by an additional 30 s of vortexing, in accordance with the technique of Trampuz et al.
      • Trampuz A.
      • Piper K.E.
      • Jacobson M.J.
      • Hanssen A.D.
      • Unni K.K.
      • Osmon D.R.
      • et al.
      Sonication of removed hip and knee prostheses for diagnosis of infection.
      To concentrate the resulting sonication fluid, centrifugation was performed in 50-ml aliquots at 2500 rpm for 5 min. The supernatant was aspirated, leaving 0.5 ml (100-fold concentration), and aliquots of 0.1 ml of concentrated sonication fluid were then plated onto aerobic sheep blood, chocolate, and anaerobic sheep blood agar; these were incubated aerobically at 37 °C for 7 days and anaerobically at 37 °C for 14 days, respectively, and inspected daily for bacterial growth. Additionally, 4 ml of the remaining concentrated sonication fluid was inoculated in 10 ml thioglycolate broth, plated as described above, and incubated aerobically at 35–37 °C in 5% CO2 for 2 days and anaerobically at 37 °C for 14 days. A 4-ml aliquot of sonication fluid and all the detected organisms were frozen at −70 °C. Colonies of isolated microorganisms growing on plates were quantitated (number of colony-forming units per milliliter sonication fluid; CFU/ml), identified, and their antimicrobial susceptibility was tested using standard microbiological techniques. Due to the addition of a concentration step to the sonication fluid culture, a cut-off of 50 CFU/plate was considered positive and used for ideal sensitivity and specificity analysis.
      • Yano M.H.
      • Klautau G.B.
      • da Silva C.B.
      • Nigro S.
      • Avanzi O.
      • Mercadante M.T.
      • et al.
      Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.
      Furthermore, for those subjects on antimicrobial therapy or those who had previously received antibiotics for at least 24 h in the 14 days prior to surgery, any growth of organism in the sonication fluid culture was considered positive. CIEDs explanted due to aseptic reasons were used as negative controls and processed in the same manner as described for the infected CIED implants retrieved.

      2.5 Statistical analysis

      Characteristics of subjects with infected and non-infected cardiovascular electronic devices were summarized as the frequency and percentage, or as the mean (range) and standard deviation (SD). Descriptive comparisons between categorical variables were performed using the Chi-square test or Fisher's exact test, as appropriate. Continuous variables were compared with the Student t-test (normally distributed) or Mann–Whitney U-test (non-normally distributed). Sensitivities, specificities, positive predictive values, and negative predictive values were compared between tissue culture and sonication fluid culture using the McNemar test of paired proportions. Ninety-five percent confidence intervals (CIs) were calculated as exact binominal confidence intervals. Differences were considered significant when the p-value was <0.05 (two-tailed). Data were analyzed using IBM SPSS Statistics for Windows version 19.0 (IBM Corp., Armonk, NY, USA).

      3. Results

      3.1 Study population and devices

      Eighty-five CIEDs from 85 subjects were retrieved consecutively. Two of the subjects were excluded from further analysis, one because a removed implant was not cultured and the second due to clear contamination being detected during implant removal. In total, 83 implants were analyzed: 72 (86.7%) were PPMs and 11 (13.3%) were ICDs. Among these, CIDAIs were diagnosed clinically in 15 subjects (18.1%) and non-infected-CIDs (NICIDs) were found in 68 (81.9%) subjects. Generator pocket infection and device-related endocarditis were found in 13 (86.7%) and four (26.7%) subjects, respectively. Demographic parameters and clinical characteristics of the study population are shown in Table 1.
      Table 1Characteristics of 83 subjects with cardiac implantable device-associated infections (CIDAI) and non-infected cardiac implantable devices (NICID)
      Subjects with CIDAI (n = 15)Subjects with NICID (n = 68)p-Value
      Patient characteristics were summarized as the frequency and percentage, or as the median and range, and were compared using the Pearson Chi-square test or Fisher's exact test as appropriate for nominal variables and the Mann–Whitney test or t-test as appropriate for continuous variables (IBM SPSS software, version 19.0). All tests were two-sided and a p-value of <0.05 was considered statistically significant.
      Demographic characteristics
      All percentages are in relation to the number of subjects with CIDAI or NICID unless indicated otherwise.
       Male sex, n (%)7 (46.6%)27 (39.7%)0.620
       Age, years, median (range)60 (40–90)66.5 (32–95)0.596
       Duration of CIED use, months, median (range)24 (1–168)84 (12–324)<0.001
      Clinical characteristics, n (%)
       Diabetes mellitus3 (15%)11 (16.2%)0.711
       Coronary diseases4 (26.7%)14 (20.6%)0.730
       Heart failure9 (60%)48 (61.8%)0.899
       Chagas disease4 (26.7%)15 (22.1%)0.738
       Chronic renal failure1 (6.7%)2 (2.9%)0.455
       Smoking3 (15%)4 (5.9%)0.107
       More than one revision6 (40%)12 (17.6%)0.057
       Previous CIED complications8 (53.3%)1 (1.5%)<0.001
       Previous CIED infections2 (13.3%)0 (0%)0.031
       Prior use of antimicrobials
      Patients who received a minimum of 1 day of antibiotic therapy within the 14 days prior to removal of the implants.
      13 (86.7%)0 (0%)<0.001
      CIED, cardiac implantable electronic device.
      a Patient characteristics were summarized as the frequency and percentage, or as the median and range, and were compared using the Pearson Chi-square test or Fisher's exact test as appropriate for nominal variables and the Mann–Whitney test or t-test as appropriate for continuous variables (IBM SPSS software, version 19.0). All tests were two-sided and a p-value of <0.05 was considered statistically significant.
      b All percentages are in relation to the number of subjects with CIDAI or NICID unless indicated otherwise.
      c Patients who received a minimum of 1 day of antibiotic therapy within the 14 days prior to removal of the implants.
      The clinical presentation of the 15 subjects diagnosed with a CIDAI included signs and symptoms of pocket inflammation, such as pocket erythema (86.7%), swelling (66.7%), pus (60%), local pain (53.3%), and warmth (53.3%), and the presence of systemic signs such as fever (53.3%) and chills (26.7%). Only one patient (6.7%) presented septic shock associated with the cardiac implant infection. The median age of the implants at the time of resection surgery was lower in the CIDAI group than in the NICID group (24 vs. 84 months, respectively; p < 0.001). The mean numbers of previous technical complications and infections associated with CIEDs were higher in the CIDAI group than in the NICID group (8 vs. 1, p < 0.001; 2 vs. 0, p < 0.031, respectively). The groups were similar with regard to comorbidities (Table 1).

      3.2 Microbiology

      Sonication fluid cultures and swab/tissue/blood cultures were performed for 83 (100%) and 31 (37.4%) subjects, respectively. For subjects with a NICID, sonication fluid cultures and swab/tissue/blood cultures were collected for 68 (100%) and 16 (23.5%), respectively. Higher numbers of microorganisms were identified by sonication fluid culture of explanted CIEDs than by peri-implant tissue and blood cultures (58 vs. 4 specimens; p < 0.001). Twelve (80%) subjects with a CIDAI received a definitive microbial diagnosis through a combination of sonication of explanted devices and tissue/blood cultures. On the other hand, tissue/blood culture identified bacteria but not sonication fluid culture in only one (6.7%) infected patient. Conversely, sonication fluid culture was the only source of pathogen identification for eight (72.7%) CIDAI subjects. Indeed, tissue and blood culture were able to detect only four Gram-negative bacilli (Escherichia coli, Enterobacter sp, Klebsiella pneumoniae, Morganella morgannii) but no Gram-positive cocci among four subjects with CIDAIs, and no pathogens were detected among 16 subjects with NICIDs. Sonication fluid cultures yielded a significantly higher rate of pathogen detection among 11 subjects with CIDAI, but also in 35 subjects with NICID (with no clinical diagnosis of infection), a result that was considered to indicate colonization. From sonication fluid of explanted CIDs, the most frequent organisms cultured were Gram-positive cocci (66.1%), of which CoNS was identified in 35.5%, followed by Streptococcus spp in 14.5% and S. aureus in 9.8%, whereas Gram-negative bacilli were cultured in 27.4%. Peculiar biofilm-forming pathogens such as Stenotrophomonas maltophilia, Corynebacterium sp, Acinetobacter baumannii, Pseudomonas sp, and Candida sp, were also identified. A polymicrobial infection was diagnosed in only one patient (S. aureus and CoNS detected on sonication fluid culture). For three (20%) subjects with CIDAIs, bacterial isolation was not possible with either sonication fluid or tissue/blood culture. Table 2 summarizes the microorganisms identified in sonication fluid cultures and tissue/blood cultures among 83 subjects with CIDAI and NICID (colonized).
      Table 2Distribution of microorganisms detected by sonication fluid culture and tissue/blood culture among the 83 subjects included in the study
      MicroorganismSonication culture

      (n = 83)
      Tissue/blood culture

      (n = 31)
      n%n%
      CoNS2226.500
      Staphylococcus aureus67.200
      Streptococcus sp910.800
      Micrococcus sp22.400
      Bacillus sp11.200
      Acinetobacter baumannii33.600
      Stenotrophomonas maltophilia33.600
      Klebsiella pneumoniae33.613.2
      Enterobacter sp22.413.2
      Pseudomonas sp22.400
      Escherichia coli11.213.2
      Morganella morgannii0013.2
      Corynebacterium sp11.200
      Chryseobacterium indologenes22.400
      Candida albicans11.200
      CoNS, coagulase-negative Staphylococcus.

      3.3 Comparison of microbiological tests and discordant results between sonication fluid and tissue/blood cultures

      Forty-six subjects (55.4%) had positive sonication fluid cultures (11 CIDAI and 35 NICID), and only four (4.8%) presented positive tissue cultures (4 CIDAI and 0 NICID) (p < 0.001). The sensitivity of sonication fluid culture for microbiological diagnosis among 15 subjects with CIDAI was higher than tissue/blood culture (73.3% vs. 26.7%, respectively; p < 0.001). Conversely, the specificity showed significantly better results for tissue/blood culture than sonication fluid culture, at 100% and 48.5%, respectively (p < 0.001). Positive and negative predictive values of tissue/blood culture and sonication tissue culture were 100% and 23.9%, and 59.2% and 89.2%, respectively (Table 3).
      Table 3Comparison between sonication fluid culture and tissue/blood culture
      Comparison among 83 subjects for whom sonication fluid culture was done and 31 subjects for whom tissue/blood culture was done.
      Sensitivity
      The sensitivities of the different culture methods were compared with McNemar's test of paired proportions (p < 0.001).
      Specificity
      The specificities of the different culture methods were compared with McNemar's test of paired proportions (p<0.001).
      PPVNPV
      (95% CI)(95% CI)(95% CI)(95% CI)
      Sonication fluid culture73.3 (11/15)48.5 (33/68)23.9 (11/46)89.2 (33/37)
      CI: 44.6–100CI: 36.6–60.4CI: 11.6–36.2CI: 77.3–100
      Tissue/blood culture26.7 (4/15)100 (16/16)100 (4/4)59.2 (16/27)
      CI: 13.8–31.2CI: 91–100CI: 70.5–100CI: 41.6–60.7
      PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.
      a Comparison among 83 subjects for whom sonication fluid culture was done and 31 subjects for whom tissue/blood culture was done.
      b The sensitivities of the different culture methods were compared with McNemar's test of paired proportions (p < 0.001).
      c The specificities of the different culture methods were compared with McNemar's test of paired proportions (p < 0.001).
      The global concordance (positive and negative results) between the tissue and sonication fluid cultures was only 51.6% (16/31). Among the subjects with CIDAIs and NICIDs, the tissue/blood and sonication fluid cultures were concordant in 40% (6/15) and 62.5% (10/16), respectively. There were 15 discordant results between the sonication fluid and tissue/blood cultures. Table 4 summarizes the discordant microbiology results between the sonication fluid and tissue cultures among subjects with CIDAI and NICID.
      Table 4Cardiac implantable device-associated infection (CIDAI) and non-infected cardiac implantable device (NICID) subjects with discordant results between sonication fluid culture and tissue/blood culture
      CaseCIDAITissue/blood cultureSonication fluid cultureNICIDTissue/blood cultureSonication fluid culture
      ICD 22YesNegativeEnterobacter spNo--
      MP 30YesNegativeCandida albicansNo--
      MP 33No--YesNegativeStreptococcus sp
      MP 36YesNegativeS. maltophiliaNo--
      MP 45No--YesNegativeS. aureus
      MP 58YesNegativeS. aureusNo--
      MP 59YesM. morganniiNegativeNo--
      ICD 60No--YesNegativeCoNS
      MP 67YesNegativeS. aureus + CoNSNo--
      MP 68No--YesNegativeA. baumannii
      MP 71No--YesNegativeK. pneumoniae
      ICD 77YesNegativeS. aureusNo--
      MP 78No--YesNegativeS. aureus
      MP 80YesNegativeStreptococcus spNo--
      MP 83YesNegativeCoNSNo--
      CoNS, coagulase-negative Staphylococcus.

      3.4 Identification of microorganisms in subjects presenting no clinical signs of CIDAI

      Thirty-five (51.5%) out of 68 non-infected subjects (NICID) were considered colonized due to the positive identification of microorganisms exclusively through sonication fluid culture. Gram-positive cocci accounted for 69.5% of microorganisms isolated, mainly CoNS, which was identified in 43.5%, followed by Streptococcus spp in 8.7%, Enterococcus sp in 8.7%, and S. aureus in 6.5%, whereas Gram-negative bacilli were cultured in 30.5% (mainly A. baumannii, 10%). No differences were found between colonized and non-colonized subjects regarding demographic parameters and clinical characteristics such as comorbidities, age of the implant, number of past surgical revisions, previous local complications, and antibiotic use prior to surgery, among the study population.

      3.5 Previous antimicrobial therapy

      Thirteen out of 15 (86.7%) subjects with a CIDAI received at least of 1 day of antibiotic therapy within 14 days prior to the surgical removal of the cardiac implants, and among them, identification of organisms occurred in 30.8% (4/13) and 76.7% (10/13) of the tissue and sonication fluid cultures, respectively. Although the percentage of microbial identification was higher for sonication fluid culture when compared with traditional tissue/blood culture, this difference did not reach statistical significance (76.7% vs. 30.8%, p = 0.07). For the two infected subjects without previous antibiotic use, sonication cultures identified organisms in only one subject, while tissue cultures identified pathogens in neither of them. None of the NICID subjects had used antibiotics before implant removal.

      4. Discussion

      Interestingly, some studies have shown an increase in the rate of CIDAIs greater than the rate of new cardiac implantable devices inserted.
      • Mond H.G.
      • Proclemer A.
      The 11th world survey of cardiac pacing and implantable cardioverter-defibrillators: calendar year 2009—A World Society of Arrhythmias Project.
      • Welch M.
      • Uslan D.Z.
      • Greenspon A.J.
      • Sohail M.R.
      • Baddour L.M.
      • Blank E.
      • et al.
      Variability in clinical features of early versus late cardiovascular implantable electronic device pocket infection.
      • Viola G.M.
      • Awan L.L.
      • Ostrosky-Zeichner L.
      • Chan W.
      • Darouiche R.O.
      Infections of cardiac implantable electronic devices: a retrospective multicenter observational study.
      Consequently, it is important to assure a prompt and accurate clinical and microbial diagnosis, which will guide the optimal treatment of the device infection. Recently published guidelines addressing the management of CIDAIs recommend a combination of different methods to achieve an adequate microbial diagnosis, including culture of blood, lead tips, lead vegetation, generator pocket tissues, and pus from the generator pocket wound.
      • Sandoe J.A.
      • Barlow G.
      • Chambers J.B.
      • Gammage M.
      • Guleri A.
      • Howard P.
      • et al.
      Guidelines for the diagnosis, prevention and management of implantable cardiac electronic device infection. Report of a joint working party project on behalf of the British Society for Antimicrobial Chemotherapy (BSAC, host organization), British Heart Rhythm Society (BHRS), British Cardiovascular Society (BCS), British Heart Valve Society (BHVS) and British Society for Echocardiography (BSE).
      Although presenting high rates of specificity, blood culture lacks sensitivity particularly among those patients with prior antimicrobial therapy.
      • Bongiorni M.G.
      • Tascini C.
      • Tagliaferri E.
      • Di Cori A.
      • Soldati E.
      • Leonildi A.
      • et al.
      Microbiology of cardiac implantable electronic device infections.
      • Margey R.
      • McCann H.
      • Blake G.
      • Keelan E.
      • Galvin J.
      • Lynch M.
      • et al.
      Contemporary management of and outcomes from cardiac device related infections.
      In the present study, at least two sets of blood cultures were collected for all infected patients, but few pathogens (13.3%) were identified. Because they are easy and simple to perform, swab culture techniques have been the preferred methodology for sample collection for many surgeons, but frequent contamination and discordant and inferior results for swabs and tissue cultures have been reported by some authors.
      • Dy Chua J.
      • Abdul-Karim A.D.
      • Mawhorter S.
      • Procop G.W.
      • Tchou P.
      • Niebauer M.
      • et al.
      The role of swab and tissue culture in the diagnosis of implantable cardiac device infection.
      Additionally, many studies addressing the diagnosis of implanted device-associated infections have demonstrated that the identification of the causative microorganisms solely by tissue culture has been unhelpful for a considerable proportion of patients, especially among those with previous use of systemic antibiotics.
      • Oliva A.
      • Nguyen B.L.
      • Mascellino M.T.
      • D’Abramo A.
      • Iannetta M.
      • Ciccaglioni A.
      • et al.
      Sonication of explanted cardiac implants device infections improves microbial detection in cardiac devices infections.
      • Yano M.H.
      • Klautau G.B.
      • da Silva C.B.
      • Nigro S.
      • Avanzi O.
      • Mercadante M.T.
      • et al.
      Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.
      • Berbari E.F.
      • Marculescu C.
      • Sia I.
      • Lahr B.D.
      • Hanssen A.D.
      • Steckelberg J.M.
      • et al.
      Culture-negative prosthetic joint infection.
      • Portillo M.E.
      • Salvadó M.
      • Alier A.
      • Martínez S.
      • Sorli L.
      • Horcajada J.P.
      • et al.
      Advantages of sonication fluid culture for the diagnosis of prosthetic joint infection.
      Among 15 infected patients included in the present study, swab cultures identified no pathogens and peri-implant tissue cultures gave just two positive results (13.3%). It is important to note that almost 90% of infected patients were on antimicrobial therapy during the swab and tissue collection, which might partially explain the low sensitivity of swab and tissue cultures. Indeed, among infected subjects with previous antibiotic use, sonication had much higher success in identifying microorganisms compared to tissue/blood culture (76.7% vs. 30.8%), although this difference did not reach statistical significance, probably due to the small sample size analyzed.
      This study group has previously studied the microbiological diagnosis of osteosynthesis-associated infections and showed the superiority of sonication of explanted orthopedic devices over tissue culture.
      • Yano M.H.
      • Klautau G.B.
      • da Silva C.B.
      • Nigro S.
      • Avanzi O.
      • Mercadante M.T.
      • et al.
      Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.
      In the present study, compared to blood/tissue culture, sonication of CIEDs identified causative pathogens in a significantly higher proportion of subjects. Tissue-positive and sonication fluid culture-negative results were seen in only one infected subject (6.7%), and among three other infected subjects presenting positive tissue/blood cultures, sonication fluid detected the same pathogens (E. coli, M. morgannii, and K. pneumoniae). In agreement with other authors, it is hypothesized that the reason for the increased detection of pathogens through sonication is that CIDAIs are typically produced by low-virulence biofilm-forming pathogens that attach to the surfaces of large stainless steel implants such as PPMs and ICDs.
      • Oliva A.
      • Nguyen B.L.
      • Mascellino M.T.
      • D’Abramo A.
      • Iannetta M.
      • Ciccaglioni A.
      • et al.
      Sonication of explanted cardiac implants device infections improves microbial detection in cardiac devices infections.
      • Trampuz A.
      • Piper K.E.
      • Jacobson M.J.
      • Hanssen A.D.
      • Unni K.K.
      • Osmon D.R.
      • et al.
      Sonication of removed hip and knee prostheses for diagnosis of infection.
      • Yano M.H.
      • Klautau G.B.
      • da Silva C.B.
      • Nigro S.
      • Avanzi O.
      • Mercadante M.T.
      • et al.
      Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.
      • Arciola C.R.
      • Montanaro L.
      • Costerton J.W.
      New trends in diagnosis and control strategies for implant infections.
      Furthermore, the explanted CIED components, including the pocket generator and leads, were all placed together into the container, thus detaching large amounts of bacteria from the biofilm. Other researchers have also advocated alternative techniques to swab and tissue culture for the recovery of microorganisms from CIEDs, and they have all concluded that low-intensity ultrasound applied to the retrieved implants to dislodge microorganisms from the biofilm represents a more sensitive technique, especially in patients receiving antimicrobial therapy.
      • Oliva A.
      • Nguyen B.L.
      • Mascellino M.T.
      • D’Abramo A.
      • Iannetta M.
      • Ciccaglioni A.
      • et al.
      Sonication of explanted cardiac implants device infections improves microbial detection in cardiac devices infections.
      • Rohacek M.
      • Weisser M.
      • Kobza R.
      • Schoenenberger A.W.
      • Pfyffer G.E.
      • Frei R.
      • et al.
      Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.
      • Mason P.K.
      • Dimarco J.P.
      • Ferguson J.D.
      • Mahapatra S.
      • Mangrum J.M.
      • Bilchick K.C.
      • et al.
      Sonication of explanted cardiac rhythm management devices for the diagnosis of pocket infections and asymptomatic bacterial colonization.
      Classically, Gram-positive bacteria (both CoNS and S. aureus) have been by far the most common pathogens isolated in CIDAIs.
      • Sandoe J.A.
      • Barlow G.
      • Chambers J.B.
      • Gammage M.
      • Guleri A.
      • Howard P.
      • et al.
      Guidelines for the diagnosis, prevention and management of implantable cardiac electronic device infection. Report of a joint working party project on behalf of the British Society for Antimicrobial Chemotherapy (BSAC, host organization), British Heart Rhythm Society (BHRS), British Cardiovascular Society (BCS), British Heart Valve Society (BHVS) and British Society for Echocardiography (BSE).
      • Tayebjee M.H.
      • Joy E.R.
      • Sandoe J.A.
      Can implantable cardiac electronic device infections be defined as ‘early’ or ‘late’ based on the cause of infection?.
      Not surprisingly in the present study, sonication fluid cultures of explanted CIEDs yielded mainly Gram-positive cocci (69.5%), mostly CoNS, followed by Streptococcus spp and S. aureus; however, other biofilm-forming pathogens such as S. maltophilia, Corynebacterium sp, A. baumannii, Pseudomonas sp, and Candida sp were also identified. Conversely, among the infected subjects with positive cultures (including two cases of endocarditis-associated CIED), the spectrum of microorganisms identified was different from that reported by other authors, in which half of the bacteria isolated were Gram-negative bacilli, suggesting a strong influence of previous antibiotic intake on the skin flora prior to device removal. Furthermore, subjects presenting late infections are likely to present with non-staphylococcal organisms that may be less frequently diagnosed in pocket infection.
      • Welch M.
      • Uslan D.Z.
      • Greenspon A.J.
      • Sohail M.R.
      • Baddour L.M.
      • Blank E.
      • et al.
      Variability in clinical features of early versus late cardiovascular implantable electronic device pocket infection.
      • Viola G.M.
      • Awan L.L.
      • Ostrosky-Zeichner L.
      • Chan W.
      • Darouiche R.O.
      Infections of cardiac implantable electronic devices: a retrospective multicenter observational study.
      • Tayebjee M.H.
      • Joy E.R.
      • Sandoe J.A.
      Can implantable cardiac electronic device infections be defined as ‘early’ or ‘late’ based on the cause of infection?.
      Asymptomatic bacterial colonization due to biofilm formation on the surface of CIEDs has been defined as a risk factor for CIDAI, with rates varying from 21% to 27% when phenotypic methods of investigation were applied.
      • Rohacek M.
      • Weisser M.
      • Kobza R.
      • Schoenenberger A.W.
      • Pfyffer G.E.
      • Frei R.
      • et al.
      Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.
      • Mason P.K.
      • Dimarco J.P.
      • Ferguson J.D.
      • Mahapatra S.
      • Mangrum J.M.
      • Bilchick K.C.
      • et al.
      Sonication of explanted cardiac rhythm management devices for the diagnosis of pocket infections and asymptomatic bacterial colonization.
      Nevertheless, authors using molecular biology methods were more successful in identifying microorganisms colonizing uninfected CIEDs, with rates ranging from 38.5% to 47.2%.
      • Pichlmaier M.
      • Marwitz V.
      • Kühn C.
      • Niehaus M.
      • Klein G.
      • Bara C.
      • et al.
      High prevalence of asymptomatic bacterial colonization of rhythm management devices.
      • Chu X.M.
      • Li B.
      • An Y.
      • Li X.B.
      • Guo J.H.
      Genetic identification and risk factor analysis of asymptomatic bacterial colonization on cardiovascular implantable electronic devices.
      In the present study, subjects presenting no clinical signs or symptoms of infection had microorganisms cultured in 51.5% of CIEDs submitted to sonication, but no growth was seen on tissue/blood culture. Whether the isolation of microorganisms on cardiac devices submitted to sonication means true colonization or future clinical pocket infection, or even simply contamination of the processes, has been the focus of literature debate.
      • Oliva A.
      • Nguyen B.L.
      • Mascellino M.T.
      • D’Abramo A.
      • Iannetta M.
      • Ciccaglioni A.
      • et al.
      Sonication of explanted cardiac implants device infections improves microbial detection in cardiac devices infections.
      • Rohacek M.
      • Weisser M.
      • Kobza R.
      • Schoenenberger A.W.
      • Pfyffer G.E.
      • Frei R.
      • et al.
      Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.
      • Chu X.M.
      • Li B.
      • An Y.
      • Li X.B.
      • Guo J.H.
      Genetic identification and risk factor analysis of asymptomatic bacterial colonization on cardiovascular implantable electronic devices.
      • Welch M.
      • Uslan D.Z.
      • Greenspon A.J.
      • Sohail M.R.
      • Baddour L.M.
      • Blank E.
      • et al.
      Variability in clinical features of early versus late cardiovascular implantable electronic device pocket infection.
      Previous studies have demonstrated that compared to conventional tissue culture, sonication of infected implanted devices achieves higher rates of sensitivity, but that specificity may be less accurate due to the nature of biofilm-associated colonization.
      • Yano M.H.
      • Klautau G.B.
      • da Silva C.B.
      • Nigro S.
      • Avanzi O.
      • Mercadante M.T.
      • et al.
      Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.
      This is especially true among subjects using antibiotics, which affects bacterial growth on tissues but not within the biofilm. The most common microorganisms cultured in colonized subjects belonged to the skin flora, predominantly Gram-positive (69.5%), particularly CoNS (43.5%). Previous authors have obtained similar results, in which ubiquitous Gram-positive organisms of the skin flora predominate over other organisms.
      • Pichlmaier M.
      • Marwitz V.
      • Kühn C.
      • Niehaus M.
      • Klein G.
      • Bara C.
      • et al.
      High prevalence of asymptomatic bacterial colonization of rhythm management devices.
      • Chu X.M.
      • Li B.
      • An Y.
      • Li X.B.
      • Guo J.H.
      Genetic identification and risk factor analysis of asymptomatic bacterial colonization on cardiovascular implantable electronic devices.
      It is argued that applying sonication routinely for every explanted cardiac device would be costly and that it is therefore unfeasible. Nevertheless, as the clinical signs of biofilm-associated infection may be undetected, sonication of the CIEDs may be useful to increase microbiological diagnosis in the case of a symptomatic or even asymptomatic subject presenting a higher risk of infection (prior CIDAI or a higher number of previous local procedures).
      Fewer non-infected subjects (23.5%) had tissue/blood cultures performed, which might have led to bias with regard to the higher sensitivity of sonication. This study had several other limitations, including the relatively small sample size involved and the high rates of chronic medical conditions such as heart failure, Chagas diseases, and diabetes, which might have influenced the high rates of colonization. In addition, the follow-up period was no longer than 6 months, and it was therefore not possible to assess the real impact of device colonization on the subsequent development of clinical infection over time. As already stated by others, the non-blinded collection of infected and non-infected subjects also limited the results.
      • Rohacek M.
      • Weisser M.
      • Kobza R.
      • Schoenenberger A.W.
      • Pfyffer G.E.
      • Frei R.
      • et al.
      Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.
      In summary, the results of this study demonstrated that sonication culture is superior to tissue/blood culture for the microbial diagnosis of cardiac implantable device infections, especially among subjects using systemic antibiotics. Since half of all CIDAIs are caused by Gram-negative bacteria, the present findings should prompt clinicians to choose a broad empiric antibiotic therapy to cover Gram-positive, but also Gram-negative bacteria, until cultures identify the causative agent(s). To be able to better understand the epidemiology of colonized cardiac implants in uninfected subjects and to assess the risk of subsequent infection, sonication enables the growth of a high proportion of latent organisms encased in the biofilm.

      Acknowledgements

      We thank Eliete Celestino, Lucia Hiromi Kawai, Iolanda Santana, and Regiane Leandro for their outstanding technical assistance, and Erika Fukunaga for her support in the statistical analysis.
      This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) under award number 2013/002039. The sponsor had no involvement in the study design, in the collection, analysis, and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.
      Conflict of interest: None.

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