- Email: [email protected]
Q fever endocarditis associated with a cardiovascular implantable electronic device
RESEARCH NOTE
10.1111/j.1469-0691.2012.03992.x
Q fever endocarditis associated with a cardiovascular implantable electronic device J. A. Oteo1, S. Pe´rez-Corte´s2, P. Santiba´n˜ez1, E. Gutie´rrez3, A. Portillo1, J. R. Blanco1 and A. de Alarco´n4 1) A´rea de Enfermedades Infecciosas, Hospital San Pedro-CIBIR, Logron˜o, 2) Unidad de Enfermedades Infecciosas, Servicio de Medicina Interna, Hospital de Jerez de la Frontera, Ca´diz, 3) Servicio de Cirugı´a Cardiaca, Hospital Virgen del Rocı´o, Sevilla and 4) Servicio de Enfermedades Infec-
ity and mortality [2]. Staphylococcal species are the main cause of CIED infections, representing 60–80% of cases in most series. However, in 8% of events there is not a causative agent [3]. Coxiella burnetii is one of the most important microorganisms implicated in blood culture-negative endocarditis in our geographical area [4,5] but to our knowledge, no cases of PPM or ICD-related endocarditis have been published. Herein we report two cases of CIED infections by the Q fever agent (one of them co-infected with Brucella melitensis).
ciosas, Hospital Virgen del Rocı´o, Sevilla, Spain
Clinical Cases Abstract Case 1
Cardiovascular implantable electronic devices (CIEDs) are frequently related to endocarditis. Most cases of intravascular CIED infections are usually related to skin flora, but a few cases may occur with negative blood culture. Coxiella burnetii is one of the main causes of blood culture-negative endocarditis in native and prosthetic valves, but to date no cases related to CIED have been published. Herein we report two cases of Q fever endocarditis related to these non-valvular cardiovascular devices.
Keywords: Cardiovascular implantable electronic device, cardioverter-defrillator device, Coxiella burnetii, endocarditis, pacemaker, Q fever. Original Submission: 26 April 2012; Revised Submission: 5 July 2012; Accepted: 11 July 2012 Editor: D. Raoult Clin Microbiol Infect
Corresponding author: J. A. Oteo, Hospital San Pedro-CIBIR, A´rea de Enfermedades Infecciosas, C/Piqueras, 98-7ª N.E., 26006 Logron˜o, La Rioja, Spain E-mail: [email protected]
Indications for cardiovascular implantable electronic devices (CIEDs) such as permanent pacemakers (PPMs) and implantable cardioverter-defibrillators (ICDs) have increased in the last decades. Infections of these electronic devices have been recognized as important challenges in clinical practice since 1971 [1]. Although most infections are limited to the pocket, involvement of intravascular wires accounts for approximately 10% of cases, with high morbid-
A 51-year-old man with a bicameral ICD developed intermittent fever, purpuric skin lesions in the legs, bone pain and wrist arthritis 4 years after its implantation. Blood cultures were positive for Brucella melitensis and seroagglutination showed titres of 5210. The patient was treated with streptomycin (21 days) plus doxycycline (45 days). The outcome was initially favourable, but 4 months later he developed similar symptoms. Relapse was considered and a similar treatment was started. He also had a favourable course but developed the same clinical picture 6 months later. Then he was treated again for Brucella infection and completely recovered. On this occasion blood cultures were negative. Five months later the patient was again symptomatic, and indirect immunofluorescence assays (IFAs) against C. burnetii showed IgG titres of 1024 (phases I and II) (Focus Diagnostics, Cypress, CA, USA). A transoesophageal echocardiography (TEE) showed vegetations on the pacemaker wires. Doxycycline plus ciprofloxacin were prescribed. The device was completely removed by percutaneous traction. DNA was extracted from the vegetations using the QIAmp DNA Tissue minikit (Quiagen, Hilden, Germany). Polymerase chain reaction (PCR) methods targeting the htp-AB gene for C. burnetii, and 16S rRNA, 31-kDa protein and omp-2 genes for Brucella spp. were positive. PCR primers and size of amplicons are shown in Table 1. Negative controls (with template DNA but without primers and with primers and containing water instead of DNA) were included. Nucleotide sequence analysis showed 100% identity with htp-AB from C. burnetii and with 16S rRNA, 31-kDa protein and omp-2 genes from Brucella melitensis, respectively. Sequences for htp-AB and 16S rRNA generated in this study were deposited in GenBank under accession numbers JX275487 and JX275486, respectively. Specimens were also analysed by IS30a quantitative PCR (qPCR) using a 7300 Real Time PCR system (Applied Biosystems, Foster City, CA,
ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases
2
Clinical Microbiology and Infection
CMI
TABLE 1. PCR primer pairs used for conventional PCR methods in this study Bacteria genus
Target gene
Primer name
Primer sequence (5¢ fi 3¢)
Amplified fragment (bp)
Reference
Coxiella
htp-AB
CB-1 CB-2 Trans-1 Trans-2 F4 R2 B4 B5 JPF JPR
ACT CAA CGC ACT GGA ACC GC TAG CTG AAG CCA ATT CGC C TAT GTA TCC ACC GTA GCC AGT C CCC AAC AAC ACC TCC TTA TTC TCG AGC GCC CGC AAG GGG AAC CAT AGT GTC TCC ACT AA TGG CTC GGT TGC CAA TAT CAA CGC GCT TGC CTT TCA GGT CTG GCG CTC AGG CTG CCG ACG CAA ACC AGC CAT TGC GGT CGG TA
257
[6]
687
[7]
905
[8]
223
[9]
193
[10]
IS1111 Brucella
16S rRNA 31-kDa antigen omp-2
TABLE 2. PCR primer pairs and probe used for the quantitative PCR method in this study Bacteria genus
Target gene
Primer and probe name
Primer and probe sequence (5¢ fi 3¢)
Coxiella
IS30a
Cbis30aF Cbis30aR IS30a
AAT GTC TGC GGG AAA TAG GC GAG GCC TTT TAC CGG AAT TC 6FAM-TCG AGA TCA-ZEN-TAG CGT CAT T-IABkFQ
USA), according to Brouqui et al. (2005) with slight modifications [11]. Primers and probe are shown in Table 2. The PCR mixture contained 10 lL of Premix Ex Taq (Probe qPCR) (Takara), 0.4 lL (10 lM) of each primer, 0.4 lL (10 lM) of a double-quenched hydrolysis probe (Integrated DNA Technologies, Coralville, IA, USA), 0.4 lL ROX reference dye, 3.4 lL of distilled water, and 5 lL of DNA. Using this technique, the sample was positive with a cycle threshold value of 32.8. Culture in cells was not undertaken. A new device was implanted 1 month later. The patient completed 12 months of treatment and, 1 year after the cessation of antimicrobial therapy, remains asymptomatic. Case 2
A 74-year-old man with a PPM was admitted for fever, malaise, anorexia, weight loss and cough during the last month. Before admission he had been treated at home with amoxicillin and then ciprofloxacin, without improvement. A chest X-ray and a computed tomography (CT) scan showed multiple cavitated nodules in both lungs. A TEE was made and vegetations on the tricuspid valve and wires were identified. Repeated blood cultures were negative. Staphylococcal infection was suspected and daptomycin plus gentamycin were administered. Ten days later, the device was extracted by right auriculotomy. PCR assays for C. burnetii intergenic spacer IS1111 from vegetations were performed as previously described (Table 1), yielding positive results. Sequencing confirmed highest identity (99.4%) with C. burnetii (GenBank accession no. JX275488). This sample was positive with a cycle threshold value of 34.2 using IS30a qPCR. IFA showed IgG titres of 1600 (phases I and II). Culture in cells was not undertaken. Doxycyclin and chloroquine were
Amplified fragment (bp)
Reference
120
[11]
administered and the patient, still on treatment, is now asymptomatic.
Discussion In the last years CIED endocarditis has been recognized as an important problem in clinical practice. As occurs for endocarditis on native or prosthetic valves, implicated microorganisms are not always recognized. Although few infections are due to microorganisms coming from a remote source that shed on wires, contamination from skin flora during surgery is the main mechanism for development of infection on these devices. Therefore, Staphylococcus spp. are the most frequent microorganisms involved [3]. When blood cultures are negative, empirical treatment is usually given for this aetiology. C. burnetti and Bartonella spp. are the main bacteria implicated in endocarditis with negative blood culture in Europe, with important series published in France and Spain [4,5,12]. Recently, a large outbreak has been described in the Netherlands and, from 2007 to 2009, 3.1% of 3264 cases of acute Q fever developed endocarditis [13]. However, no Q fever infections on CIEDs have been reported. In our first case, molecular biological tools also demonstrated chronic infection by B. melitensis. Brucella endocarditis is a rare and life threatening complication of brucellosis. Only seven cases on CIEDs have been reported [14]. Our patient had three relapses despite appropriate treatment. Co-infection with C. burnetii could have been the cause because this agent usually requires 12–24 months of treatment [3,4]. Doxycycline and hydroxychloroquine were not prescribed in order to avoid photosensitivity problems, because the patient works
ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI
Research Note
CMI
outdoors in a sunny area of southern Spain. Surgical removal of the device and accurate diagnosis were essential to achieve the cure. We think that molecular biological techniques may be an important aid also in this setting, as has been demonstrated for blood culture-negative endocarditis [15].
Acknowledgements This work was presented in part at the XVI Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) Conference held in Bilbao, Spain, May 2012.
Transparency Declaration The authors do not have any potential conflicts of interests related particularly to this manuscript.
References 1. Schwartz IS, Pervez N. Bacterial endocarditis associated with a permanent transvenous cardiac pacemaker. JAMA 1971; 218: 736–737. 2. Baddour LM, Epstein AE, Erickson CC, Knight BP, Levison ME, Lockhart PB. Update on cardiovascular implantable electronic device infections and their management. Circulation 2010; 121: 458–477. 3. Sohail MR, Uslan DZ, Khan AH et al. Management and outcome of permanent and implantable cardioverter-defibrillator infections. J Am Coll Cardiol 2007; 49: 1851–1859.
3
4. Fenollar F, Fournier PE, Carrieri MP, Habib G, Messana T, Raoult D. Risk factors and prevention of Q fever endocarditis. Clin Infect Dis 2001; 33: 312–316. 5. Mogollo´n MV, Anguita MP, Aguado JM et al. Q fever endocarditis in Spain. Clinical characteristics and outcome. Enferm Infecc Microbiol Clin 2011; 29: 109–116. 6. Stein A, Raoult D. Detection of Coxiella burnetii by DNA amplification using PCR. J Clin Microbiol 1992; 30: 2462–2466. 7. Willems H, Thiele D, Fro¨lich-Ritter R, Krauss H. Detection of Coxiella burnetii in cow’s milk using the polymerase chain reaction (PCR). J Vet Med 1994; 41: 580–587. 8. Romero C, Gamazo C, Pardo M, Lo´pez-Gon˜i I. Specific detection of Brucella DNA by PCR. J Clin Microbiol 1995; 33: 615–617. 9. Baily GG, Krahn JB, Drasar BS, Stoker NG. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 1992; 95: 271–275. 10. Leal-Klevezas DS, Martı´nez-Va´zquez IO, Lo´pez-Merino A, Martı´nezSoriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol 1995; 33: 3087–3090. 11. Brouqui P, Rolain JM, Foucault C, Raoult D. Q fever and Plasmodium falciparum malaria co-infection in a patient returning from the Comoros Archipielago. Am J Trop Med Hyg 2005; 73: 1028–1030. 12. Million M, Thuny F, Richet H, Raoult D. Long-term outcome of Q fever endocarditis: a 26-year personal survey. Lancet Infect Dis 2010; 10: 527–535. 13. Dijkstra R, van der Hoek W, Wijers N et al. The 2007–2010 Q fever epidemic in the Netherlands: characteristics of notified acute Q fever patients and the association with dairy goat farming. FEMS Immunol Med Microbiol 2012; 64: 3–12. 14. Al-Majid FM. Pacemaker lead endocarditis due to Brucellosis. Saudi Med J 2010; 31: 448–450. 15. Fournier PE, Thuny F, Richet H et al. Comprehensive diagnostic strategy for blood culture-negative endocarditis. A prospective study of 819 new cases. Clin Infect Dis 2010; 51: 131–140.
ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI
10.1111/j.1469-0691.2012.03992.x
Q fever endocarditis associated with a cardiovascular implantable electronic device J. A. Oteo1, S. Pe´rez-Corte´s2, P. Santiba´n˜ez1, E. Gutie´rrez3, A. Portillo1, J. R. Blanco1 and A. de Alarco´n4 1) A´rea de Enfermedades Infecciosas, Hospital San Pedro-CIBIR, Logron˜o, 2) Unidad de Enfermedades Infecciosas, Servicio de Medicina Interna, Hospital de Jerez de la Frontera, Ca´diz, 3) Servicio de Cirugı´a Cardiaca, Hospital Virgen del Rocı´o, Sevilla and 4) Servicio de Enfermedades Infec-
ity and mortality [2]. Staphylococcal species are the main cause of CIED infections, representing 60–80% of cases in most series. However, in 8% of events there is not a causative agent [3]. Coxiella burnetii is one of the most important microorganisms implicated in blood culture-negative endocarditis in our geographical area [4,5] but to our knowledge, no cases of PPM or ICD-related endocarditis have been published. Herein we report two cases of CIED infections by the Q fever agent (one of them co-infected with Brucella melitensis).
ciosas, Hospital Virgen del Rocı´o, Sevilla, Spain
Clinical Cases Abstract Case 1
Cardiovascular implantable electronic devices (CIEDs) are frequently related to endocarditis. Most cases of intravascular CIED infections are usually related to skin flora, but a few cases may occur with negative blood culture. Coxiella burnetii is one of the main causes of blood culture-negative endocarditis in native and prosthetic valves, but to date no cases related to CIED have been published. Herein we report two cases of Q fever endocarditis related to these non-valvular cardiovascular devices.
Keywords: Cardiovascular implantable electronic device, cardioverter-defrillator device, Coxiella burnetii, endocarditis, pacemaker, Q fever. Original Submission: 26 April 2012; Revised Submission: 5 July 2012; Accepted: 11 July 2012 Editor: D. Raoult Clin Microbiol Infect
Corresponding author: J. A. Oteo, Hospital San Pedro-CIBIR, A´rea de Enfermedades Infecciosas, C/Piqueras, 98-7ª N.E., 26006 Logron˜o, La Rioja, Spain E-mail: [email protected]
Indications for cardiovascular implantable electronic devices (CIEDs) such as permanent pacemakers (PPMs) and implantable cardioverter-defibrillators (ICDs) have increased in the last decades. Infections of these electronic devices have been recognized as important challenges in clinical practice since 1971 [1]. Although most infections are limited to the pocket, involvement of intravascular wires accounts for approximately 10% of cases, with high morbid-
A 51-year-old man with a bicameral ICD developed intermittent fever, purpuric skin lesions in the legs, bone pain and wrist arthritis 4 years after its implantation. Blood cultures were positive for Brucella melitensis and seroagglutination showed titres of 5210. The patient was treated with streptomycin (21 days) plus doxycycline (45 days). The outcome was initially favourable, but 4 months later he developed similar symptoms. Relapse was considered and a similar treatment was started. He also had a favourable course but developed the same clinical picture 6 months later. Then he was treated again for Brucella infection and completely recovered. On this occasion blood cultures were negative. Five months later the patient was again symptomatic, and indirect immunofluorescence assays (IFAs) against C. burnetii showed IgG titres of 1024 (phases I and II) (Focus Diagnostics, Cypress, CA, USA). A transoesophageal echocardiography (TEE) showed vegetations on the pacemaker wires. Doxycycline plus ciprofloxacin were prescribed. The device was completely removed by percutaneous traction. DNA was extracted from the vegetations using the QIAmp DNA Tissue minikit (Quiagen, Hilden, Germany). Polymerase chain reaction (PCR) methods targeting the htp-AB gene for C. burnetii, and 16S rRNA, 31-kDa protein and omp-2 genes for Brucella spp. were positive. PCR primers and size of amplicons are shown in Table 1. Negative controls (with template DNA but without primers and with primers and containing water instead of DNA) were included. Nucleotide sequence analysis showed 100% identity with htp-AB from C. burnetii and with 16S rRNA, 31-kDa protein and omp-2 genes from Brucella melitensis, respectively. Sequences for htp-AB and 16S rRNA generated in this study were deposited in GenBank under accession numbers JX275487 and JX275486, respectively. Specimens were also analysed by IS30a quantitative PCR (qPCR) using a 7300 Real Time PCR system (Applied Biosystems, Foster City, CA,
ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases
2
Clinical Microbiology and Infection
CMI
TABLE 1. PCR primer pairs used for conventional PCR methods in this study Bacteria genus
Target gene
Primer name
Primer sequence (5¢ fi 3¢)
Amplified fragment (bp)
Reference
Coxiella
htp-AB
CB-1 CB-2 Trans-1 Trans-2 F4 R2 B4 B5 JPF JPR
ACT CAA CGC ACT GGA ACC GC TAG CTG AAG CCA ATT CGC C TAT GTA TCC ACC GTA GCC AGT C CCC AAC AAC ACC TCC TTA TTC TCG AGC GCC CGC AAG GGG AAC CAT AGT GTC TCC ACT AA TGG CTC GGT TGC CAA TAT CAA CGC GCT TGC CTT TCA GGT CTG GCG CTC AGG CTG CCG ACG CAA ACC AGC CAT TGC GGT CGG TA
257
[6]
687
[7]
905
[8]
223
[9]
193
[10]
IS1111 Brucella
16S rRNA 31-kDa antigen omp-2
TABLE 2. PCR primer pairs and probe used for the quantitative PCR method in this study Bacteria genus
Target gene
Primer and probe name
Primer and probe sequence (5¢ fi 3¢)
Coxiella
IS30a
Cbis30aF Cbis30aR IS30a
AAT GTC TGC GGG AAA TAG GC GAG GCC TTT TAC CGG AAT TC 6FAM-TCG AGA TCA-ZEN-TAG CGT CAT T-IABkFQ
USA), according to Brouqui et al. (2005) with slight modifications [11]. Primers and probe are shown in Table 2. The PCR mixture contained 10 lL of Premix Ex Taq (Probe qPCR) (Takara), 0.4 lL (10 lM) of each primer, 0.4 lL (10 lM) of a double-quenched hydrolysis probe (Integrated DNA Technologies, Coralville, IA, USA), 0.4 lL ROX reference dye, 3.4 lL of distilled water, and 5 lL of DNA. Using this technique, the sample was positive with a cycle threshold value of 32.8. Culture in cells was not undertaken. A new device was implanted 1 month later. The patient completed 12 months of treatment and, 1 year after the cessation of antimicrobial therapy, remains asymptomatic. Case 2
A 74-year-old man with a PPM was admitted for fever, malaise, anorexia, weight loss and cough during the last month. Before admission he had been treated at home with amoxicillin and then ciprofloxacin, without improvement. A chest X-ray and a computed tomography (CT) scan showed multiple cavitated nodules in both lungs. A TEE was made and vegetations on the tricuspid valve and wires were identified. Repeated blood cultures were negative. Staphylococcal infection was suspected and daptomycin plus gentamycin were administered. Ten days later, the device was extracted by right auriculotomy. PCR assays for C. burnetii intergenic spacer IS1111 from vegetations were performed as previously described (Table 1), yielding positive results. Sequencing confirmed highest identity (99.4%) with C. burnetii (GenBank accession no. JX275488). This sample was positive with a cycle threshold value of 34.2 using IS30a qPCR. IFA showed IgG titres of 1600 (phases I and II). Culture in cells was not undertaken. Doxycyclin and chloroquine were
Amplified fragment (bp)
Reference
120
[11]
administered and the patient, still on treatment, is now asymptomatic.
Discussion In the last years CIED endocarditis has been recognized as an important problem in clinical practice. As occurs for endocarditis on native or prosthetic valves, implicated microorganisms are not always recognized. Although few infections are due to microorganisms coming from a remote source that shed on wires, contamination from skin flora during surgery is the main mechanism for development of infection on these devices. Therefore, Staphylococcus spp. are the most frequent microorganisms involved [3]. When blood cultures are negative, empirical treatment is usually given for this aetiology. C. burnetti and Bartonella spp. are the main bacteria implicated in endocarditis with negative blood culture in Europe, with important series published in France and Spain [4,5,12]. Recently, a large outbreak has been described in the Netherlands and, from 2007 to 2009, 3.1% of 3264 cases of acute Q fever developed endocarditis [13]. However, no Q fever infections on CIEDs have been reported. In our first case, molecular biological tools also demonstrated chronic infection by B. melitensis. Brucella endocarditis is a rare and life threatening complication of brucellosis. Only seven cases on CIEDs have been reported [14]. Our patient had three relapses despite appropriate treatment. Co-infection with C. burnetii could have been the cause because this agent usually requires 12–24 months of treatment [3,4]. Doxycycline and hydroxychloroquine were not prescribed in order to avoid photosensitivity problems, because the patient works
ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI
Research Note
CMI
outdoors in a sunny area of southern Spain. Surgical removal of the device and accurate diagnosis were essential to achieve the cure. We think that molecular biological techniques may be an important aid also in this setting, as has been demonstrated for blood culture-negative endocarditis [15].
Acknowledgements This work was presented in part at the XVI Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) Conference held in Bilbao, Spain, May 2012.
Transparency Declaration The authors do not have any potential conflicts of interests related particularly to this manuscript.
References 1. Schwartz IS, Pervez N. Bacterial endocarditis associated with a permanent transvenous cardiac pacemaker. JAMA 1971; 218: 736–737. 2. Baddour LM, Epstein AE, Erickson CC, Knight BP, Levison ME, Lockhart PB. Update on cardiovascular implantable electronic device infections and their management. Circulation 2010; 121: 458–477. 3. Sohail MR, Uslan DZ, Khan AH et al. Management and outcome of permanent and implantable cardioverter-defibrillator infections. J Am Coll Cardiol 2007; 49: 1851–1859.
3
4. Fenollar F, Fournier PE, Carrieri MP, Habib G, Messana T, Raoult D. Risk factors and prevention of Q fever endocarditis. Clin Infect Dis 2001; 33: 312–316. 5. Mogollo´n MV, Anguita MP, Aguado JM et al. Q fever endocarditis in Spain. Clinical characteristics and outcome. Enferm Infecc Microbiol Clin 2011; 29: 109–116. 6. Stein A, Raoult D. Detection of Coxiella burnetii by DNA amplification using PCR. J Clin Microbiol 1992; 30: 2462–2466. 7. Willems H, Thiele D, Fro¨lich-Ritter R, Krauss H. Detection of Coxiella burnetii in cow’s milk using the polymerase chain reaction (PCR). J Vet Med 1994; 41: 580–587. 8. Romero C, Gamazo C, Pardo M, Lo´pez-Gon˜i I. Specific detection of Brucella DNA by PCR. J Clin Microbiol 1995; 33: 615–617. 9. Baily GG, Krahn JB, Drasar BS, Stoker NG. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 1992; 95: 271–275. 10. Leal-Klevezas DS, Martı´nez-Va´zquez IO, Lo´pez-Merino A, Martı´nezSoriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol 1995; 33: 3087–3090. 11. Brouqui P, Rolain JM, Foucault C, Raoult D. Q fever and Plasmodium falciparum malaria co-infection in a patient returning from the Comoros Archipielago. Am J Trop Med Hyg 2005; 73: 1028–1030. 12. Million M, Thuny F, Richet H, Raoult D. Long-term outcome of Q fever endocarditis: a 26-year personal survey. Lancet Infect Dis 2010; 10: 527–535. 13. Dijkstra R, van der Hoek W, Wijers N et al. The 2007–2010 Q fever epidemic in the Netherlands: characteristics of notified acute Q fever patients and the association with dairy goat farming. FEMS Immunol Med Microbiol 2012; 64: 3–12. 14. Al-Majid FM. Pacemaker lead endocarditis due to Brucellosis. Saudi Med J 2010; 31: 448–450. 15. Fournier PE, Thuny F, Richet H et al. Comprehensive diagnostic strategy for blood culture-negative endocarditis. A prospective study of 819 new cases. Clin Infect Dis 2010; 51: 131–140.
ª2012 The Authors Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI